external scientific report - ChemService

Supporting Publications 2015:EN-478

EXTERNAL SCIENTIFIC REPORT

Collate literature data on toxicity of Chromium (Cr) and Nickel (Ni) in experimental animals and humans1 Carlotta Casalegno, Onofrio Schifanella, Eleonora Zennaro, Sandro Marroncelli, Robert Briant ChemService S.r.l. Controlli e Ricerche Novate Milanese (MI), Italy

ABSTRACT Literature data on Chromium show that trivalent chromium has low acute and long term toxicity whilst hexavalent chromium is acutely toxic and produces long term effects on hematological parameters and liver. Continuous exposure to high concentrations of hexavalent chromium in drinking water results in intestinal tumors in mice but not rats. However, evidence of the carcinogenic potential of chromium has been demonstrated in rats but not consistently in mice. Cr (III) organic complexes (chromium picolinate) did not show evident adverse effects after repeated oral exposure. Both in vitro and in vivo data show that trivalent chromium is not genotoxic whilst hexavalent chromium is genotoxic. Chromium has been shown to affect sperm, estrous cycle and fetal development. Human toxicity data reveals mixed results but there is some evidence that hexavalent chromium can increase the risk of cancer. Data on Nickel show that nickel soluble compounds (nickel sulphate, nickel chloride or nickel nitrate) have acute and long term toxicity and produce oxidative stress and long term effects on liver. Less soluble compounds (nickel sulfides or nickel oxides) are less toxic. Nickel shows genotoxic effects both in vitro and in vivo. Only a limited number of studies on carcinogenic effects after oral exposure to nickel compounds are available. These studies showed no neoplastic effects in rats after oral administration. Nickel has been shown to affect sperm, live litter size and post-implantation loss. Teratogenic effects are reported on Amphibian embryos. Nickel can affect neurodifferentiation, the T-cell system and suppress the activity of natural killer cells. Human data reveals that nickel is excreted in the urine following oral exposure and that this increases with increasing age. There is some evidence that nickel might promote oral cancer etiology but no clear evidence that nickel can increase the risk of respiratory cancer. © ChemService S.r.l. Controlli e Ricerche, 2015

KEY WORDS Chromium, Nickel, oral toxicity, humans, experimental animals

DISCLAIMER The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender procedure. The present document is published complying with the transparency principle to which the Authority is subject. It may not be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 1

Question No EFSA-Q-2012-00662.

Any enquiries related to this output should be addressed to [email protected] Suggested citation: Carlotta Casalegno, Onofrio Schifanella, Eleonora Zennaro, Sandro Marroncelli, Robert Briant; Collate literature data on toxicity of Chromium (Cr) and Nickel (Ni) in experimental animals and humans. Supporting Publications 2015:EN-478. [287 pp.]. Available online: www.efsa.europa.eu/publications

© European Food Safety Authority, 2015

Chromium and Nickel oral toxicity in experimental animals and humans

SUMMARY Exposure to Nickel and Chromium for the general non-smoking population is primarily from food and drinking water and to a lesser extent through inhalation of ambient air. Regulation 1881/2006/EC2 and its amendments, establishes the maximum levels (MLs) for certain contaminants in foodstuffs including lead, cadmium, mercury and inorganic tin. There are not MLs set for nickel and chromium in food. There are however, maximum permissible levels for drinking water of 20μg nickel/L and of 50μg chromium/L as laid down in Council Directive 98/83/EC3. In March 2012 EFSA received a mandate from the Hellenic Food Authority (EFET) for a scientific opinion on estimation of the risk to human health from the presence of Nickel (Ni) and Chromium (Cr) in vegetables and hexavalent chromium (CrVI) in bottled water. The CONTAM Panel of EFSA accepted the mandate and proposes to deliver two separate Scientific Opinions: the first one on the risks to human health related to the presence of Chromium in vegetables and hexavalent Chromium in bottled water (EFSA-Q-2012-00379); and a second one on the risk to human health related to the presence of Nickel in food (EFSA-Q-2012-00378). In order to facilitate the CONTAM panel in this task, the preparatory work of collecting scientific information within the public domain on the toxicity of Chromium (including hexavalent Chromium) and Nickel in experimental animals and humans was outsourced. To identify the relevant literature, i.e. data on toxicity and toxicokinetics of chromium and nickel after oral exposure in experimental animals and humans, an extensive literature search was conducted and the results of the search screened for relevance. The scientific literature on studies in experimental animals and humans available in the public domain and identified to be relevant to assess oral toxicity of Chromium (Cr), including hexavalent chromium (CrVI), and Nickel (Ni) is documented in the present report under Section I and Section II, respectively. The scientific information was gathered from different sources of the available public information (scientific literature, EU/US/OECD/National authorities’ various sources, industry associations). Even though priority was given to information from peer reviewed sources, other available data, e.g. grey literature, was also taken into account. The key data reported for Chromium (Section I) and for Nickel (Section II), e.g. NOAEL or dose (in mg/kg bw/day), are always those of the authors of the papers or those reported in relevant sources of information (e.g. ECHA web site). The authors of the present report have neither calculated any parameter nor added any personal interpretation of the results. Section I – Data on Chromium (including hexavalent Chromium). The time-frame for the extensive literature search is from 2000 to January 2013. More than 3100 abstracts were screened to select the relevant studies to be analysed in depth to retrieve the studies to be documented in this report. The first gross screening identified 349 abstracts on Chromium toxicity and, after a second screening, the abstracts identified to be relevant to assess oral toxicity of Chromium (Cr) including hexavalent Chromium were 141. 2

Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. OJ L 364, 20.12.2006, p. 5-24.

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Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption. OJ L 330, 5.12.98, p. 32-54.

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The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender procedure. The present document is published complying with the transparency principle to which the Authority is subject. It may not be considered as an output adopted by the Authority. The European food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.


Cr(III) when administered orally (gavage) either as inorganic salt or organic complex was not acutely toxic to rats, with LD50>2000 mg/kg bw. On the contrary, water-soluble Cr(VI) compounds (chromium trioxide, sodium chromate, sodium dichromate, potassium dichromate) were acutely toxic to rats, with LD50 ranging from 46 to 113 mg/kg bw/day (male/female rats), and mice, with LD50 ranging from 135 to 175 mg/kg bw (male/female mice). The repeated oral administration of Cr(VI) lead to various adverse effects for example on body weight gain (reduction), on hematological parameters and on liver. Signs of oxidative stress (increases in GSH and GSSG levels, SOD and CAT activity) after repeated oral exposure to Cr(VI) are clearly reported by various authors. Altered hepatic functions (e.g. increased bile acid concentrations) and increased nonneoplastic lesions (focal ulceration, regenerative epithelial hyperplasia, and squamous epithelial metaplasia) in the glandular stomach of male and female rats exposed to sodium dichromate dihydrate administered via drinking water for 90 days were reported. In the NTP 90-day toxicity study used as a sighting study for the subsequent carcinogenicity, the administration of sodium dichromate in drinking water to rats for 90 days caused effects on the red blood cell (microcytic anaemia) and liver. Local irritant effects on the non-glandular gastric mucosa were also apparent. In some cases, repeated dose toxicity studies have been conducted to investigate the modes of action underlying oral mucosa tumors after exposure to Cr (VI). After 90 days oral administration of potassium dichromate, histopathological findings in the duodenum, biochemical analyses related to oxidative status and increase of chromium content in target tissues (oral cavity, duodenum, jejunum) were reported. Adverse effects on fertility of female rats and on pups through lactation were reported in a combined repeated dose and reproduction / developmental screening study where potassium dichromate was administered through drinking water to lactating maternal rats, and via maternal milk to pups. There was a significant decrease of body and uterus weight and a dose-dependent increase of Chromium (CrVI) concentration in the serum and uterus. Both serum and uterus Cr(VI) levels gradually decreased with the increase in age. The onset of puberty was delayed in a dose-dependent manner and extension in diestrous and metestrous stage was recorded. Continuous exposure to high concentrations of hexavalent chromium in drinking water resulted in intestinal tumors in mice but not rats. A study of the concentration-dependent gene expression effects in rat and mice duodenal and jejunal epithelia following 7 and 90 days of exposure to sodium dichromate dehydrate showed that the phenotypic anchoring of rat differential gene expression responses was related to gene expression for oxidative stress response, to cell growth that stabilizes the gastrointestinal mucosa and provides a physical barrier against toxic agents, to immune response, DNA damage and modification genes. Cr(III) organic complexes (chromium picolinate, a commonly used nutritional supplement in humans) did not show evident adverse effects after repeated oral exposure. A significant number of relevant publications report data on genotoxicity in vitro and in vivo. In vitro data confirm that hexavalent chromium is genotoxic but that trivalent chromium is not genotoxic. Alternate results exist for chromium picolinate that was genotoxic when tested on mouse lymphoma L5178Y cells and not genotoxic when tested on chinese hamster ovary cells. Chromium trispicolinate was not genotoxic


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when tested on chinese hamster ovary cells. Overall, the weight of evidence suggests that chromium (III) and (IV) are not genotoxic in vivo whilst genotoxicity has been reported for chromium (VI). Evidence of the carcinogenic potential of chromium has been demonstrated in a number of studies in rats but not consistently in mice. The incidence of preputial gland adenoma was significantly increased in a two year study in Fischer 344 rats administered chromium picolinate monohydrate in the feed. However, the authors reported that no neoplastic or non-neoplastic lesions were attributed to the test item when fed to mice. Sodium dichromate dihydrate supplied in the drinking water for two years to Fischer 344 rats resulted in significantly increased incidences of highly aggressive neoplasms of the squamous epithelium that lines the oral cavity (oral mucosa and tongue) and in significant increases in histiocytic cell infiltration in duodenum, mesenteric lymph nodes, liver and pancreatic lymph nodes. Administration of the test item to B6C3F1 mice caused small intestine neoplasms and hyperplasia and significant increases in histiocytic cell infiltration in duodenum, mesenteric lymph nodes, jejunum, liver and pancreatic lymph nodes. Carcinogenicity was reported in rats and mice administered sodium dichromate dihydrate in drinking water for 90 days. In a 90-day study, drinking water administration to B6C3F1 mice resulted in an overall gene expression pattern induced by Cr(VI) that was more similar to non mutagenic carcinogens, however Cr(VI) clustered separately from the four non mutagenic compounds. In another study it was concluded that with the exception of Cyp1b1, expression of all drug metabolism genes was unaffected by Cr(VI) exposure via sodium dichromate dihydrate in the drinking water for two months. It was reported that CRL: SK1-hrBR mice that were given potassium dichromate in drinking water for six months showed no visible skin tumors whereas when they were also exposed to UV irradiation there was a dose dependent increase in tumors compared to mice exposed to UV and no test item. In an in vitro mechanistic study with sodium dichromate using human lung epithelial cancer cell line NCI-H460, it was demonstrated that Cr(VI) treatment caused a dose dependent increase in cell apoptosis, induced hydroperoxide and O2 production in a dose-dependent manner and induced both caspase-8 and caspase-9 activation. There was also a dose-dependent decrease in Bcl-2 expression level. The results suggest that the mitochondrial pathway is the major apoptotic pathway induced in response to Cr(VI) exposure. There are only a very limited number of studies that examined the effects of chromium on reproduction parameters. Effects on sperm and the estrous cycle of Fischer 344 rats given chromium picolinate monohydrate in the diet for 93 days were reported. Daily intraperitoneal injections of potassium dichromate for 26 days to male Sprague-Dawley rats decreased epididymal sperm number and accessory sex organ weight. A study report on potassium dichromate refers to three studies in mice in which there were effects on fertility when the test item was administered in the drinking water. Potassium chromate administered to male monkeys in drinking water resulted in decreased sperm count and motility. The treatment also caused suppression of superoxide dismutase and catalase activity, decreased glutathione levels and increased H2O2 production in seminal plasma and sperm. Simultaneous administration of vitamin C and withdrawal of chromium treatment after six months both resulted in a reversal of the effects. The effects of chromium on developmental parameters have been reported in studies in rats and mice. Following dietary administration of chromium(III) propionate cation in the form of nitrate salt to Wistar rats, maternal tissue mineral levels were altered; chromium levels were increased in liver and kidneys whilst copper and zinc levels in liver were decreased. No adverse affects were observed on fetal viability or body and organ weights and there were no malformations but fetal liver zinc levels increased and fetal kidney copper levels decreased. The effects of administration of potassium dichromate via the drinking Supporting publications 2015:EN-478

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water were reported in three studies. Renal disfunctions were recorded in the form of increased urinary volume, creatinine, urea and uric acid and levels of kidney malondialdehyde and lactate dehydrogenase. The effects of Cr(VI) on bone maturity of suckling rats were reported as decreased femur weight and length, decline in calcium and phosphorus levels in bone, increased calcium and decreased phosphorus in plasma and decreased calcium and increased phosphorus in urine, increased lipid peroxidation in bone and decreased femur NPSH, GSH and vitamin C levels and antioxidant enzyme activities, increased ACP and decreased ALP in plasma. In the third study, decreased relative liver weight of pups was recorded together with a significant increase in liver malondialdehyde in dams and pups. Liver anitoxidant activities were altered; there were significant decreases in CAT and GPx activities in dams and pups while SOD was increased significantly in dams but decreased in pups. In both dams and pups the hepatotoxicity biomarkers, plasma transaminases (AST and ALT) acitivities and bilirubin level were significantly increased while LDH activity increased in plasma and decreased in liver. Histological changes were also recorded in liver but were more pronounced in dams than in pups. There are three studies in mice in which chromium was administered as chromium picolinate. Increased incidence of bifurcated arches was reported in one study. An increase in the number of resorbed or dead foetuses was reported in a second study and no maternal or fetal toxicity was observed in the third study. Reviews of the human toxicokinetic literature provided evidence that exposure to trivalent chromium resulted in significant increases in urinary concentrations of creatinine and increased levels of chromium in the blood and urine. Increased creatine levels were observed in red blood cells, plasma and urine in a man who had been exposed to potassium dichromate. Elimination from serum followed an open twocompartment model. Chromium kinetics was shown to be independent of the oxidation state of the administered chromium, except for the fraction absorbed from the intestine. Workers exposed to chromium dust (mixtures of trivalent chromium, metallic chromium, chromite, ferrochromium, and contaminants) during grinding, pelletizing and sintering of concentrated chromite, and crushing of cooled ferrochromium castings were studied. Cough or dyspnoea and shortness of breath were significantly more frequent. The authors concluded that the reason for the symptoms was irritation which was not related to smoking. In a further report, it was shown that there were no statistically significant differences in the frequency of centromere-negative or centromere-positive micronuclei and no differences in relation to either nasal diseases and symptoms, mucociliary clearance of the nasal cavity, or incidence of cell atypia. Volunteers exposed to sodium dichromate showed an increase in DNA strand breaks. There was substantial interindividual variation but there was a statistically significant positive correlation between levels of formamidopyrimidine glycosylase-dependent oxidative DNA damage and antioxidant capacity. Analyses of data obtained on cancer mortalities in Chinese populations in an area where contamination of groundwater by Cr(VI) had occurred due to disposal of chromium waste into the river by a nearby plant showed no correlation between cancer mortality rates and degree of exposure. On the contrary, in a region of Greece where water had been contaminated with hexavalent chromium there was a slight increase in cancer deaths but it was not statistically significant. However, for primary liver cancer, deaths were eleven-fold higher than the expected number of deaths among both males and females and deaths associated with kidney and other genitourinary organ cancers were more than threefold higher than expected in women. Elevated mortality ratios were also observed for other cancer sites. It was noted that three out of the six deaths associated with primary liver cancer and two out of the five deaths associated with female kidney and other genitourinary organ cancers came from a small village and the highest concentration of hexavalent chromium was found in a well close to that village. The mortality of workers involved in the production of stainless and alloyed steel in France for at least one year during the years 1968-1991 was studied in order to investigate the risk of lung cancer due to exposure to chromium and/or Supporting publications 2015:EN-478

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nickel compounds or iron oxides. No lung cancer excess was observed for exposure to chromium. A cohort of 2357 workers employed at a chromate production plant was studied for lung cancer occurrence in relation to exposure to trivalent or hexavalent chromium. Proportional hazards models were used to assess the relationship between cumulative chromium exposure and lung cancer mortality risk. Cumulative trivalent chromium exposure and duration of work were not found to be associated with a risk of lung cancer whilst cumulative hexavalent chromium exposure, on the other hand, showed a strong dose-response relationship. Elevated chromium levels in blood samples in patients in Central Taiwan demonstrated a strong association with oral cancer. The results of a clinical trial of chromium picolinate administration via capsule showed no changes in FPG, 2-hour plasma glucose levels, fasting or 2-hour post–oral glucose tolerance test insulin levels, or HOMA-IR as compared with placebo after 6 months of chromium use. There were no significant changes in glycohemoglobin, weight, waist circumference, BMI, blood pressure, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, or urine microalbumin. An asophagogastroduodenal endoscopy of a patient who was admitted to hospital after oral ingestion of potassium dichromate revealed necrotic and bleeding lesions of the gastric mucosa. Laboratory findings on day 3 showed kidney and liver failure. In a clinical case study of a patient who had taken various oral vitamin and mineral preparations, subacute dermatitis with erythema and scaling was present in scattered patches primarily on the lower legs, ankles, hands, and wrists. Several in vitro studies have been performed using human cells. The role of NER in the removal of chromium-DNA adducts using potassium dichromate has been reported. In another study, a 50% decrease in (H3)-thymidine incorporation was observed after 24 hours incubation of human blood lymphocytes with Cr(V) complexes and it was dose dependent. Under identical conditions, the decrease in (H3)thymidine incorporation was nearly 90% with Cr(VI) complexes. In a further study, it was shown that treatment of lymphocytes with Na[Cr(V)O(ehba)(2)] and K (2)Cr(2)O(7) leads to the activation of the Srcfamily protein tyrosine kinases namely, p56(lck), p59(fyn), and p56/53(lyn), which then activates caspase-3, both of which are under the partial influence of reactive oxygen species. Responses of human stem cells CD34 + hematopoietic progenitor cells to hexavalent chromium were studied and cell loss and inhibition of cluster formation was observed together with marked alterations in subcellular structures. Potassium dichromate increased the frequency of aneuploid cells. Section II – Data on Nickel. The time-frame for the extensive literature search is from 2000 to April 2013 except for Area 1 where the search timeframe was extended up to 1993 due to the limited number of results retrieved in this area for Nickel. 1284 abstracts were screened to select the relevant studies to be analysed in depth to retrieve the studies to be documented in this report. The first gross screening identified 176 abstracts on Nickel toxicity and after a second screening the abstracts identified to be relevant to assess oral toxicity of Nickel (Ni) was 157. For completness, the literature included in EU RAR on Nickel and relevant to Area 2 has been checked and a summary of studies relevant to oral toxicity of Nickel given in Appendix 1. When administered orally, the absorption of nickel soluble compounds (nickel sulphate, nickel chloride, nickel nitrate) was higher than Ni insoluble compounds (carbonates, sulfides, oxides). ASingle or repeated oral administration of soluble nickel compounds in rodents lead to elevated nickel concentrations in liver and kidney while insoluble compounds (metallic nickel) accumulated in lungs and pancreas. Data on nickel metabolism and elimination in rodents confirmed that nickel was poorly absorbed from diets and was eliminated mainly in the faeces. Absorbed nickel was rapidly cleared from serum and excreted in Supporting publications 2015:EN-478

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urine. The mechanism of toxicity of metals involves a common cascade of events which entails an oxidative stress and production of reactive oxygen species and a single oral administration of nickel sulphate through drinking water in male rats led to increase the hepatic lipid peroxidation and to decrease the antioxidant ezyme activities. Nickel soluble compounds (nickel sulphate or nickel chloride) were acutely toxic to rats, with LD50 > 300 < 2000 mg/kg bw, while less soluble compounds were not acutely toxic to rats, with LD50 > 2000 mg/kg bw (nickel dihydroxide) or even higher, ranging from 8796 to >11000 mg/kg bw/day (female rats) (nickel oxide black, nickel oxide green). Studies on repeated dose toxicity of nickel sulphate, nickel chloride and nickel bis (2-ethylhexanoate) are available for rodents (rats, mice), hens, chicken, rabbits and fish (Coregonus spp.). The studies were conducted according to OECD 408 -Repeated Dose 90-Day Oral Toxicity in Rodents, OECD 453 combined chronic/ carcinogenicity, OECD 451 Carcinigenicity or were performed as non-guideline studies with exposure durations ranging from 8/10 weeks up to 6 months. Available data on nickel sulphate in rats showed that after 104 weeks exposure the NOAEL was equal to 2.2 mg/kg bw/day and the LOAEL ranged from 6.7 mg/kg bw/day (104 weeks exposure) to 30 mg/kg bw/day (90 days exposure), based on reduced body weight. The repeated oral administration of nickel sulphate through drinking water led to significant increase of concentrations of nickel in liver tissue and of oxidative stress (increases in hepatic lipid peroxidation, catalase, glutathione peroxidase, glutathione reductase, superoxide dismutase activity, and glutathione-S-transferase) (Sidhu 2005). It was also observed that the repeated oral administration of nickel sulphate through drinking water in mice lead to nickel accumulation in the interstitial tissue of the testes and to a decrease in the seminal vesicle weight, diameter, and activity of epithelium. This appears to be in accord with the theory that nickel influences the production of testosterone. A repeated oral administration (subacute -10 d, subchronic -31 d, and chronic -104 d) of nickel sulphate through diet in fish (Coregonus clupeaformis) led to histopathological lesions in liver (areas of focal necrosis and altered bile ducts) and in kidney (lesions in glomeruli, tubules, collecting ducts, and hematopoietic tissue). Significant increases of Metalliothionein in intestine (on day 10) and lipid peroxide concentration in plasma (on day 31) were also observed. A LOAEL of 10 mg/kg bw/day, based on decrease of the body weight at 30 mg/kg bw, was reported for rats exposed to nickel bis (2-ethylhexanoate) for 104 weeks through drinking water, showing that the oral toxicity after repeated exposure to nickel bis (2-ethylhexanoate) is comparable to that of nickel sulphate. The repeated oral ingestion of nickel chloride through drinking water (1200 ppm, nominal in water) led to reduced body weight gain and to an increase of lung and brain weights in rats. The same author also observed that nickel induced the iron uptake by serum and some organs. The repeated oral administration of nickel chloride through diet (500 mg Ni/kg nominal in diet) led to a reduced body weight gain and to an increase of enzyme concentrations (i.e., triglycerides, alanine transferase, gamma-glutamyltransferase, and cholinesterase) in broiler chickens and rabbits. Detrimental effects were also reported in hens where nickel chloride led to decreases in egg production and weight. Several reports on the effects of repeated oral administration on sensitization to nickel were retrieved. These data showed that the repeated oral administration of water enriched with nickel chloride in mice prevented subsequent sensitization to this common contact allergen and that iNKT cells are required for the induction of oral tolerance toward nickel, but not for nickel sensitization. There is clear evidence for the in vitro genotoxicity of nickel salts. Although most of the classical bacterial and mammalian cell culture mutagenicity tests yielded negative results, positive effects were Supporting publications 2015:EN-478

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generally seen in studies on chromosomal effects (chromosomal aberrations, sister chromatid exchanges, micronuclei), and tests for DNA damage and repair. Data on in vitro genotoxicity confirm that nickel (tested as nickel compounds zero valent, tetrakis(tritolyl phosphite)nickel and molibdenum nickel tetraoxide) was not genotoxic on bacteria cells (various strains of S.typhimurium and E. coli) with and without metabolic activation. Conflicting results were reported on mammalian cell mutagenicity. Both negative and positive effects were observed in chromosome aberration assays (OECD 473 Chromosome Aberration Assay and micronucleus test), on gene mutation assays (OECD 476) and on DNA damage or repair assays. Nickel sulphate showed positive effects both on DNA damage or repair and on chromosomal aberrations (micronucleus test). Nickel dihydroxide and nickel dichloride showed positive effects in a mammalian cell gene mutation assay and in DNA damage or repair, respectively. Nickel oxide was genotoxic. Nickel hidroxycarbonate was negative in a mammalian cell gene mutation assay and tetrakis(tritolyl phosphite)nickel was negative in chromosome aberration assays and ambiguous in cell gene mutation assay. Retrieved data suggests that nickel is genotoxic in vivo and show that nickel-induced responses involved cell toxicity in all gene mutation studies using mammalian cells. Positive effects in rats and mice were reported for nickel chloride, nickel dichloride and nickel oxide in DNA damage and repair, Comet assay and FADU assay. Positive effects were also reported for nickel dichloride and nickel sulphate in Drosophila melanogaster when tested for DNA damage and for gene mutation. A limited number of studies on carcinogenic effects after oral exposure to nickel compounds were retrieved. These studies showed no neoplastic effects in rats after oral administration of nickel bis (2ethylhexanoate) and nickel sulphate for 104 weeks. These findings are in accordance with previous knowledge showing that no increase of tumour incidence was observed in lifetime exposed rats to drinking-water and feed containing nickel and in dogs exposed for 2 years. Mice drinking nickel in water had significantly higher skin concentration compared with mice having no nickel in water, and the cocarcinogenic effect of oral nickel with UVR as a matter of skin cancer yield and incidence was directly correlated with the nickel concentration in the skin. Dietary administration of ten doses of nickel sulphate to male Wistar rats resulted in significant reduction in the activities of the testicular steroidogenic enzymes and plasma testosterone concentration accompanied by a significant elevation in cholesterol and ascorbic acid levels. Gavage doses of nickel sulphate (5 days/week for 35 days) to male mice resulted in adverse sperm effects (reduced motility and count and abnormalities). A NOAEL of 5 mg NiSO4/ kg bw/day was identified. In a two-generation study in Sprague-Dawley rats given gavage doses of nickel sulphate hexahydrate the NOAEL was 2.2 mg Ni/kg bw, since there were no effects on reproduction at this highest exposure level used in the study. In a onegeneration gavage study in the same strain, the incidence of dead pups on lactation day 0 was significantly increased and mean live litter size was significantly decreased at 75 mg NiSO4/kg/day. Mean post-implantation loss was significantly increased at dosage levels ≥ 30 mg/kg/day. The main teratogenic effects on Rhinella arenarum (Amphibia, Anura) embryos treated with NiCl2 6H2O for 24 hours were retarded growth and development, extremely severe axis incurvations, persistent yolk plug, asymmetry, microcephaly and mouth and gill agenesia, and limited neuromuscular activity. Ciliated cells were not functional. EC50 (malformation) early embryo-larval developmental toxicity values of 0.42 mg Ni/L for Xenopus laevis, 3.94 mg Ni/L for Bufo terrestris and 0.61 mg/L Ni for Gastrophryne carolinensis exposed to NiCl2 for 4 days were reported.


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In vitro data on neuronotypic PC12 cells assessing gene transcription involved in the cyclic AMP pathway showed that Ni can produce outcomes by targeting cell signaling pathways involved in neurodifferentiation. Voltage dependent block effects on NR channels and voltage independent inhibition causing a decrease of Po (Po= probability that at least one channel is open) was reported in Xenopus laevis oocytes exposed to Ni2+. Potentiation of NMDA current in rat cerebellar granules cells (from 8-day old rats) exposed in vitro for 16 days and inhibitory effects on NMDA current in rat embryo cortical neurons exposed in vitro for 35 days was also reported. Nickel salts were reported to affect the T-cell system and suppress the activity of natural killer cells in rats and mice. Dietary exposure of male and female Wistar rats to NiCl 2 resulted in dose- and time-dependent immunosuppression effects on T-lymphocyte proliferation and Th1 (IFN-gamma) and Th2 (IL-10) cytokine production. Production of the pro-inflammatory cytokine TNF-alpha was inhibited in a dosedependent manner. There was a dose-dependent increase in the production of the anti-inflammatory cytokine IL-10 from lipopolysaccharide (LPS) stimulated cultures. Minimal plasma concentrations of nickel (209-585 ng/mL) were required to provoke immunosuppression. Effects on natural killer cells were also recorded in vitro. The viability of murine macrophages was reduced following in vitro exposure to NiCl2. In vitro exposure of NiCl2 for 2 hours to spleen cells of female Sprague-Dawley rats and male/female cynomolgus monkeys resulted in statistically significant decrease in NK cell activity of both species. Reviews of the human toxicokinetic literature show that nickel is excreted in the urine following oral exposure and that this increases with increasing age. Ni excretion in urine significantly increased with increasing age, ingestion of dietary supplements, drinking of stagnant tap water, and consumption of nickel-rich food. There was a strong correlation between age and Ni bioaccumulation in a population drinking water and eating fish from a dam in Sao Paulo, Brazil containing high levels of heavy metals; bioaccumulation was more intense in the childhood period and was followed by low clearance in contrast to other heavy metals. Concentrations in hair and exhaled breath condensate are increased following occupational exposure. Occupational contact of Russian chemical plant workers with Ni was reflected in a pronounced increase of the content in hair and urine but not in the blood and plasma profiles. Ni concentration in scalp hair was also increased in patients admitted to hospital in Hyderabad, Pakistan suffering with myocardial infarction and that levels were higher in third-attack patients as compared to first- and second-attack patients. There was a significant increase of Ni concentrations in exhaled breath condensate from welders at three German companies compared to control subjects. However, there was no correlation between Ni in the air and in post-shift urine of Japanese workers at a battery plant using nickel hydroxide, probably due to the use of respiratory protection; however, the authors suggest that the results showed that exposure to nickel hydroxide yields lower urine Ni concentrations than very soluble nickel salts. Residents from areas of moderate fighting in Croatia had significantly higher serum levels of Ni than participants from areas of heavy fighting, though there were no significant differences in urine or hair levels of Ni between the two study groups. Ni concentrations in urine of the three groups of workers (plant, laboratory and administrative) at a Spanish incinerator were significantly lower than those of the baseline survey suggesting that there was no occupational Ni exposure. Allergic contact dermatitis is the most prevalent effect of nickel in the general population and several papers were reviewed concerning the effects of oral exposure to nickel in patients affected by nickel induced systemic contact dermatitis. Ingested Ni may induce flare-up of cutaneous reactions in some nickel-allergic patients, independently of the degree of sensitization and the intake of metal. Nickel Supporting publications 2015:EN-478

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stimulates the immune system, inducing maturation of T lymphocytes from virgin into memory cells; these latter cells seem to accumulate in the intestinal mucosa. However, the evidence is inconclusive that dermatitis may occur in allergic patients in a dose-dependent manner. A study in Taiwan of mortality due to oral cancer showed that some metals (Cr, Cu, Ni, and Zn) in soils might act as promoters in oral cancer etiology. However, the evidence from four studies indicates that exposure to nickel did not increase the risk of respiratory cancer. A study on reproductive effects observed in female workers in a Russian refinery was reported in a series of papers from the same authors. Maternal exposure to water-soluble nickel was not correlated with the observed reproductive impairment or the increased incidence of perinatal effects in infants (spontaneous abortions, small-for-gestational-age newborns and musculosketal effects in newborns). In a clinical case study involving a two-year old infant who had a previous history of asthma and dermatitis from contact with sleeper snaps, edematous red papules occurred on the trunk, extremities, face, and ears after he had swallowed two coins. One year later the child developed acute generalised dermatitis again and another coin was found in his stomach. A number of in vitro mechanistic studies in human cells have been reported. Nickel induces changes in gene expression but does not affect the replication fidelity of human cells. Nuclear factor etythroid-2 related factor 2 (Nrf2) has a protective role in the suppression of mutagenicity and carcinogenicity resulting from environmental nickel exposure. Reactive oxygen species (ROS) have an important role in the induction of histone hypoacetylation caused by nickel. Nickel reduces cellular anti-oxidative defence activities. Nickel chloride from dental alloys can induce alterations in the cellular redox state of oral epithelium at concentrations which do not cause evident cell viability. In this context nickel alters nuclear levels of several subtypes of the NFjB pathway when the pathway is activated by LPS (an Escherichia coli serotype which is a broad activator of monocytes. Nickel-induced changes in cytokine secretion by monocytes are diverse and may be influenced by Nrf2.


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TABLE OF CONTENTS Abstract ......................................................................................................................................................... 1 Summary ....................................................................................................................................................... 2 Table of contents ......................................................................................................................................... 11 List of tables ................................................................................................................................................ 13 Background ................................................................................................................................................. 16 Terms of reference....................................................................................................................................... 16 Acknowledgement ....................................................................................................................................... 17 Introduction and objectives ......................................................................................................................... 18 Materials and methods................................................................................................................................. 18 Search strategy ........................................................................................................................................ 18 Selection criteria ..................................................................................................................................... 19 Relevant results ....................................................................................................................................... 20 Description of the studies........................................................................................................................ 21 Results section I – Data on Chromium ........................................................................................................ 23 1 Toxicokinetics (Absorption, Metabolism, Distribution and Elimination) in Experimental Animals and Humans (Area 1) .................................................................................................................... 23 1.1 Summary ................................................................................................................................... 23 1.2 Non-human information............................................................................................................. 26 1.3 Human data from toxicokinetic studies ..................................................................................... 36 1.4 Human data from occupational exposure and poisoning incidents ............................................ 39 1.5 Data on dental alloys (non-relevant data) .................................................................................. 41 2 Toxicity in Experimental Animals (Area 2) ....................................................................................... 42 2.1 Acute toxicity ............................................................................................................................. 42 2.1.1 Rats ........................................................................................................................................ 42 2.1.2 Mice ....................................................................................................................................... 45 2.1.3 Other Animals........................................................................................................................ 48 2.1.4 Metal mixtures ....................................................................................................................... 50 2.2 Repeated dose toxicity ............................................................................................................... 50 2.2.1 Rats ........................................................................................................................................ 52 2.2.2 Mice ....................................................................................................................................... 64 2.2.3 Other animals......................................................................................................................... 66 2.3 Genotoxicity............................................................................................................................... 66 2.3.1 Genotoxicity in vitro.............................................................................................................. 66 2.3.1.1 Bacteria cells ................................................................................................................. 67 2.3.1.2 Mammalian cells ........................................................................................................... 71 2.3.1.3 Data on mechanism of action ........................................................................................ 79 2.3.2 Genotoxicity in vivo............................................................................................................... 84 2.3.2.1 Mammalian ................................................................................................................... 85 2.3.2.2 Other animals ................................................................................................................ 92 2.3.3 Carcinogenicity...................................................................................................................... 92 2.3.3.1 Rats ............................................................................................................................... 94 2.3.3.2 Mice .............................................................................................................................. 99 Supporting publications 2015:EN-478

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2.3.3.3 Data on mechanism of action ...................................................................................... 114 2.3.4 Toxicity to reproduction ...................................................................................................... 115 2.3.4.1 Toxicity to reproduction ............................................................................................. 115 2.3.4.2 Developmental toxicity ............................................................................................... 124 2.3.5 Immunotoxicity ................................................................................................................... 134 3 Data on observations in Humans (Area 3) ........................................................................................ 136 3.1 Summary .................................................................................................................................. 136 3.2 Health surveillance data ........................................................................................................... 138 3.2.1 Toxicokinetic data from health surveillance........................................................................ 138 3.2.2 Repeated dose toxicity data in humans from health surveillance ........................................ 141 3.2.3 Genotoxicity in humans from health surveillance ............................................................... 142 3.2.4 Carcinogenicity in humans (oral exposure) ......................................................................... 144 3.2.5 Carcinogenicity in humans (inhalation exposure) ............................................................... 148 3.2.6 Carcinogenicity in humans (retrospective studies) .............................................................. 151 3.2.7 Clinical trials in human subjects with metabolic syndromes (retrospective studies) .......... 153 3.3 Epidemiological data ............................................................................................................... 155 3.4 Clinical cases, poisoning incident ............................................................................................ 155 3.5 In vitro data .............................................................................................................................. 156 References ................................................................................................................................................. 165 Abbreviations ............................................................................................................................................ 174 Results section II – Data on Nickel ........................................................................................................... 177 4 Toxicokinetics (Absorption, Metabolism, Distribution and Elimination) in Experimental Animals and Humans (Area 1) .................................................................................................................. 177 4.1 Summary .................................................................................................................................. 177 4.2 Non-human information........................................................................................................... 180 4.3 In vitro data on toxicokinetics.................................................................................................. 188 4.4 Human data from toxicokinetic studies ................................................................................... 191 4.5 Human data from occupational exposure................................................................................. 193 4.6 Other animals ........................................................................................................................... 194 5 Toxicity in Experimental Animals (Area 2) ..................................................................................... 195 5.1 Acute toxicity ........................................................................................................................... 195 5.1.1 Rats ...................................................................................................................................... 195 5.2 Repeated dose toxicity ............................................................................................................. 199 5.2.1 Rats ...................................................................................................................................... 200 5.2.2 Mice ..................................................................................................................................... 203 5.2.3 Other animals....................................................................................................................... 205 5.3 Genotoxicity............................................................................................................................. 207 5.3.1 Genotoxicity in vitro............................................................................................................ 207 5.3.1.1 Bacteria cells ............................................................................................................... 209 5.3.1.2 Mammalian cells ......................................................................................................... 211 5.3.1.3 Data on mechanism of action ...................................................................................... 220 5.3.2 Genotoxicity in vivo............................................................................................................. 221 5.3.2.1 Mammalian ................................................................................................................. 221 5.3.2.2 Other animals .............................................................................................................. 223 5.3.3 Carcinogenicity.................................................................................................................... 224 Supporting publications 2015:EN-478

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5.3.3.1 Rats ............................................................................................................................. 225 5.3.3.2 Mice ............................................................................................................................ 226 5.3.3.3 Data on mechanism of action ...................................................................................... 227 5.3.4 Toxicity to reproduction ...................................................................................................... 227 5.3.4.1 Toxicity to reproduction ............................................................................................. 227 5.3.4.2 Developmental toxicity ............................................................................................... 230 5.3.5 Neurotoxicity ....................................................................................................................... 234 5.3.5.1 In vitro data ................................................................................................................. 234 5.3.6 Immunotoxicity ................................................................................................................... 237 5.3.6.1 Rats ............................................................................................................................. 237 5.3.6.2 In vitro data ................................................................................................................. 239 6 Data on observations in Humans (Area 3) ........................................................................................ 240 6.1 Summary .................................................................................................................................. 240 6.2 Health surveillance data ........................................................................................................... 243 6.2.1 Toxicokinetic data ............................................................................................................... 243 6.2.2 Ocupational data on dermal exposure.................................................................................. 250 6.2.3 Effects of nickel oral exposure in nickel-sensitive patients ................................................. 251 6.2.4 Genotoxicity in humans from epidemiological studies ....................................................... 256 6.2.5 Carcinogenicity in humans (oral exposure) ......................................................................... 257 6.2.6 Carcinogenicity in humans (inhalation exposure) ............................................................... 258 6.2.7 Developmental toxicity in humans ...................................................................................... 261 6.2.8 Clinical case studies in human ............................................................................................. 262 6.3 In vitro data on human cells ..................................................................................................... 263 References ................................................................................................................................................. 269 Abbreviations ............................................................................................................................................ 277 Appendix 1 ................................................................................................................................................ 281 LIST OF TABLES SECTION I – Data on Chromium Table 1: Toxicokinetics-non-human information........................................................................................ 26 Table 2: Human data from toxicokinetic studies ......................................................................................... 36 Table 3: Human data from occupational exposure and poisoning incidents ............................................... 39 Table 4: Data on dental alloys (non-relevant data) ..................................................................................... 41 Table 5: Acute toxicity in rats ..................................................................................................................... 42 Table 6: Acute toxicity in mice ................................................................................................................... 45 Table 7: Acute Toxicity on Other Animals ................................................................................................. 48 Table 8: Acute toxicity of metal mixtures ................................................................................................... 50 Table 9: Repeated dose toxicity in rats........................................................................................................ 52 Table 10: Repeated dose toxicity in mice.................................................................................................... 64 Table 11: Genetic toxicity in vitro-bacterical cells ..................................................................................... 67 Table 12: genetic toxicity in vitro–mammalian cells .................................................................................. 71 Table 13: Genetic toxicity in vitro–mecchanism of action.......................................................................... 79 Table 14: Genetic toxicity in vivo-mammalian ........................................................................................... 85 Table 15: Genetic toxicity in vivo-other animals ........................................................................................ 92 Table 16: Carcinogenicity in rats ................................................................................................................ 94 Supporting publications 2015:EN-478

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Table 17: Carcinogenicity in mice .............................................................................................................. 99 Table 18: Mechanism of action ................................................................................................................. 114 Table 19: Toxicity to reproduction in rats ................................................................................................. 115 Table 20: Toxicity to reproduction in mice ............................................................................................... 117 Table 21: Toxicity to reproduction in primates ......................................................................................... 118 Table 22: Developmental toxicity in rats .................................................................................................. 125 Table 23: Developmental toxicity in mice ................................................................................................ 130 Table 24: Developmental toxicity in other animals .................................................................................. 133 Table 25: Developmental toxicity- In vitro Data ...................................................................................... 134 Table 26: In vitro and in vivo immunotoxicity .......................................................................................... 134 Table 27: Toxicokinetic data from health surveillance ............................................................................. 138 Table 28: Repeated dose toxicity data in humans from health surveillance ............................................. 141 Table 29: Genotoxicity in humans from health surveillance ..................................................................... 142 Table 30: Carcinogenicity in humans (oral exposure) .............................................................................. 144 Table 31: Carcinogenicity in humans (inhalation exposure) ..................................................................... 148 Table 32: Carcinogenicity in humans (retrospective studies) ................................................................... 151 Table 33: Clinical trials in human subjects with metabolic syndromes (retrospective studies) ................ 153 Table 34: Clinical cases, poisoning incident ............................................................................................. 155 Table 35: Exposure related observations-in vitro data .............................................................................. 156 SECTION II – Data on Nickel Table 36: Toxicokinetics-non-human information (oral exposure) .......................................................... 180 Table 37: Toxicokinetics-non-human information (other exposure routes) .............................................. 186 Table 38: In vitro data on toxicokinetics (bioaccessibility as surrogate for bioavailability) ..................... 188 Table 39: Human data from toxicokinetic studies related to chronic intake of dietary supplement ......... 191 Table 40: Human data from occupational exposure (inhalation) .............................................................. 193 Table 41: Toxicokinetic data on other animals ......................................................................................... 194 Table 42: Acute toxicity in rats ................................................................................................................. 195 Table 43: Repeated dose toxicity in rats.................................................................................................... 200 Table 44: Repeated dose toxicity in mice.................................................................................................. 203 Table 45: Repeated dose toxicity in other animals .................................................................................... 205 Table 46: Genetic toxicity in vitro-bacterical cells ................................................................................... 209 Table 47: genetic toxicity in vitro–mammalian cells ................................................................................ 211 Table 48: Genetic toxicity in vitro–mecchanism of action........................................................................ 220 Table 49: Genetic toxicity in vivo-mammalian ......................................................................................... 221 Table 50: Genetic toxicity in vivo-other animals ...................................................................................... 223 Table 51: Carcinogenicity in rats .............................................................................................................. 225 Table 52: Carcinogenicity in mice ............................................................................................................ 226 Table 53: Toxicity to reproduction in rats ................................................................................................. 228 Table 54: Toxicity to reproduction in mice ............................................................................................... 230 Table 55: Developmental toxicity in rats .................................................................................................. 231 Table 56: Developmental toxicity in mice ................................................................................................ 232 Table 57: Developmental toxicity in other animals .................................................................................. 232 Table 58: Neurotoxicity – in vitro data ..................................................................................................... 234 Table 59: Immunotoxicity in rats .............................................................................................................. 237 Table 60: Immunotoxicity - in vitro data .................................................................................................. 239 Supporting publications 2015:EN-478

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Table 61: Toxicokinetic data from health surveillance ............................................................................. 243 Table 62: Toxicokinetic data from occupational exposure (inhalation and dermal exposure mainly)...... 245 Table 63: Dermal exposure in workers ..................................................................................................... 250 Table 64: Effects of nickel oral exposure in nickel sensitive patients ....................................................... 251 Table 65: Genotoxicity in humans ............................................................................................................ 256 Table 66: Carcinogenicity in humans (oral exposure) .............................................................................. 257 Table 67: Carcinogenicity in humans (inhalation exposure) ..................................................................... 258 Table 68: Developmental toxicity in human subjects (retrospective studies) ........................................... 261 Table 69: Clinical case studies in human subjects with previous nickel allergic syndromes (respiratory and dermal sensitisation) .................................................................................................................................. 262 Table 70: in vitro data on human cells – genotoxicity and carcinogenicity .............................................. 264 Table 71: in vitro data on human cells – general cytoxicity...................................................................... 264 Table 72: in vitro data on human cells - immunotoxicity ......................................................................... 264


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BACKGROUND Exposure to Nickel and Chromium for the general non-smoking population is primarily from food and drinking water and to a lesser extent through inhalation of ambient air. Regulation 1881/2006/EC4 and its amendments, establishes the maximum levels (MLs) for certain contaminants in foodstuffs including lead, cadmium, mercury and inorganic tin. There are not MLs set for nickel and chromium in food. There are however, maximum permissible levels for drinking water of 20μg nickel/L and of 50μg chromium/L as laid down in Council Directive 98/83/EC5. In March 2012 EFSA received a mandate from the Hellenic Food Authority (EFET) for a scientific opinion on estimation of the risk to human health from the presence of Nickel (Ni) and Chromium (Cr) in vegetables and hexavalent chromium (CrVI) in bottled water. The CONTAM Panel of EFSA accepted the mandate and proposes to deliver two separate Scientific Opinions: the first one on the risks to human health related to the presence of Chromium in vegetables and hexavalent Chromium in bottled water (EFSA-Q-2012-00379); and a second one on the risk to human health related to the presence of Nickel in food (EFSA-Q-2012-00378).

TERMS OF REFERENCE In order to facilitate the CONTAM Panel in its task of delivering two separate Scientific Opinions: the first one on the risks to human health related to the presence of Chromium in vegetables and hexavalent Chromium in bottled water (EFSA-Q-2012-00379); and a second one on the risk to human health related to the presence of Nickel in food (EFSA-Q-2012-00378), EFSA outsourced preparatory work of collecting scientific information within the public domain on the toxicity of Chromium (including hexavalent Chromium) and Nickel in experimental animals and humans. The objective of present procurement procedure was to collect, compile and summarise scientific information within the public domain by extensive literature search on the oral toxicity of Chromium (including hexavalent Chromium) and Nickel. Information on the toxicity of the two elements by routes of exposure other than the oral route was not part of the objectives. Scientific information for the two metals has been collected, compiled and synthesised in the following three toxicology areas:   

Data on toxicokinetics (absorption, distribution, metabolism, excretion) in experimental animals and humans (Area 1); Data on toxicity in experimental animals, including acute, repeat dose toxicity, immunotoxicity, developmental and reproductive toxicity, carcinogenicity, and other effects (Area 2); Data on observation in humans, including epidemiology, case reports and biomarkers of exposure (Area 3)

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Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. OJ L 364, 20.12.2006, p. 5-24. 5

Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption. OJ L 330, 5.12.98, p. 32-54.


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Scientific information gathered from the extensive literature review of each metal has been collected, compiled and summarised in two distinct sections of this report: section I covering toxicological data of Chromium (including hexavalent Chromium); section II covering toxicological data of Nickel. Each section (I and II) outline the key findings in the toxicology areas 1, 2 and 3 (classified into tabular form) as well as an executive summary. For each metal, a IUCLID 5 database with the robust study summaries of relevant data on each of the 3 toxicity areas and a full reference list in an EndNote library file have been also provided.

ACKNOWLEDGEMENT This contract was awarded by EFSA to: ChemService S.r.l. Controlli e Ricerche, Novate Milanese (MI), Italy Contract title: Collate literature data on toxicity of Chromium (Cr) and Nickel (Ni) in experimental animals and humans Contract number: NP/EFSA/CONTAM/2012/01


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INTRODUCTION AND OBJECTIVES This is the final report for the project titled “Collate literature data on toxicity of Chromium (Cr) and Nickel (Ni) in experimental animals and humans” (NP/EFSA/CONTAM/2012/01). The final report is divided in “Section 1” and “Section 2” with human and experimental animal data on Chromium (including hexavalent Chromium) and Nickel, respectively. Exposure to Nickel and Chromium for the general non-smoking population is primarily from food and drinking water and to a lesser extent through inhalation of ambient air. Regulation 1881/2006/EC1 and its amendments, establishes the maximum levels (MLs) for certain contaminants in foodstuffs including lead, cadmium, mercury and inorganic tin. There are not MLs set for nickel and chromium in food. There are however, maximum permissible levels for drinking water of 20μg nickel/L and of 50μg chromium/L as laid down in Council Directive 98/83/EC. In March 2012 EFSA received a mandate from the Hellenic Food Authority (EFET) for a scientific opinion on estimation of the risk to human health from the presence of Nickel (Ni) and Chromium (Cr) in vegetables and hexavalent chromium (CrVI) in bottled water. The CONTAM Panel of EFSA accepted the mandate and proposes to deliver two separate Scientific Opinions: the first one on the risks to human health related to the presence of Chromium in vegetables and hexavalent Chromium in bottled water (EFSA-Q-2012-00379); and a second one on the risk to human health related to the presence of Nickel in food (EFSA-Q-2012-00378). In order to facilitate the CONTAM panel in this task, the preparatory work of collecting scientific information within the public domain on the toxicity of Chromium (including hexavalent Chromium) and Nickel in experimental animals and humans was outsourced. It was the scope of this project to collect scientific information within the public domain on the oral toxicity of Chromium (including hexavalent Chromium) and Nickel in experimental animals and humans. The scientific literature identified to be relevant to assess oral toxicity of Chromium (Cr) including hexavalent chromium (CrVI) and Nickel (Ni) is documented in the present report under Section I and Section II respectively. The scientific information was gathered from different sources of the available public information (scientific literature, EU/US/OECD/National authorities’ various sources). Even though priority was given to information from peer reviewed sources, also other available data as for e.g. grey literature was taken into account.

MATERIALS AND METHODS SEARCH STRATEGY Status of the literature search: from January 2000 up to January 2013 for Chromium As requested in the tender offer the search was limited to the years 2000 onwards unless only limited or incomplete data were retrieved on all 3 areas from 2000 onwards. For Nickel the timeframe was from January 2000 up to April 2013 for Area 2 and Area 3. For Area 1 the timeframe was extended up to 1993 in order to include additional relevant data. For completness, the literature included in EU RAR on Nickel and relevant to oral toxicity of Nickel in experimental animals (Area 2) was checked and main results summarized in Appendix 1. Language limit considered English as well as Italian and, even though priority was given to information from peer reviewed sources, this did not prevented to mention other available data (grey literature) which was considered relevant.


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The publically available literature database Pubmed was used to search the peer reviewed scientific literature for relevant data and to establish a substance specific search strategy. Additionally the scientific databases Toxline, Web of Science, Current Contents and Proceedings were used to refine and supplement the searches from peer reviewed sources. International (ECHA, WHO, JRC, US FDA; EU databases CORDIS and OAIster) and National (Italian Superior Health Institute ISS) authorities web sites were searched to gather relevant grey literature. Google scholar was also searched for any other relevant data. The search strategy for Chromium and Nickel is reported below. The search was built combining search terms taken from Reg (EC) 429/2008 with the following operators:  boolean operators AND (to search both term A and term B), OR (to search term A or term B or both)  proximity operator ADJ (to search term A and B as a phrase in the specified order)  wildcard symbol * that represents zero to n characters in a term that can be used at beginning, middle, end of a term. In this case was used to search “biomarker” and “biomarkers”. Search strategy for Chromium: “Chromium” OR “Cr” OR “Chromate” OR “Bichromate” AND “oral” AND the following terms (based on Reg (EC) 429/2008) combination: “toxicity” OR “toxicokinetics” OR “absorption” OR “distribution” OR “metabolism” OR “excretion” OR “(acute ADJ toxicity)” OR “(repeated ADJ dose)” OR “(repeat ADJ dose)” OR “immunotoxicity” OR “(developmental ADJ toxicity)” OR “(reproductive ADJ toxicity)” OR “carcinogenicity” OR “neurotoxicity” OR “genotoxicity” OR “mutagenicity” OR “clastogenicity” OR “subacute” OR “subchronic” OR “chronic” OR “teratogenicity” OR “epidemiology” OR “(case ADJ reports)” OR “(biomarker*)” OR “exposure” Search strategy for Nickel “Nickel” OR “Ni” AND “oral” AND the same search terms combination used for Chromium reported above SELECTION CRITERIA Chromium: for the years 2000 to January 2013 more than 3100 publications have one of the search terms combinations used for Chromium, however in most cases the searches with the term “Cr” were not relevant to Chromium (Cr referred to e.g. “Creatinine” “Creatine” “Complete Response”, “Complete Remission”, “Calory Restriction”, “cajal-retsius cells”). Nickel: more than 1280 publications have one of the search terms combinations used for Nickel. To identify the relevant studies from this huge amount of publications a screening for relevance based on title and abstracts was performed. The screen for relevance was conducted following the below step-wise procedure, as agreed with EFSA: 1. First gross screening of big search results, applying general criteria. Compile a list of included and excluded papers into End Note. Results on Chromium: 349 papers were included as relevant. Results on Nickel: 176 papers were included as relevant. Supporting publications 2015:EN-478

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2. Second screening to refine the search. Compile a list of included and excluded papers into End Note. Results on Chromium: 140 papers were included as relevant. Results on Nickel: 157 papers were included as relevant In case of doubts about relevance, the title/abstract was discussed with the team and if still uncertain about relevance the full text was purchased to allow a definitive decision. Chromium After the first gross screening, a first list of 349 papers with data on Chromium (including complexes, ions) effects after oral exposure (food, diet, drinking water, gavage, other oral ingestion) on humans, experimental animals or in vitro effects as mutagenicity on bacteria or mammalian cells, and other in vitro effects was compiled. As agreed with EFSA, a definitive list of 141 relevant papers was obtained through a second screening to exclude the papers on invertebrates, on effects of Chromium in diabetic subjects (humans, rats), on effects when administered in combination with other substances (as for e.g. anti diabetic agents) or as a mixture (e.g. oral toxicity of metal mixtures) and on the effects of orthodontic dental alloys containing Chromium on oral mucosa cells. These papers were excluded because in these cases it was not possible/straightforward to infer about toxic effects of chromium. Papers with in vitro data on lung, bronchial or dermal cells together with review papers reporting data already included in the definitive list were also excluded. Nickel After the first gross screening, a first list of 176 papers with data on Nickel (including complexes, ions) effects after oral exposure (food, diet, drinking water, gavage, other oral ingestion) on humans, experimental animals or in vitro effects as mutagenicity on bacteria or mammalian cells, and other in vitro effects was compiled. A definitive list of 157 relevant papers was obtained through a second screening to exclude the papers on invertebrates, on effects of Nickel when administered through other routes (e.g. parenteral route), in combination with other substances or as a mixture (e.g. oral toxicity of metal mixtures). These papers were excluded because in these cases it was not possible/straightforward to infer about toxic effects of chromium. Papers with in vitro data on lung, bronchial or dermal cells considered not relevant however in case of observations in humans (inhalation or dermal occupational exposure) or in vitro data on human cells (Area 3) these were included in the list, summarized and discussed in the present report in view of their relevance to human toxicity. Several data on the effects of orthodontic dental alloys containing Nickel on oral mucosa cells were retrieved however these were excluded from the list of relevant studies with the first gross screening. RELEVANT RESULTS Only studies containing information relevant to toxic effects due to oral exposure to Chromium and Nickel were selected for the report. As the data collection should contribute to the evaluation of the risks to human health related to the presence of Chromium in vegetables and bottled water and to the presence Supporting publications 2015:EN-478

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of Nickel in food, studies dealing with other exposures than oral (e.g. inhalation, dermal, parenteral etc.) were excluded from the further evaluation except for human data (Area 3) where in some cases also inhalation data were considered relevant. Data on wildlife, invertebrates, plants, algae, non genotoxic effects on bacteria were also excluded from the further evaluation. The relevant results (140 relevant results for Chromium; 158 relevant results for Nickel) passing the first and second screening with data on Chromium and Nickel effects after oral exposure on humans, experimental animals or in vitro were searched for full text and, after having grouped papers with same experimental data to avoid bias from double-counting (e.g. data from same experiment published in different journals, different years, by different sub-groups of authors) and excluded review papers reporting data already included in the definitive list, the the papers were summarized as robust study summaries into a IUCLID 5 database. Among groups of papers with same experimental data only the most recent and/or complete papers were included into the IUCLID 5 database which in the end contained 99 robust study summaries for Chromium and 114 for Nickel. Only publications in English and Italian were considered. DESCRIPTION OF THE STUDIES To simplify the reading of the report the relevant studies are described in tabular form using Chemical Safety Report (CSR) template. The CSR template is obtained from a IUCLID plug-in which was implemented during 2009 for version 5.1 to help in the preparation of sections 1 to 7 of the CSR. Considering that the relevant data were entered into IUCLID 5 through copy and paste from pdf files and that the tables of relevant studies were created with IUCLID plug-in, no typing errors are expected. Deviating from this procedure, when the studies were of limited relevance for the overall evaluation, e.g. studies on the effects of Chromium in diabetic subjects, or findings of a study were confirmed by a second study of the same authors using identical study design, these are only reported in short paragraphs of this report and are not included into IUCLID database. Chromium As expected, 90% of relevant studies entered into IUCLID were assigned reliability 2 (Klimisch et al., 1997), being well documented and meeting basic scientific principles but no GLP and no guideline study. Reliability 1 (GLP and guideline study) was assigned to 10% of relevant studies (9 study reports extracted from IUCLID dossiers available on ECHA web site and 1 peer reviewed paper). The studies assigned reliability 1 were all relevant to oral toxicity of Chromium in experimental animals (area 2), reporting data on acute toxicity (2 study reports) and repeated dose toxicity (2 study reports) in rats, in vitro genotoxicity (3 study reports), in vivo genotoxicity (1 study report) and carcinogenicity in mouse and rats (2 study reports). None of the relevant studies was assigned reliability 3 or 4 and a low reliability was not applied as exclusion criteria (no studies were excluded because of reliability 3 or 4). The 60% of relevant studies was on toxicity in experimental animals (area 2: 59 studies) while toxicokinetics data in experimental animals and humans (area 1: 20 studies) and data on humans (area 3: 20 studies) accounted for the remaining 40 %.


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Animal data were restricted to observations in rats, mice, hamster. Few data were available for other animals as for e.g. primates, fish, amphibian and chickens. No relevant data on rabbits, dogs, cats or Guinea Pig were retrieved. The publications dealing with observations in wildlife (e.g. mallard duck), aquatic or terrestrial invertebrates (e.g. Caenorabditis elegans, Ciona intestinalis), plants, algae, and non genotoxicity effects on bacteria (e.g. gut bacteria) were excluded. Nickel 83% of relevant studies entered into IUCLID were assigned reliability 2 (Klimisch et al., 1997), being well documented and meeting basic scientific principles but no GLP and no guideline study. Reliability 1 (GLP and guideline study) was assigned to 17% of relevant studies (19 study reports extracted from IUCLID dossiers available on ECHA web site and 1 peer reviewed paper). The studies assigned reliability 1 were all relevant to oral toxicity of nickel in experimental animals (area 2), reporting data on acute toxicity (8 study reports) and repeated dose toxicity (3 study reports) in rats, in vitro genotoxicity (6 study reports), in vivo genotoxicity (1 study report) and carcinogenicity (2 study reports). None of the relevant studies was assigned reliability 3 or 4 and a low reliability was not applied as exclusion criteria (no studies were excluded because of reliability 3 or 4). The 52% of relevant studies reported data on toxicity in experimental animals (area 2: 61 studies) while toxicokinetics data in experimental animals and humans (area 1: 20 studies) and data on humans (area 3: 37 studies) accounted for the remaining 48 %. Animal data were restricted to observations in rats, mice, hamster. Few data were available for other animals as for e.g. primates, rabbit, fish, amphibian and chickens. No relevant data on dogs, cats or Guinea Pig were retrieved. The publications dealing with observations in wildlife, aquatic or terrestrial invertebrates (e.g. Caenorabditis elegans), plants, algae, and non genotoxicity effects on bacteria were excluded.


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RESULTS SECTION I – Data on Chromium 1 Toxicokinetics (Absorption, Metabolism, Distribution and Elimination) in Experimental Animals and Humans (Area 1)

1.1 Summary Several studies on absorption, distribution, metabolism and elimination (ADME) after single or repeated oral administration of Chromium (including hexavalent Chromium) are available for rodents (rats and mice). Within these studies, Chromium was administered to rodents as inorganic salt (Cr (III) or Cr (VI) salts) or as organic complex (e.g. Cr Picolinate). The administration was mainly via drinking water. However, administrations through diet and gavage were also reported. Data on Cr (VI) reduction capacity of the stomach are available from in vivo and ex vivo experiments in rats and mice. In vitro data on solubility of a Cr pigment in simulated human body fluids are also available. Based on a review of the literature and of NTP (2008) study (Thompson et al., 2011), the saturation of reduction capacity of the upper alimentary tract is hypothesized to be a key event in the Mechanism Of Action (MOA) for the Cr (VI) induced intestinal cancers. However, there does not appear to be any clear evidence to support a hypothesis that the tumours in the mouse small intestine, in the NTP (2008) study, resulted from the Cr (VI) overwhelming the reduction capacity of the gastrointestinal tract. In contrast, both the mouse and human pharmacokinetic data support the conclusion that in both mice and humans, even low, environmentally relevant doses of Cr(VI) are likely to escape reduction in the stomach, due to the ability of absorption and gastric emptying to successfully compete with reduction (Stern et al., 2010). Less data in humans are available. These studies are toxicokinetics studies, where Chromium was orally administered to adult human volunteers through drinking water, occupational data recording toxicokinetic parameters from exposed workers and poisoning incident data where toxicokinetic parameters were recorded after accidental oral exposure. A Physiologically Based (PB) kinetic model based on human data was also developed. A huge amount of toxicokinetics studies on dental alloys containing Chromium have been published. These studies are focused on the release of metals from metal alloy particles (e.g. Cr and FeCr and FeSiCr alloy particles) in artificial body fluids (in vitro studies) or in human volunteers (in vivo studies). As agreed with EFSA these studies were considered not relevant to assess oral toxicity of Chromium and were not included in the IUCLID database. However, an example of an in vitro study on dental alloys is reported under Section 1.4. Absorption NTP conducted a 2-year bioassay of the Cr (III) dietary supplement, chromium picolinate in parallel with the Cr (VI) sodium dichromate bioassay (NTP, 2008). Comparing the concentration of total Cr retained in various tissues after 25 weeks of dosing, with either chromium picolinate or sodium dichromate, the concentration of total Cr was1.4–16.7 times larger for the rats ingesting Cr (VI), and 2.1–38.6 times larger for the mice ingesting Cr (VI) despite a 1.8 and 2.8 times larger Cr (III) dose in rats and mice, Supporting publications 2015:EN-478

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respectively, (Collins et al., 2010). It seems clear that despite the assumed capacity of the gastrointestinal tract to reduce Cr (VI), in this study, Cr was absorbed as Cr (VI) rather than as Cr (III) (Stern et al., 2010). The rapid uptake of Cr (VI) from the gastro intestinal tract appears to result from the transport of anionic chromate and dichromate complexes across cell membranes by the SO42- and PO43- anion transport system. Cr (III), on the other hand, crosses cell membranes only by passive diffusion (Cohen et al., 1993). The absorption of the inorganic salt of Cr (III), chromium acetate hydroxide, after a single oral gavage administration to male rats was approximately 0.8% of the administered dose (Juturu V., 2003). This value is confirmed by O’Flaherty et al. (2001) in a PB kinetic model based on human data where the absorption of Chromium(III) was in the range of 0.7% - 2%. A poor (1-3 %) absorption after oral administration of Cr (VI) was also reported by the European Chemical Bureau Risk Assessment Report on Chromium (RAR, 2005). Higher absorption values are reported by Febel et al., 2001, for rats fed for 3 days with two different inorganic Cr (III) and Cr (VI) compounds, namely chromic oxide (Cr2O3), as non-absorbable form of Cr (III), and sodium chromate (Na2CrO4), as water-soluble and more absorbable form of Cr (VI). In this study, the retained dose was equal to 13% and 17% for Cr (III) and Cr (VI), respectively. However, the experiment failed to detect that Cr (III) is poorly absorbed and no increment of retained dose was observed after an oral dose of more soluble sodium chromate. Several reviews on bioaccumulation potential of various trivalent and hexavalent Cr compounds reported that various Cr (III) and Cr (VI) salts, including Cr (III) oxide showed a low bioaccumulation potential (ECB RAR, 2005; International Chromium Development Association, 2006; Agency for Toxic Substances and Disease Registry (2008a)). A low bioaccumulation potential is also reported for the organic complex of Cr (III) chromium picolinate in a study conducted in accordance with OECD Guideline 417 (Toxicokinetics). As a conclusion, the retrieved studies show that, when administered orally, inorganic Cr is absorbed as Cr (VI) rather than as Cr (III). The PB kinetic model developed by O'Flaherty et al., 2001 confirmed that, in cases of administration of inorganic Cr III and Cr VI with water, the amount adsorbed is greater for Cr VI. However, irrespective of under inorganic or organic form, Chromium is poorly absorbed and shows a low bioaccumulation potential. In human subjects, the absorption of organic complexes Cr picolinate and Cr D-Phen3 (administered as aqueous solutions) was substantially higher in humans than in rats, but also lower than so far recognised (3-5 times lower). Despite the absolute values, for both organic Cr complexes, the profile of absorption, urine excretion and retention seems similar between humans and rats (Laschinsky et al., 2012). Distribution Elevated chromium concentrations were observed in liver and kidneys after a single oral administration of the inorganic salt of Cr (III), chromium acetate hydroxide (Juturu V., 2003). High mean Chromium concentrations in kidney and liver of rodents (rats and mice) were reported also after 2-years oral exposure to inorganic salts of Cr (VI) and organic complex of Cr (III) through drinking water and feed (Collins et al., 2010) and after 44 weeks exposure through drinking water (Sutherland et al., 2000). In the same study, the Cr concentration in testis, ovaries, brain and whole blood was below the Limit Of Detection (LOD = 0.05-0.1 microg Cr/g). Supporting publications 2015:EN-478

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Data on male rats fed for 28 days with inorganic salt and organic complex of Cr (III) (chromium chloride hexahydrate as an inorganic salt and chromium picolinate as an organic complex), confirmed the increase of Cr levels in the liver and kidney and, in addition, reported an increase of Cr levels in the femur. Interestingly, while no influence of different Cr species was seen on distribution in the liver and kidney, in the femur the inorganic Cr (III) salt caused significantly higher Cr accumulation than the organic Cr (III) complex (Yoshida et al., 2010). A different tissue distribution of Chromium depending on whether it was given as inorganic salt or as a complex with organic anions was reported in the PB kinetic model developed by O’Flaherty ey al., 2001. A significant increase of bone Cr concentration in male/female rats (no differences between males and females) treated with Cr(VI) was reported also by Sutherland et al., 2000. In a study designed to support Cr(VI) risk assessment using US EPA risk assessment guidance (Kirman et al., 2012) it is reported that after administration of Cr (VI) to rats and mice through drinking water for 90 days, the duodenum showed higher levels of Chromium than liver; and that, for a given exposure level, there were higher levels of Chromium in mice (responsive species) than in rats (non responsive species). The same study also reported that the concentration ratios for Chromium in erythrocytes:plasma were above a value of 1 in both rats and mice treated with 20–180 mg Cr/L and 60–180 mg Cr/L, respectively. The data show that after single, repeated or chronic oral administration in mice and rats, levels of Cr were increased in the liver and kidney. Increased Cr levels were also observed in the femur, duodenum and erythrocytes. A different tissue distribution of Chromium depending on whether it is given as inorganic salt or as a complex with organic anions is reported. For a given exposure level, there were higher levels of Chromium in mice (responsive species) than in rats (non responsive species). Metabolism and Elimination Data on Chromium metabolism confirm existing data on the rapid reduction of Cr (VI) in the gastrointestinal tract, in the plasma and intracellularly to Cr (III) by reaction with ascorbic acid, glutathione and by cytochrome P450 and following exposure to Cr (VI), Chromium is excreted in the form of Cr (III) complexes with glutathione (ECB RAR, 2005). Goullè at al. (2012) reported that in an adult male, immediately after an incidental oral ingestion of a 30 g/L solution of potassium dichromate (day 0), significant levels in plasma, Red Blood Cells (RBC) and urine were recorded and after 49 days these concentrations remained elevated in plasma and urine and even more in RBC (149 micrograms/L). The Chromium elimination from serum followed an open twocompartment model and the metal-half life in plasma calculated (5.6 h first metal half-life; 191 h second half-life) were higher than previously published of 3.2 h and 50 h. Calculated half-life in RBC was 440 h which was more than double the plasma half-life. Collected data also confirm that Cr (III) is not metabolised (ECHA, 2008, Study Report on Chromium picolinate monohydrate; International Chromium Development Association, 2006). Several data on Cr (VI) reduction capacity of the stomach are available. According to Kirman et al. (2012), the reductive capacity of the Gastro Intestinal (GI) lumen was depleted at doses > 1 mg Cr(VI)/kg bw day (drinking water concentrations 5–6 mg Cr(VI)/L in rodents). In ex vivo studies using stomach contents collected from the fore stomach and glandular stomachs of rats and mice, the capacities for Supporting publications 2015:EN-478

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Cr(VI) reduction were exceeded at drinking water concentrations >21 mg/L and >60 mg/L in mice and rats, respectively (Proctor et al., 2012). In the 2-year NTP study conducted on rats and mice (Collins et al., 2010), after oral administration of Sodium Dichromate Dihydrate (SDD) as the most water soluble salt of Cr (VI) through drinking water, 50% of the administered Cr dose was excreted in faeces (49.2% in rats, 48.8% in mice) and less than 3% in urine (0.58-2.4% for two lowest exposure concentrations; 0.19-0.95% for two highest in rats). As for Cr(VI), Chromium Picolinate Monohydrate (CPM), an organic complex with Cr(III), administered through diet was mainly excreted in faeces (42.2% of administered dose in rats; 20.2% in mice). Data presented by Febel at al. (2001) confirmed that, after oral exposure, faeces were the predominant route of excretion for both trivalent (chromium oxide) and hexavalent Chromium (potassium chromate). Faecal Cr excretion accounted for 80.66% of the dose for potassium chromate and 85.58% of the dose for chromium oxide. Rats given chromate had significantly higher urinary Cr as a percentage of the oral dose. Rats fed for 28 days with the Cr (III) organic complex Chromium Picolinate (CrPic) showed significantly higher daily urinary Cr excretion than those given the Cr (III) inorganic salt CrCl3, particularly at a dietary Cr level of 100 microg/g. The rate of urinary Cr excretion of CrPic was constant, irrespective of the dietary Cr level. The rate for CrCl3 fell with the increase of the dietary Cr level (Yoshida et al., 2010). Laschinsky et al. (2012) reported that up to 98% of administered dose of Cr (III) organic complexes was excreted with faeces (70-98% of administered dose). The retrieved data on metabolism and excretion confirm that, after oral exposure, Cr (VI) is rapidly reduced in the gastrointestinal tract, in the plasma and intracellularly to Cr (III) by reaction with ascorbic acid, glutathione and by cytochrome P450 while Cr (III) is not metabolised. Faeces are the predominant route of excretion for both inorganic and organic forms of trivalent (chromium oxide) and hexavalent Chromium (potassium chromate). The papers on absorption, metabolism, distribution and elimination in experimental animals and humans are summarised in the following tables. Overall, 19 studies were retrieved: 14 studies with non-human information; 6 studies with human data. Among these, 3 papers and 2 review papers are included twice in the current report and their references are identified with “*”in the tables below (1 paper with both human and non-human toxicokinetics data, included under Tables 1 and 2; 2 review papers with toxicokinetics data from occupational exposure and 1 paper with toxicokinetics data from exposure poisoning incident included under Table 3 and in chapter 3 on human data, under Table 27; 1 paper on human kinetic model included under Table 2 and in chapter 3 on human data, under Table 27). All papers were assigned Klimisch score 2. 75% (15 papers) were peer reviewed papers, 25% (5 papers: 3 on animals; 2 on humans) grey literature. 1.2

Non-human information

Table 1: Toxicokinetics - non-human information Method rat (Sprague-Dawley)

Results Main ADME results:


Remarks 2 (reliable with

Reference Juturu V. (2003) 26



Method male oral: gavage Exposure regime: single dose Doses/conc.: 1000 mg/kg Evaluation of absorption and excretion of orally administered chromium acetate in rats.

Results

Remarks restrictions)

Reference

2 (reliable with restrictions)

Collins Bradley J. , Matthew D. Stout, Keith E. Levine, Grace E. Kissling (2010)

absorption: Approximately 0.8% of the administered dose were absorbed experimental (calculated from excretion and tissue result distribution). distribution: Statistically significant Test material (Common name): elevated chromium concentrations were observed in liver, heart, kidneys chromium acetate hydroxide and pancreas. excretion: Elevated urine chromium levels were observed in chromium acetate administered animals compared to controls (93 ± 56 mg/L versus 0.28 ± 0.17 mg/L).

GLP compliance Statics: one-way analysis of variance. (p value < 0.05) rats and mice (F344/N male rats; B6C3F1 female mice) male/female

Main ADME results:

absorption: concentration of total Cr 1.4–16.7 times larger for the rats ingesting CrVI, and 2.1–38.6 times larger for the mice ingesting CrVI SDD through drinking despite a 1.8 and 2.8 times larger water; CPM through CrIII dose in rats and mice, feed respectively. Exposure regime: 2excretion: SDD in faeces: 49.2% in year bioassay (NTP 2 rats, 48.8% in mice. SDD in urine: years studies) 0.58-2.4% for two lowest exposure concentrations; 0.19-0.95% for two Doses/conc.: highest in rats. Insufficient data in mice. SDD (CrVI) excretion: CPM in faeces: 42.2% in Administered in rats; 20.2% in mice. CPM in urine at drinking water to 48 h: up to 31 micrograms of groups of 40 male rats chromium for rats. Insufficient data and female mice at for mice.. concentrations of 0, distribution: SDD: rats and mice, 14.3, 57.3, 172, and highest mean chromium 516 mg/L. Supporting publications 2015:EN-478

experimental result Test material (Common name): Sodium dichromate dihydrate (SDD) as the most water soluble salt of Cr(VI) and chromium picolinate monohydrate (CPM), an organic complex with Cr(III).

27



Method

Results concentrations in the glandular CPM (CrIII) stomach, kidney, and liver. Tissue Administered in feed concentrations increased with to groups of 30 male increasing exposure concentration. rats and female mice Linear or supralinear shapes of at concentrations of 0, exposure. 2000, 10000, and Distribution: CPM: rats and mice, 50000 ppm. highest mean chromium Analytical verification concentrations in the glandular of doses: yes ( 25 mg/kg bw/day (nominal) based on: test mat. chromium picolinate (No maternal toxicity was observed)


Bailey MM, Sturdivant J, Jernigan PL, Townsend MB, Bushman J, Ankareddi (2008)

15 mg/kg bw/d Cr3complex (providing 3.3 mg Cr/kg/day) (based on predicted food consumption)

NOAEL (developmental toxicity): > 25 mg/kg bw/day (nominal) based on: test mat. chromium picolinate (No adverse effects were observed)

120 mg/kg bw/d Cr3-complex (providing 26 mg Cr/kg/day) (based on predicted food consumption)

NOAEL (maternal toxicity): > 26 mg/kg bw/day (nominal) based on: test mat. (Cr3) (No maternal toxicity was


experimental result Test material (Common name): water soluble complexes of Cr (III) chromium picolinate and [Cr3O(O2CCH2CH3)6(H2O)3]+ (Cr3) Chromium(III) picolinate

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Method Exposure: Females were exposed from Gestation Day 6-17 (Continuous (dietary) Statistics: data from each study replicate calculated independently, tested for homogeneity of variance by the Levene statistic, and then the replicates pooled and analyzed together. All tabular data are presented as the mean ± SEM, and the mean value for each parameter was calculated as the mean of the litter means. Data analyzed by ANOVA, followed by an LSD post-hoc test to determine specific significant differences (Pr ≤ 0.05). mouse (CD-1) oral: feed 200 mg/kg body mass per day (25 mg Cr per kg body mass per day)

Results observed)

Remarks

Reference

(fertility): 95.7 %


(fetal developmental): increase in number of total resorbed or dead fetuses

experimental result

McAdory, D. Rhodes, N. R. Briggins, F. Bailey, M. M. Di Bona, K. R (2011)

NOAEL (developmental toxicity): > 26 mg/kg bw/day (nominal) based on: test mat. (Cr3) (No adverse effects were observed)

Test material (Common name): chromium picolinate

Exposure: 4 weeks (daily) Statistics: data from each study replicate calculated Supporting publications 2015:EN-478

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Method independently, tested for homogeneity of variance by the Levene statistic, and then the replicates pooled and analyzed together. All tabular data are presented as the mean ± SEM, and the mean value for each parameter was calculated as the mean of the litter means. Data analyzed by ANOVA, followed by an LSD post-hoc test to determine specific significant differences (Pr ≤ 0.05). 2.3.4.2.3

Results

Remarks

Reference

Other Animals

Table 24: Developmental toxicity in other animals Method frog embryo teratogenesis assayXenopus (FETAX). Xenopus embryos at blastula stage Doses: 0 (control), 0.025, 0.1, 1, 25, 100 and 500 μM of 51Cr(VI). (nominal conc.) Exposure: 120 h post fertilization

Results LC50 (embryotoxicity): 890 microM based on: test mat. (The embryolethality reach a value of 100% at 1.5 mM.)

Remarks 2 (reliable with restrictions) experimental result Test material (Common name): Sodium Chromate Tetrahydrate

Reference Bosisio Stefano, Salvador Fortaner, Sonia Bellinetto, Massimo Farina (2009)

TC50 (teratogenicity): 260 microM based on: test mat. (The teratogenicity reached a value of 100% at 1.5 mM.)

Chromium Supporting publications 2015:EN-478

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Method uptake/release, body distribution, 51CrDNA adducts were also assessed.

Results

Remarks

Reference

Remarks 2 (reliable with restrictions)

Reference Stummann T.C. (2007)

Statistics: lethality and malformation data were analysed by using a PriProbit program and median lethal (LC50) and teratogenic (TC50) concentrations were obtained. 2.3.4.2.4

In vitro data

Table 25: Developmental toxicity- In vitro Data Method in vitro screening study Embryonic stem cell test (EST)

Results The assay demonstrated that chromium trichloride is not embryotoxic in EST.

experimental result Test material (Common name): chromium trichloride

Statistics: no data

2.3.5

Immunotoxicity

A literature review on immunotoxicity is summarised in the following table. Overall, in vitro (humana and murine cells) and in vivo data (mice) were assigned Klimisch score 2 and were from peer rewieved papers. No data from grey literature. Table 26: In vitro and in vivo immunotoxicity Method In vitro Human chronic myelogenous leukemic K562 cells and murine macrophage J774A. (57BL/6Ntac and p53

Results After chromium (VI) treatment of Human peripheral blood mononuclear (HPBM) for 48 h, we observed a 1.4- and 1.7-fold



Reference Bagchi, D. Bagchi, M. Stohs, S. J. (2001)

experimental result Test material 134



Method deficient) In vivo: C57BL/6TSG p53 mice (4 weeks old)/ female oral: gavage in vitro: 12.5 and 25 μM sodium dichromate for 24h (Human peripheral blood mononuclear (HPBM) cells and K562 cells in vivo: 95 mg/kg body wt (=0.50 LD50 dose of sodium dichromate) Exposure: in vitro: 24, 48h in vivo: one single dose in vitro an in vivo studies on effects of CrVI on enhanced production of superoxide anion and hydroxyl radicals, DNA fragmentation and apoptotic cell death in human peripheral blood mononuclear cells. Effects of chromium (VI) on chronic myelogenous leukemic K562 cells and J774A.1 murine macrophage cells. Effect of a single oral LD50 dose of chromium (VI) on female C57BL/6Ntac and p53-deficient

Results increases in cytochrome c reduction. Following treatment of the human chronic myelogenous leukemic K562 cells for 24 h, approximately 2.7- and 4.8-fold increases in cytochrome c reduction were observed, respectively. Increases in cytochrome c reduction were observed in the liver and brain tissues of both strains of mice.

Remarks (Common name): Sodium dichromate

Reference

Following chromium (VI) treatment of the HPBM cells, approximately 2.4-and 2.5-fold increases in hydroxyl radical production were observed, respectively, and under these same conditions 2.7-and 3.2fold increases in hydroxyl radical production were observed after 48 h, respectively. After 24h of treatment with chromium (VI) approximately 1.0- and 1.3-fold increases in DNA fragmentation were observed in HPBM cells, respectively. Following treatment of the human K562 cells for 24 h, approximately 2.2- and 3.0-fold


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Method C57BL/6TSG p53 mice. Statistics: the presence of significant differences between groups was determined using analysis of variance (ANOVA), with Scheffe’s S method as the post hoc test. Each value is the mean ± standard deviation from 4–6 experiments. The level of statistical significance employed in all cases was p < 0.05.

Results increases in DNA fragmentation were observed, respectively.

Remarks

Reference

Due to extensive cell necrosis at 48 h, DNA fragmentation could not be measured at 48 h. Concentration dependent increases in DNA fragmentation were observed following incubation of cultured J774A. Genomic DNA fragmentation was determined in the liver and brain tissues obtained from both C57BL/6Ntac and p53deficient C57BL/6TSG p53 mice.) Following a single acute oral 0.50 LD50 dose of chromium (VI) approximately 3.3- and 3.5-fold increases in lipid peroxidation were observed in the liver and brain tissues of C57BL/6Ntac mice,

3

Data on observations in Humans (Area 3)

3.1 Summary Reviews of the human toxicokinetic literature provided evidence that exposure to trivalent chromium resulted in significant increases in urinary concentrations of creatinine and increased levels of chromium in the blood and urine. Increased creatine levels were observed in red blood cells, plasma and urine in a man who had been exposed to potassium dichromate. Elimination from serum followed an open twocompartment model. Chromium kinetics was shown to be independent of the oxidation state of the administered chromium, except for the fraction absorbed from the intestine. Supporting publications 2015:EN-478

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Workers exposed to chromium dust (mixtures of trivalent chromium, metallic chromium, chromite, ferrochromium, and contaminants) during grinding, pelletizing and sintering of concentrated chromite, and crushing of cooled ferrochromium castings were studied. Cough or dyspnoea and shortness of breath were significantly more frequent. The authors concluded that the reason for the symptoms was irritation which was not related to smoking. In a further report, it was shown that there were no statistically significant differences in the frequency of centromere-negative or centromere-positive micronuclei and no differences in relation to either nasal diseases and symptoms, mucociliary clearance of the nasal cavity, or incidence of cell atypia. Volunteers exposed to sodium dichromate showed an increase in DNA strand breaks. There was substantial interindividual variation but there was a statistically significant positive correlation between levels of formamidopyrimidine glycosylase-dependent oxidative DNA damage and antioxidant capacity. Analyses of data obtained on cancer mortalities in Chinese populations in an area where contamination of groundwater by Cr(VI) had occurred due to disposal of chromium waste into the river by a nearby plant showed no correlation between cancer mortality rates and degree of exposure. On the contrary, in a region of Greece where water had been contaminated with hexavalent chromium there was a slight increase in cancer deaths but it was not statistically significant. However, for primary liver cancer, deaths were eleven-fold higher than the expected number of deaths among both males and females and deaths associated with kidney and other genitourinary organ cancers were more than threefold higher than expected in women. Elevated mortality ratios were also observed for other cancer sites. It was noted that three out of the six deaths associated with primary liver cancer and two out of the five deaths associated with female kidney and other genitourinary organ cancers came from a small village and the highest concentration of hexavalent chromium was found in a well close to that village. The mortality of workers involved in the production of stainless and alloyed steel in France for at least one year during the years 1968-1991 was studied in order to investigate the risk of lung cancer due to exposure to chromium and/or nickel compounds or iron oxides. No lung cancer excess was observed for exposure to chromium. A cohort of 2357 workers employed at a chromate production plant was studied for lung cancer occurrence in relation to exposure to trivalent or hexavalent chromium. Proportional hazards models were used to assess the relationship between cumulative chromium exposure and lung cancer mortality risk. Cumulative trivalent chromium exposure and duration of work were not found to be associated with a risk of lung cancer whilst cumulative hexavalent chromium exposure, on the other hand, showed a strong dose-response relationship. Elevated chromium levels in blood samples in patients in Central Taiwan demonstrated a strong association with oral cancer. The results of a clinical trial of chromium picolinate administration via capsule showed no changes in FPG, 2-hour plasma glucose levels, fasting or 2-hour post–oral glucose tolerance test insulin levels, or HOMA-IR as compared with placebo after 6 months of chromium use. There were no significant changes in glycohemoglobin, weight, waist circumference, BMI, blood pressure, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, or urine microalbumin. An asophagogastroduodenal endoscopy of a patient who was admitted to hospital after oral ingestion of potassium dichromate revealed necrotic and bleeding lesions of the gastric mucosa. Laboratory findings on day 3 showed kidney and liver failure. In a clinical case study of a patient who had taken various oral


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vitamin and mineral preparations, subacute dermatitis with erythema and scaling was present in scattered patches primarily on the lower legs, ankles, hands, and wrists. Several in vitro studies have been performed using human cells. The role of NER in the removal of chromium-DNA adducts using potassium dichromate has been reported. In another study, a 50% decrease in (H3)-thymidine incorporation was observed after 24 hours incubation of human blood lymphocytes with Cr(V) complexes and it was dose dependent. Under identical conditions, the decrease in (H3)thymidine incorporation was nearly 90% with Cr(VI) complexes. In a further study, it was shown that treatment of lymphocytes with Na[Cr(V)O(ehba)(2)] and K (2)Cr(2)O(7) leads to the activation of the Srcfamily protein tyrosine kinases namely, p56(lck), p59(fyn), and p56/53(lyn), which then activates caspase-3, both of which are under the partial influence of reactive oxygen species. Responses of human stem cells CD34 + hematopoietic progenitor cells to hexavalent chromium were studied and cell loss and inhibition of cluster formation was observed together with marked alterations in subcellular structures. Potassium dichromate increased the frequency of aneuploid cells. 3.2

Health surveillance data

The health surveillance data in humans are summarised in the following tables. Overall, 12 relevant papers were retrieved: 2 papers and 2 review papers with toxicokinetics data (included twice in the report being summarized also in chapter 1 and identified with “*” near reference); 1 paper on repeated dose toxicity; 2 papers on genotoxicity; 5 papers on carcinogenicity and 1 paper with clinical trial data. All papers were assigned Klimisch score 2 and were peer rewieved. 3.2.1

Toxicokinetic data from health surveillance

Table 27: Toxicokinetic data from health surveillance Method Study type: reviews of various studies in the literature

Results Ferrochromium production workers principally exposed to trivalent chromium oxide showed a statistically significant increase in urinary concentrations of Details on study creatinine. An increase in urinary design: Various chromium concentrations were also seen studies are covered by for sub-contractors while values were the reviews stable for clerks. Endpoint addressed: basic toxicokinetics Human target: ferrochromium production, electroplating shop

Uptake of metallic/trivalent chromium were estimated from workers' urinary chromium in the United Kingdom. Increased levels of creatinine were detected in different thermal spraying processes.


Remarks 2 (reliable with restrictions) Test material (Common name): chromium (III) compounds including chromium (III) oxide and chromium (III) sulphate

Reference Finnish Institute of Occupational Health (2006)* Agency for Toxic Substances and Disease Registry (2008b)*

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Method workers Processes:

Results In an industry report, urinary chromium concentrations in workers of one electroplating shop employing trivalent chromium varied from 0.23 to 0.85 ug/g creatinine.

Remarks

Reference


J P Goullé, E Saussereau, J Grosjean, C Doche, L Mahieu, J M Thouret (2012)*

thermal spraying processes, production of chromium (III) No indication was found that exposure to oxide or chromium chromium (III) resulted in stomach (III) sulphate. disorders in workers employed in two factories that produced chromium (III) Statistics: no data oxide or chromium (III) sulphate. No renal impairment was found in 236 workers employed in the ferrochromium production industry. It has been suggested that very poorly water-soluble chromium (III) oxide dust played a role in cases of pneumoconiosis reported among ferrochromium alloy production workers and metal dressers in steelworks. However, these workers had mixed dust exposure, precluding any evaluation of the involvement of trivalent chromium. Workers handling hides soaked in chromium (III) sulphate solution during leather tanning had increased levels of chromium in the blood and urine (HSE review, 1989). Study type: poisoning Day 0: the first results in plasma, RBC incident and urine were significant, with creatinine levels of: 2088 micrograms/L Type of population: in plasma, 631 micrograms/L in RBC and 58-year old man 3512 mic rograms/gram in urine, compared to physiological concentrations Subjects: 58-year old of 76% available Nickel were expected to have an LD50 between 300 and 2000 mg/kg. Distribution Single or repeated oral administration of soluble Nickel compounds in rodents lead to elevated Nickel concentrations mainly in liver and kidney while insoluble compounds such as metallic Nickel accumulated in lungs and pancreas. In rats administered a single dose of Ni-metal, significantly higher Nickel concentrations were retrieved in the lungs, kidneys, and pancreas while in rats administered NiCl2 and NiSO4, 84-87% of the Ni was found in the kidneys.The ratio of Ni in the kidneys depended on the solubility of the administered Ni compound. A distinct transfer of Nickel (significantly different than control) was observed in kidney and lung (Ishimatsu et al., 1995). Observations performed on ex vivo intestinal perfusion models in anesthetized rats (Arnich et al. 2004), confirmed the accumulation of nickel chloride in the kidney and showed a transfer of nickel chloride via gastric mucosa in the small intestine (about 82% retention). Muller at al. (2003) reported that in ex vivo intestinal perfusion models the Nickel secretion via gastric mucosa (mucosa to serosa transport) significantly increased in iron-deficient rats. Iron deficiency increased intestinal Nickel absorption in vitro and in vivo, indicating that Nickel is partially absorbed by the active transfer system for iron absorption in the intestinal mucosal cells (Tallkvist et al., 1994). Adedara et al., 2011 reported an accumulation of Ni in liver and an increase in the activities of antioxidant enzymes (SOD, CAT, GPx and GST) after 7 days oral administration of Bonny light crude oil (BLCO) through gavage. These findings are confirmed by Severa et al (1995) who reported that highest concentrations of Nickel were found in the liver of both male and female rats after long term exposure (2 years) and that Nickel levels decreased in the order: liver > kidney = whole blood = serum > urine.


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Interestingly, after intramuscular injection of nickel monosulphide (NiS) in rats, distinct transfers from muscle to liver and muscle to pancreas were recorded but no muscle to kidney, leading to the conclusion that injected NiS is taken up by the liver and pancreas (Novelli et al., 1995). Administration of nickel sulphate (NiSO4) through diet in fish (Coregonus sp.) for 10, 31 and 104 days led to high Ni concentrations in intestine and pyloric caeca on day 10, decreasing on subsequent sampling days, possibly due to protective mechanisms. Ni accumulated in stomach, kidney, liver, gill, skin, and scales in a dose-duration-dependent manner. The tissues that best assess dietary Ni bioavailability were kidney and scales. Moreover, exposure to Ni altered the concentrations of Cu and Zn in tissues (Ptashynski and Klaverkamp, 2002). Metabolism and Elimination Data on Nickel metabolism and elimination in rodents confirmed that Nickel was poorly absorbed from diets and was eliminated mainly in the faeces. Absorbed Nickel was rapidly cleared from serum and excreted in urine. After oral administration of Nickel, no renal failure was detected (Koizumi et al 2005), the increase in urinary Nickel concentration occurred in a dose dependent manner, and soluble Ni compounds were excreted with urine within 72 hours after a single oral administration (Ishimatsu et al., 1995; Koizumi et al 2005; Janicka and Cempel, 2003). This is confirmed by Nielsen et al., (1993) who reported that up to 20 h after single oral administration of doses comparable to the human dietary intake, Nickel retention was highest in the kidneys. However, Ni deposited in the kidneys was rapidly excreted, whereas the elimination from the lungs and liver was relatively slow, resulting in a higher Nickel content in the liver than that in the kidney at 40 h after administration. Heim et al. (2007) reported that urinary Nickel levels increased with exposure and correlated with blood levels in rats and that Nickel is rapidly excreted in the urine. In addition, the same authors reported that Nickel levels in faeces increased in an exposure-dependent manner and relatively high faecal levels were retrieved compared to the blood and urinary Nickel levels, demonstrating that the majority of the Nickel was not systemically absorbed, but was excreted in the faeces. Human data from toxicokinetic studies related to chronic intake of dietary supplements in adut females with and without atopic eczema, pointed to significantly increased urinary Nickel excretion with increasing age, ingestion of dietary supplements, drinking of stagnant tap water, and consumption of Nickel-rich food. Age and dietary supplements remained significant predictors of high Nickel excretion. Patients with atopic eczema showed urine Nickel concentrations similar to those in non-atopic controls. (Darsow et al., 2012). The papers on absorption, metabolism, distribution and elimination on experimental animals and humans are summarised in the following tables. Overall, 20 relevant papers were retrieved: 13 studies with nonhuman information (12 on rodents, 1 study in fish), 3 studies with human data and 4 in vitro studies on bioaccessibility. Among these, 5 papers are included twice in the current report and their references are identified with “*”in the tables below (2 rodent studies summarized in the current chapter, under Table 36, and in chapter 6, under Table 43 with data on repeated dose toxicity in rats, and Table 51 with data on Supporting publications 2015:EN-478

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carcinogenicity in rats; 3 human studies summarized also in chapter 6, under Tables 61 and 62). All papers were assigned Klimisch score 2 and peer rewieved. 4.2

Non-human information

Table 36: Toxicokinetics-non-human information (oral exposure) Method rat (Wistar) male oral: gavage

Results Main ADME results:


Reference Ishimatsu S, Kawamoto T, Matsuno K, Kodama experimental result Y. (1995)

absorption: 0.09% for the Ni-Metal group and 10-34% for the NiSO4, NiCl2, and Ni(NO3)2 groups. NiCl2 Test material absorption was 9.8% (scarcely Doses/conc.: 10 mg of soluble compounds) (chemical Ni administered as formula) NiCl2, various Ni compounds. distribution: significantly higher Ni(NO3)2, NiMetal, NiSO4. Sampling and analysis: concentrations in the lungs, kidneys, tissues and body fluids and pancreas for the Ni-metal group. (lungs, liver, kidneys, spleen, pancreas, heart, Ni concentrations in all organs in the brain, and blood) were rats given NiSO4, NiCl2, and Ni(NO3)2 were significantly higher sampled for the determination of Ni 24 than those in the control rats. h after the About 84-87% of the Ni found in the administration. kidneys for the NiCl2 and NiSO4. Statistics: Student’s t The ratio in the kidneys for the Ni(NO3)2 and Ni-M groups was 73, test. and 51%, respectively. Exposure regime: single dose

Ni amount in the liver was higher than that in the kidneys for the NiO(G) group, showing similar results as the control group. The ratio of Ni in the kidneys depended on the solubility of the administered Ni compound. Transfer: distinct transfer (significantly different than control) in kidney and lung. Excretion: soluble Ni compounds were excreted within 72 hours after Supporting publications 2015:EN-478

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Method

Results the oral administration. 24 hours after oral administration NiCl2 was measured in the urine at 914 ug.

Remarks

Reference


Koizumi, C. Usuda, K. Hayashi, S. Dote, T. Kono, K. (2005)

Conclusions: absorbed fraction was 11%, 24 hours after oral administration. rat (Wistar) male oral: unspecified Exposure regime: single administration Doses/conc.: 0.025 250 mg/kg (equivalent to 0.0015% - 15% of LD50) Sampling and analysis: urine samples were collected in following 24 hours and the nickel concentration was measured by ICPAES.

Main ADME results: excretion: The increase in urinary nickel concentration occurred in a dose dependent manner.

experimental result

Test material (Common name): nickel nitrate Other results: hexahydrate, (chemical renal failure: Increasing the dose of formula) nickel had no significant effect on Ni(NO3)2 6H2O NAG, 2- microglobulin, albumin Form: solution and protein in urine samples (no renal failure).

Statistics: ANOVA with Stat View software. mouse (CBA) male

Main ADME results:

orally (gavage) or intraperitoneally

Absorption: After oral administration, the WBRs decreased within 45-75 hr to 0.020.36% of the dose administered After intraperitoneal administration, the WBR decreased within 20-50 hr to 1-6% of the dose administered The estimated instestinal absorption ranged from 1.7 to 10% of the dose administered

Exposure regime: Single oral gavage /intraperitoneal administration Doses/conc.: comparable to the human dietary intake 13 Mbq/kg bw 57Ni orally

2 (reliable with restrictions) experimental result

Nielsen GD, Andersen O and Jensen M (1993)

Test material (Common name): nickel chloride Radio labelled 57NiCl2

relation between dose and WBR: The WBRs were independent of the dose, both when nickel was given


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Method 1.3 Mbq/kg bw 57Ni intraperitonally.

Results orally and intraperitoneally

Remarks

Reference

Distribution: 20 h after administration (% of the equivalent or similar to body burden of nickel): OECD Guideline 417 Intestine: 80% (oral); 8% (ip) (Toxicokinetics) WBR Liver: 20% (oral); 4% (ip) (Whole-Body Kidneys: 15% (oral); 30% (ip) Retention) of 57Ni2+ Carcass: >50% (both oral and ip) labeled nickel chloride. Statistics: KruskalWallis one-way analysis of variance, Mann-Witney U test for comparison of unpaired groups. Retentions at 30, 55, 80 and 110 h determined by loglinear interpolation.

Counting of the residual body burden of Ni after oral administration: -At 8 hr: 87% in the carcass, 10% in the kidneys, 2% in the liver, carcass > lungs > testicles > liver > spleen -At 20 hr: kidneys > lungs > liver > carcass. Conclusion: After oral administration Ni up to 20 h retention was highest in the kidneys. However, Ni deposited in the kidneys was rapidly excreted, whereas the elimination from the lungs and liver was relatively slow, resulting in a higher nickel content in the liver than that in the kidney at 40 h after administration. After ip injection, no nickel was transported via the portal vein to the liver, resulting in a lower nickel content in the liver and a higher content in the kidneys compared to oral administration.

rat (Wistar) male oral: gavage

Main ADME results: heavy metal accumulation in tissues:



Isaac A Adedara, Azubike P Ebokaiwe and Ebenezer O experimental result Farombi (2011) 182



Method Exposure regime: 7 days Doses/conc.: 0, 200 and 800 mg/kg body weight of Bonny light crude oil (BLCO) Sampling and analysis: heavy metal concentrations in the tissues as well as the effect of BLCO on the antioxidant status of erythrocytes of rats.

Results accumulation of Ni in liver erythrocytes antioxidant status:

Remarks

Reference

Test material (Common name): nickel metallic

increase in the activities of antioxidant enzymes SOD, CAT, GPx and GST

Statistics: ANOVA, Student’s t test. Values less than 0.05 were considered statistically significant mouse (C57BL) female feed with high Nickel (Nickel high mice) and without Nickel (Nickel low/very low mice) oral: diet exposure regime: 4 weeks

Main ADME results:


Xianzhu Wu, Karin Roelofs-Haarhuis, Distribution and excretion: Jianhong Zhang, experimental result Michael Nowak nickel found in the tissues and (2007) excretions from Nihigh mice obtained Test material: directly after oral NiCl2 treatment far (chemical exceeded those found in Nilow and formula) NiCl2 Nivery low mice.

When comparing the latter two mouse subsets, statistically Observations: significant differences were only excretions and body organs were obtained 1 found in the feces and spleen. In both day after cessation of cases, the values measured in Nilow the 4- week period of mice exceeded those in the Nivery low mice. oral nickel administration. Statistics: no data rat (Wistar) male

Main ADME results

oral: drinking water

Excretion



Janicka, K and M. Cempel (2003)

experimental result 183



Method

Results

Exposure regime: 90 days

Urine volume was significantly increased at 90 days in the 1200 ppm Ni group. Doses/conc.: 0, 300, Nickel excretion was significantly 1200 ppm Ni as NiCl2. increased at 30 and 90 days in the Rats: 14 rats per dose 300 and 1200 ppm Ni groups. Urine volume and nickel, zinc, copper, calcium and inorganic phosphorus were measured in 24 h urine after 45 and 90 days of exposure Statistics: Student's ttest rat (Wistar) male

Remarks

Reference

Test material (Common name): Nickel chloride hexahydrate

Zinc excretion was significantly increased at 90 days in the 1200 ppm Ni group. Copper excretion was significantly decreased at 30 and 90 days in the 1200 ppm Ni group. Inorganic phosphorous excretion was significantly decreased at 90 days in the 1200 ppm Ni group Main ADME results


Organ and serum Fe levels in rat organs: Exposure regime: 90 d the Fe levels were affected by Ni ad libitum exposure. Doses/conc.: 1 control -300 ppm: increase of Fe concentration in the liver, lungs and group (tap water); 2 in the serum. No changes were exposed groups (ad observed in the kidney, spleen, and libitum) to water solution of nickel(II) brain. - 1200 ppm: maximum amounts of Fe chloride in the liver. Increase also in the Doses: 300 ppm (Ni- kidney, in the spleen and in the 300) and 1200 ppm Ni serum. (Ni-1200). Lungs: at 1200 ppm Fe level was The iron content in serum, liver, kidney, about 24% higher than that in control lung, spleen, and brain rats but no consistent pattern. was analyzed 30 and Brain: 20% increase of Fe level noted only at the final exposure time (90 d). 90 d post-exposure. The hemoglobin, hematocrit, and body Body weight gain: significantly lower at1200-ppm Ni. and organ weights Organ weights: were also measured. -at 1200 ppm lungs were larger than Supporting publications 2015:EN-478


Cempel, M. (2004)*

experimental result Test material (Common name): nickel(II) chloride (chemical formula) NiCl2 · 6H2O)

184



Method

Results Statistics: Student’s t- those in the control animals. The ratios of lung to body weights were test. significantly higher. -Brain weight: brain/body weight ratios were significantly increased.

Remarks

rat (Wistar) male/female



Main ADME results

Reference

Severa, J., A. Vyskocil, Z. Fiala, absorption: reaching of a steady state and M. Cizkova. experimental result of nickel during long-term exposure. (1995)

Exposure regime: 3 to distribution: highest concentrations of 6 months nickel found in the liver of both male Doses/conc.: 100 mg and female rats. In male rats nickel levels decreased in Ni/L (as nickel the order: liver > kidney = whole sulphate). blood = serum > testes > urine. In female rats the decreasing order - Tissues and body fluids sampled: urine, was similar: liver > kidney = whole blood, plasma, organs blood = serum = plasma > urine > ovaries. 3. No significant differences - Time and frequency were found between nickel of sampling: urine concentrations in organs (except collected over 24 hours ovaries). at 3 and 6 months Toxicokinetic parameters: Ni At 3 and 6 months ten absorption was 1% of oral dose. rats of each sex were sacrificed and Nickel Conclusions: Nickel sulphate administration was associated with an concentrations in increased concentration of nickel in blood, urine and organs were measured. body fluids and organs.

Test material (common name) Nickel sulphate (CAS number): 7786-81-4

Statistics: Student's T- No significant differences were found between nickel concentrations in test. organs (except ovaries), blood and urine of rats exposed for 3 months and those exposed for 6 months. rat (Fischer 344) male/female

Main ADME results


Distribution: steady state levels of experimental result nickel in blood increased with exposure.The steady state mean blood Exposure regime: 103 nickel level between the 0.5 h value Test material weeks (2 year) (peak level after exposure) and the 24 (common name) Nickel sulphate oral: gavage


Heim KE, Bates HK, Rush RE, and Oller AR (2007)

185



Method

Results h value (low level expected right Doses/conc.: 0, 10, 30, before the next exposure was due). 50 mg/kg day Carcinogenicity study Excretion: 24-hour urinary nickel levels increased with exposure and Statistics: no data correlated with blood levels.

Remarks

Reference

(chemical formula) NiSO4 (CAS number): 7786-81-4

Nickel levels in feces increased in an exposure-dependent manner in the treated males and females. Relatively high fecal levels compared to the blood and urinary nickel levels demonstrating as the majority of the nickel was not systemically absorbed, but was excreted in the feces. The toxicokinetic results of this study demonstrate that the nickel blood levels exceeded the control levels by a factor of approximately 100 for the low exposure group and over 350 in the two highest exposure groups. High levels of nickel reached the target tissues after absorption from the gastrointestinal tract. No notable differences were observed between controls and treated animals for the hematology, biochemistry and urinalysis parameters measured during the toxicokinetic satellite study.

Table 37: Toxicokinetics-non-human information (other exposure routes) Method

Results

Remarks

Reference

rat (Wistar) male

Main ADME results

intramuscular


Distribution: significant levels of Ni measured in liver and pancreas, but not experimental result kidney. Test material

Novelli ELB, Rodrigues NL, Ribas BO (1995)

Exposure regime: single injection


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Method

Results

Remarks

Reference

Transfer (muscle to liver): distinct (common name) Doses/conc.: 7 mg transfer (10.6 +/- 3.1 ug Ni/g (treated), nickel Ni/kg in the form of p < 0.05 compared to control) (Test monosulfide NiS No.: #1) (CAS number): Ni content was Transfer (muscle to pancreas): distinct 16812-54-7 measured in the transfer (7.5 +/- 1.3 ug Ni/g (treated), p liver, pancreas, and < 0.05 compared to control) (Test No.: kidney 72 hours after #2) injection. Transfer (muscle to kidney): no transfer Statistics: Student's t- detectable (2.9 +/- 0.7 ug Ni/g (treated), test. p > 0.05 compared to control) (Test No.: #3) Evaluation of results: bioaccumulation potential cannot be judged based on study results. Conclusions: injected NiS is taken up by the liver and pancreas. rat (Wistar) male luminal ex vivo perfusion Exposure regime: 60 min perfusion Doses/conc.: Nickel tested solutions: 0.17 mM (3.4x103 nmoles); 0.9 mM (18x103nmoles); 4.7 mM (94x103 nmoles) perfused at a rate of 0.6 mL/min in situ intestinal perfusion model in anesthetized rats. Statistics: ANOVA, Fisher test. The null hypothesis was

Main ADME results:

2 (reliable with Arnich, N. Cunat, L. restrictions) Lanhers, M. C. absorption: metal accumulated in the Burnel, D. (2004) tissues increased according to the experimental result amount in the perfusion solution absorption: input % in tissues (small Test material (chemical intestine, blood, target organs) decreased (from 14% to 2.7%) when formula): NaCl: NiCl2·6H2O the amount perfused increased absorption: absorption % the blood and target organs decreased (from 2.1% to 0.4%) with increased perfusion rate absorption: amount retained in the small intestine was greater than that absorbed regardless of the amount perfused. distribution: nickel accumulated in the kidney

Transfer (secretion via gastric mucosa): distinct transfer (in the small intestine (about 82% retention). Nickel


187



Method

Results

rejected at p < 0.05.

accumulated in the kidney.

rat (SpragueDawley) male

Main ADME results:

luminal ex vivo perfusion Exposure regime: 2 hours perfusion Doses/conc.: 1, 10, 50, 100, 200, and 500 micromo1/l

Jejunal tissue load: increased significantly in iron-deficiency jejunal tissue load: showed a saturable fraction and a marked linear component

Remarks

Reference


Muller-Fassbender, M. Elsenhans, B. McKie, A. T. experimental result Schumann, K. (2003) Test material (chemical formula):

Transfer (secretion via gastric mucosa): mucosa to serosa transport significantly 63Ni-NiC12 (0.02 increased in iron-deficient segments at microCi/ ml) all six luminal concentrations.

ex vivo perfusion of Transfer (secretion via gastric mucosa): proximal jejunal serosa to mucosa transport reached a segments from iron- steady state over time after 30 min deficient and ironadequate rats at six different concentrations (1/500 micromo1/l) under steady state conditions. Statistics: ANOVA, Student-t test. The null hypothesis was rejected at p < 0.05.

4.3

In vitro data on toxicokinetics

Table 38: In vitro data on toxicokinetics (bioaccessibility as surrogate for bioavailability) Method in vitro study Aim: data on bioaccessibility of nickeloxyhydroxide in body fluid simulants as a

Results

Remarks

Reference

Bioaccessibility results


ECHA (2011)

Simulated Gastric Fluid (2 h):

experimental result

µg Nickel/ g Sample: 188,047; 186,265 Test material (dup) (Common name): Nickel-oxy


188



Method

Results

surrogate for bioavailability.

% Nickel/ g Sample: 18,8; 18,6.

Remarks

Reference

hydroxide

Nickeloxyhydroxide % Nickel Release/ Nickel content: 29,38; 29,10. was extracted in leaching fluids at: Simulated Interstitial Fluid (72 h) -2hrs in simulated µg Nickel/ g Sample: 7,178; 8,033 gastric fluid (dup) - 72 hrs in simulated interstitial fluid and % Nickel/ g Sample: 0,72; 0.8. lysosomal fluid. % Nickel Release/ Nickel content: . 1,12; 1,26. The extracts were analyzed for soluble Simulated Lysosomal Fluid (72 h) nickel using EPA Method #200.7 µg Nickel/ g Sample: 542,406; 524,306 (ICP). (dup) Results were reported as ug Ni/g sample, % Nickel/g sample and as % of total available Ni released.

% Nickel/ g Sample: 54,2; 52,4. % Nickel Release/ Nickel content: 84.75; 81,92.

Statistics: no data in vitro study

Bioaccessibility

Aim: data on bioaccessibility in body fluid simulants as a surrogate for bioavailability.

Gastric bioaccessibility (2 h): Ni sulfate hexahydrate 90.55%; Ni metal 73.5%; Ni chloride/acetate: 88-89%. Ni hydroxycarbonate/sulfamate: 8384%; Nickeloxyhydroxide Ni oxide (black)/ hydroxide / was extracted in subsulphide: 22-29% ; leaching fluids at: 2, 5, 24, 72 hrs. Intestinal bioaccessibility (24 h): Ni sulfate hexahydrate 58%; The extracts were analyzed for soluble Ni metal 0.2%; nickel using ICP/MS. Ni chloride/acetate: 38%. Ni hydroxycarbonate/sulfamate: 1Results were 11%; Supporting publications 2015:EN-478


Henderson RG, Capellini D, Seilkop SK, Bates HK, Oller experimental result AR (2012) Test material (Common name): various nickel substances and metallic nickel

189



Method

Results

reported as ug Ni/g sample and as % of total available Ni released.

Ni oxide (black)/ hydroxide / subsulphide: 0.2-0.3% ;

Statistics: no data

Remarks

Reference


ECHA (2008a)

Interstitial bioaccessibility (72 h): Ni sulfate hexahydrate 57%; Ni metal 0.44%; Ni chloride/acetate: 33-44%. Ni hydroxycarbonate: 3.45% Ni sulfamate: 49%; Ni oxide (black): 75% Ni hydroxide / subsulphide: 0.044.8% ; Lysosomal bioaccessibility (72 h): Ni sulfate hexahydrate 96%; Ni metal 47%; Ni chloride/acetate/ sulfamate: 104%. Ni hydroxycarbonate: 97% Ni oxide (black): 32% Ni hydroxide: 102% Ni subsulphide: 37% Sweat bioaccessibility (24 h): Ni sulfate/ chloride/acetate/ sulfamate: 91-93%. Ni metal: 0.62%; Ni hydroxycarbonate: 6.8% Ni oxide (black): 2% Ni hydroxide: 0.03% Ni subsulphide: 5% ;

in vitro study % Nickel release from simulated gastric and alveolar fluids. Artificial perspiration. Extraction times: 2, 5, 24 and 72 h. Internale Guideline from Nipera

% Nickel release in simulated gastric fluid: 2 h: 81-83% 5 h: 82-84% 24 h: 81-84% 72 h: 85-87%

experimental result

Test material (Common name): % Nickel release in simulated alveolar nickel fluoride fluid: 2 h: 36% 5 h: 35-40% 24 h: 40-41% 72 h: 41-42%


190



Method

Results

Statistics: no data

% Nickel release in artificial perspiration: 2 h: 95-98% 5 h: 94-97% 24 h: 96-97% 72 h: 98-99%

in vitro study % Nickel release from simulated interstitial fluid, lysosomal fluid and intestinal fluids.. Extraction times: 2, 5, 24 and 72 h. Internal Nipera Guideline Statistics: no data

4.4

% Nickel release in simulated interstitial fluid: 2 h: 44-46% 5 h: 44-51% 24 h: 45-46% 72 h: 49-50%

Remarks

Reference


ECHA (2008b)

experimental result

Test material (Common name): % Nickel release in simulated intestinal nickel difluoride fluid: 2 h: 60-64% 5 h: 77% 24 h: 73-74% 72 h: 76-80% % Nickel release in simulated lysosomal fluid: 2 h: 98-109%

Human data from toxicokinetic studies

Table 39: Human data from toxicokinetic studies related to chronic intake of dietary supplement Method

Results

human female

Main ADME results:

Remarks

Doses/conc.: Various

2 (reliable with excretion: A nickel detection limit of restrictions) 0.2 microg/l was exceeded by all experimental samples result excretion: The 95th percentiles of urine nickel concentration were 3.77 Test material microg/l (age 18-30 years) and 3.98 (Common name): Nickel microg/l (age 31-46 years).

Examination of nickel excretion in the urine of 164 female patients with and without

excretion: Bivariate analyses pointed to significantly increased nickel excretion with increasing age, ingestion of dietary supplements,

oral: feed Exposure regime: Chronic intake of dietary supplement


Reference Darsow, U. Fedorov, M. Schwegler, U. Twardella, D. Schaller, K. H (2012)*

191



Method nickel contact dermatitis (CD). The associations between age, atopic dermatitis, nickel contact dermatitis and nickel exposure through nutrition (e.g. dietary supplements) and by patch tests were investigated prospectively. Statistics: Bivariate and multivariate analysis.

Results

Remarks

Reference


Arruda-Neto J.D.T. , Geraldo L.P. , Prado G.R. , Garcia F. , Bittencourt-Ol (2010)*

drinking of stagnant tap water, and consumption of nickel-rich food. excretion: In the multivariate analysis, age and dietary supplements remained significant predictors of high nickel excretion. excretion: A non-significant increase in the median concentration of nickel was observed after the administration of conventional nickel patch tests excretion: Patients with atopic eczema showed urine nickel concentrations similar to those in non-atopic controls.

human male/female

Strong correlation between individual age and bioaccumulation for all metals oral: drinking water investigated: from Guarapiranga a) 7-10 years accumulation peaks for dam (Sau Paulo, all metals, Brasil) b) metal concentrations sharply decrease between 10-20 years, Exposure regime: Age followed by the formation of a plateau range of humans from 20-50 years, and another decrease involved in the study: for ages over 60 years. 7-64 years

experimental result Test material (common name) nickel

The overall trend of all metals indicates that bioaccumulation is more intense in the childhood period followed by a sharp decrease (clearance) and the formation of a plateau at ages between 20 and 50 years old. The average concentrations of Pb, However, this sharp decrease is not Cd, Fe, Zn, Mn, Ni and verified particularly for Chromium; its plateau is only 10% lower than the Cr were determined bioaccumulation peak, indicating that with an Atomic clearance of this metal is negligible. Absorption Nickel also has small clearance. Spectrophotometer. Fortunately, Cr and Ni are not as Statistics: no data harmful as Pb and Cd. A total of 59 teeth from individuals 7 to 60 years old were collected.


192



4.5

Human data from occupational exposure

Table 40: Human data from occupational exposure (inhalation) Method Study type: biological exposure monitoring Type of population: occupational exposure in a battery plant using nickel hydroxide

Results Clinical observations: - 2 workers had skin itching and one had an itchy red rash on the hands Air and urine correlations:

- A linear correlation was observed between timeDetails on study design: weighted average (TWA) estimate the relationship concentrations of Ni and Co in between Ni concentrations in the air. the ambient air and in the Subjects were exposed to urine higher levels of Ni than Co. Workers were exposed to a - No statistical differences mixture of metallic cobalt, between pre- and post-shift cobalt, oxyhydroxide and urine samples except for nickel hydroxide dust. urinary Co concentrations on - 16 male workers the first day. participated in the study (mean age = 39 years, mean - No correlation was found between Ni in the air and in employment = 3.5 years) post-shift urine.

Remarks 2 (reliable with restrictions) Test material (common name) nickel hydroxide,

Reference Yokota K, Johyama Y, Kunitani Y, Michitsuji H, Yamada S (2007)*

(chemical formula) Ni(OH)2 (CAS number): 12054-48-7

Air sampling and analysis:

Day 1, Pre Shift: 17.5 +/- 10.7 -breathing zone samples (5.0-39) ug Ni/l - Ni and Co in total airborne Day 1, Post Shift: 21.5 +/- 17.4 dust were analyzed (5.0-67.5) ug Ni/l Day 2, Pre Shift: 20.1 +/- 13.1 Urine sampling and analysis: (6.3-39.4) ug Ni/l - 2 two urine samples/day for Day 2, Post Shift: 20.9 +/- 16.7 two consecutive days (4.7-52.9) ug Ni/l - Ni and Co concentrations were determined - A good correlation was found Statistics: Student’s paired t- between Co and Ni in posttest and regression analysis shift urine: Ni (ug/ml) = 9.9 + were employed as necessary 0.343 Co (ug/ml), r= 0.833.


193



4.6 Other animals Table 41: Toxicokinetic data on other animals Method Coregonus clupeaformis oral: feed Exposure regime: Fish were feed three times a week with Nickel in diet for 10, 31, and 104 days. Doses/conc.: 0, 10, 100, and 1000 microg Ni/g Lake whitefish (Coregonus clupeaformis) were fed diets three times a week containing 0, 10, 100, and 1000 microg Ni/g (as NiSO(4)) for 10, 31, and 104 days. Various organs were analyzed to evaluate the accumulation and distribution of Ni.

Statistics: no data

Results Main ADME results: Accumulation: At medium and high dose fish accumulated significant amounts of Ni in a majority of the tissues sampled, even after only 10 days of exposure

Remarks 2 (reliable with restrictions) experimental result

Reference Ptashynski, M. D. Klaverkamp, J. F. (2002)

Test material (chemical formula):

distribution: Ni concentrations were highest in intestine and pyloric caeca at 1000 microg Ni/g on day 10, but NiSO(4) decreased on subsequent sampling days, possibly due to protective mechanisms. Accumulation: Ni accumulation in stomach, kidney, liver, gill, skin, and scales was dose and durationdependent distribution: Ni concentrations measured in bone, gall bladder, gonad, and muscle of fish fed the control diet for 10 days and fish fed the high dose diet for all durations appeared to increase in a durationdependent manner Interaction with Cu and Zn: Exposure to Ni altered the concentrations of Cu and Zn in tissues . However, Cu and Zn concentrations did not follow a common pattern or trend Bioavailability: The tissues that best assess dietary Ni bioavailability are kidney and scales. Transfer (accumulation): dose and duration-dependent Ni accumulation in stomach, kidney, liver, gill, skin, and scales


194



5

Toxicity in Experimental Animals (Area 2)

The time-frame for the extensive literature search is from 2000 to April 2013. In order to give a comprehensive overview of published data, also the literature included in EU RAR on Nickel (EU RAR, 2008) relevant to oral toxicity of Nickel in experimental animals and published before 2000 was checked and the main results summarized under Appendix 1. 5.1

Acute toxicity

Data on acute toxicity were retrieved for rats. No data on acute toxicity were retrieved for mice or other animals. Relevant data on rats showed that, when administered orally (gavage) Nickel soluble compounds (nickel sulphate or nickel chloride) were acutely toxic to rats, with LD50 > 300 < 2000 mg/kg bw. On the contrary, less soluble compounds were not acutely toxic to rats, with LD50 > 2000 mg/kg bw (nickel dihydroxide) or even higher, ranging from 8796 to >11000 mg/kg bw/day (female rats) (nickel oxide black, nickel oxide green). The mechanism of toxicity of metals involves a common cascade of events which entails an oxidative stress and production of reactive oxygen species. Das et al. (2001) reported that a single oral administration of nickel sulphate through drinking water led to an increase of hepatic lipid peroxidation and to a decrease of antioxidant ezyme activities in male rats. The papers on acute toxicity in experimental animals are summarised in the following tables. Overall, 12 relevant papers on acute oral toxicity in rats were retrieved. Among these, 8 papers were assigned Klimisch score 1 (Reliable without restrictions) being conducted according to GLP and officially accepted guidelines, while remaining 4 were assigned Klimisch score 2. The 75% (9 papers) were study report obtained from ECHA website, the 25% (3 papers) were were peer rewieved papers. 5.1.1

Rats

Table 42: Acute toxicity in rats Method Results Remarks rat (Sprague-Dawley LD50: 1 (reliable without derived, albino) female 5000 mg/kg bw (female) restriction) oral: gavage

experimental result

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Test material nickel dihydroxide


Reference ECHA (2009a)

195



Method Statistics: no data rat (Sprague-Dawley derived, albino) female oral: gavage

Results

Remarks

Reference

LD50: 361.9 mg/kg bw (female)

1 (reliable without restriction)

ECHA (2009c)

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure) Statistics: no data rat Sprague-Dawley derived, albino female

experimental result Test material (Common name): Nickel sulfate hexahydrate

LD50: 500 mg/kg bw (female)


oral: gavage

experimental result

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Test material (Common name): Nickel chloride hexahydrate

Statistics: no data rat (Sprague-Dawley) female oral: gavage

LD50: > 300 < 2000 mg/kg bw (female)

OECD Guideline 423 (Acute Oral toxicity Acute Toxic Class Method)


ECHA (2010d)

ECHA (2010c)

experimental result Test material (Common name): Molybdenum nickel tetraoxide

EU Method B.1 tris (Acute Oral Toxicity Acute Toxic Class Method) Statistics: no data rat (Wistar) female oral: gavage

LD50: > 300 < 2000 mg/kg bw (female)

OECD Guideline 423 (Acute Oral toxicity Acute Toxic Class Method) Supporting publications 2015:EN-478


ECHA (2008d)

experimental result Test material (EC name): tetrakis(tritolyl phosphite )nickel 196



Method

Results

Remarks

Reference

EPA OPPTS 870.1100 (Acute Oral Toxicity) Statistics: no data rat (Sprague-Dawley derived, albino) female oral: gavage

LD50: 1 (reliable without 9990 mg/kg bw (female) restriction) (in water) experimental result

equivalent or similar to OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure) Statistics: no data rat (Sprague-Dawley) female oral: gavage equivalent or similar to OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure) Statistics: no data rat (Sprague-Dawley) female

Test material (Common name): Nickel oxide black N105 Form: solid LD50: > 11000 mg/kg bw (female) (test 1)


LD50: > 11000 mg/kg bw (female) (test 2)

experimental result

Form: powder/granules LD50: 1 (reliable without 8796 mg/kg bw (female) restriction) experimental result

equivalent or similar to OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Test material (Common name): nickel oxide black

oral: gavage OECD Guideline 423 (Acute Oral toxicity -

ECHA (2008c)

Test material (Common name): nickel oxide green

oral: gavage

Statistics: no data rat (Sprague-Dawley) male/female

Henderson RG, Durando J, Oller A, Merkel DJ, Marone PA, and Bates HK. (2012)

ECHA (2009b)

Form: black solid limit test: 2000 mg/kg bw (male/female). One animal tested at limit dose - died


main test: 200 mg/kg

Test material (Common name):


ECHA (2003b)

experimental result

197



Method Acute Toxic Class Method) Statistics: no data rat (Sprague-Dawley) male/female oral: gavage OECD Guideline 423 (Acute Oral toxicity Acute Toxic Class Method) Statistics: no data rat (Wistar) male oral: drinking water effect of oral ascorbic acid treatment on nickel sulfate-induced lipid peroxidation in the liver of Wistar strain male albino rats Statistics: no data mouse (C57BL) male/female for Ni tolerance induction: in drinking water for Ni immunization: i.d. injectionin both flanks for tolerance induction: 10 mM NiCl2 in their drinking water for immunization: i.d. injections into both flanks(50 microl each) of 10 mM NiCl2

Results bw (male/female) 6 animals exposed. One animal died on day 4, all others survived. limit test: 2000 mg/kg bw (male/female) 1 male, 1 female tested both died main test: 200 mg/kg bw (male/female) 6 animals exposed, no mortality hepatic lipid peroxidation: increase (male) antioxidant ezyme activities: decrease (male)

effects of Ni on B cells: In WT Nihigh mice, B cells are numerically decreased and prone to apoptosis (male/female) effects of Ni on Fas: Fas-defective mice are resistant to oral tolerance induction toward nickel (male/female) Appearance of FasLexpressing NKT cells in the draining lymph nodes and spleen is required for tolerance transfer by Nihigh B cells (male/female)


Remarks nickel nitrate solution

Reference


ECHA (2003a)

experimental result Test material (Common name):nickel nitrate cryst. 6H2O 2 (reliable with restrictions)

Das KK, Das SN, DasGupta S. (2001)

experimental result Test material (Common name): nickel sulfate

2 (reliable with restrictions) experimental result Test material (chemical formula): NiCl2

Gleichmann Michael Nowak, Frank Kopp, Karin RoelofsHaarhuis, Xianzhu (2006)

198



Method

Results

Statistics: no data

apoptosis: induction in Nihigh B cells before transfer replaces iNKT cells for tolerance induction (male/female)

5.2

Remarks

Reference

Repeated dose toxicity

Studies on repeated dose toxicity of nickel sulphate, nickel chloride and nickel bis (2-ethylhexanoate) are available for rodents (rats, mice), hens, chicken, rabbits and fish (Coregonus spp.). The studies were conducted according to OECD 408 - Repeated Dose 90-Day Oral Toxicity in Rodents, OECD 453 combined chronic/ carcinogenicity, OECD 451 Carcinigenicity or were performed as non-guideline studies with exposure durations ranging from 8/10 weeks up to 6 months. Available data on nickel sulphate in rats showed that after 104 weeks exposure the NOAEL was equal to 2.2 mg/kg bw/day and the LOAEL ranged from 6.7 mg/kg bw/day (104 weeks exposure) to 30 mg/kg bw/day (90 days exposure), based on reduced body weight. The repeated oral administration of nickel sulphate through drinking water led to significant increase of concentrations of Nickel in liver tissue and of oxidative stress (increases in hepatic lipid peroxidation, catalase, glutathione peroxidase, glutathione reductase, superoxide dismutase activity, and glutathione-S-transferase) (Sidhu 2005). It was also observed that the repeated oral administration of nickel sulphate through drinking water in mice lead to a Nickel accumulation in the interstitial tissue of the testes and to a decrease in the seminal vesicle weight, diameter, and activity of epithelium (Pandey and Singh, 2001). This seems to be in accord with the theory that Nickel influences the production of testosterone. A repeated oral administration (subacute - 10 d, subchronic - 31 d, and chronic - 104 d) of nickel sulphate through diet in fish (Coregonus clupeaformis) led to histopathological lesions in liver (areas of focal necrosis and altered bile ducts) and in kidney (lesions in glomeruli, tubules, collecting ducts, and hematopoietic tissue). Significant increases of metalliothionein in intestine (on day 10) and lipid peroxide concentration in plasma (on day 31) were also observed. A LOAEL of 10 mg/kg bw/day, based on decrease of the body weight at 30 mg/kg bw, was reported for rats exposed to nickel bis (2-ethylhexanoate) for 104 weeks through drinking water (ECHA, 2007c), showing that the oral toxicity after repeated exposure to nickel bis (2-ethylhexanoate) is comparable to that of nickel sulphate. The repeated oral ingestion of nickel chloride through drinking water (1200 ppm, nominal in water) led to reduced body weight gain and to an increase of lung and brain weights in rats (Cempel, 2004). The same author also observed that Nickel induced iron uptake by serum and some organs. Bersenyi et al. (2004) reported that the repeated oral administration of nickel chloride through diet (500 mg Ni/kg nominal in diet) led to a reduced body weight gain and to an increase of enzyme concentrations (i.e., triglycerides, alanine transferase, gamma-glutamyltransferase, and cholinesterase) in broiler chickens and rabbits. Supporting publications 2015:EN-478

199



Detrimental effects were also reported in hens where nickel chloride led to decreases in egg production and weight (Arpasova et al., 2007). Several data on the effects of repeated oral administration on sensitization to Nickel were retrieved. These data showed that the repeated oral administration of water enriched with nickel chloride in mice prevented subsequent sensitization to this common contact allergen (Atrik et al., 2001) and showed that iNKT cells are required for the induction of oral tolerance toward Nickel, but not for Nickel sensitization (RoelofsHaaruis et al., 2004). The papers on repeated dose toxicity in experimental animals are summarised in the following tables. Overall, 12 relevant papers were retrieved: 5 papers on rats (1 study identified with “*”included twice, being summarized also in chapter 4 under Table 36); 3 papers on mice; 4 papers on other animals (hens, chickens, fish and rabbits). Among these, 3 papers were assigned Klimisch score 1 (Reliable without restrictions) being conducted according to GLP and officially accepted guidelines; remaining 9 papers were assigned Klimisch score 2. 9 papers were peer rewieved papers and 3 were study reports obtained from ECHA websitee. 5.2.1

Rats

Table 43: Repeated dose toxicity in rats Method rat (Fischer 344) male/female subchronic (oral: gavage) 0, 5, 7.5, 10, 12.5, 15 mg/ml (nominal in water)

Results LOAEL: 30 mg NiSO4.6H2O/kg bw/day (male/female) Based on reduced body weight)

Remarks 1 (reliable without restriction)

Reference ECHA (2002)

experimental result Test material (common name): nickel sulfate hexahydrate

Vehicle: reverse osmosis deionized water Exposure: 90 days (daily) OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents) EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents) Supporting publications 2015:EN-478

200



Method

Results

Statistics: one-way Analysis of Variance. If significance was observed, control-totreatment comparisons were made using the Tukey-Kramer method. The level of significance was 5% (p50 years passed away. In these subjects, the level of As, Cd, Ni, and Pb were increased by Scalp hair samples 10.6, 19.5, 15.7, and 9.8% in the scalp were taken and lysized hair as compared to those who tolerated for As, Cd, Ni, and Pb third MI attack (p=0.12). Random selection of patients.

The study was carried It should be noted that all the third MI patients also had higher weight and higher Supporting publications 2015:EN-478

244



Method out 130 patients

Results BMI, potentially contributing to the Control Population: 61 higher concentrations observed in these patients. In addition, controls were not healthy subjects matched for health (e.g. diabetes, Statistics: Unpaired hypotension, etc) with MI patients. student t tests s. critical value at 95% confidence intervals

Remarks

Study type: biological strong correlation between individual age 2 (reliable with exposure monitoring and bioaccumulation for all metals: restrictions) Type of population: general male/female drinking water and eating fish from Guarapiranga dam (San Paulo, Brasil) where levels of heavy metals (lead, copper, zinc and cadmium) are above internationally accepted limits.

a) between 7 and 10 years accumulation peaks for all metals,

experimental result

Test material b) Metal concentrations sharply decrease (Common for ages between 10 and 20 years, name): nickel followed by the formation of a plateau from 20 to 50 years, and another decrease for ages over 60 years.

Reference

Arruda-Neto J.D.T., Geraldo L.P., Prado G.R., Garcia F., Bittencourt-Ol (2010)*

The overall trend of all metals indicates that bioaccumulation is more intense in oral: unspecified the childhood period followed by a sharp Exposure regime: Age decrease (clearance) and the formation of a plateau at ages between 20 and 50 years range of humans involved in the study: old. 7-64 years However, this sharp decrease is not A total of 59 teeth verified particularly for Chromium; its from individuals were plateau is only 10% lower than the collected and bioaccumulation peak, indicating that concentrations of Pb, clearance of this metal is negligible. Cd, Fe, Zn, Mn, Ni Nickel also has small clearance. and Cr were determined. Statistics: no data

Table 62: Toxicokinetic data from occupational exposure (inhalation and dermal exposure mainly) Method Results Study type: biological Clinical observations Supporting publications 2015:EN-478

Remarks 2 (reliable with

Reference Yokota K, 245



Method Results exposure monitoring at - Two workers had skin itching and one a battery plant using had an itchy red rash on the hands nickel hydroxide Air and urine correlations Type of population: - A linear correlation was observed occupational between time-weighted average (TWA) concentrations of Ni and Co in the air: Study design: relationship between Subjects were exposed to higher levels of Ni than Co Ni concentrations in ambient air and in - There were no statistical differences urine between pre- and post-shift urine samples except for urinary Co concentrations on Subjects: 16 male Workers exposed to a the first day. - No correlation was found between Ni in the air and in post-shift urine. Day 1, Pre Shift: 17.5 +/- 10.7 (5.0-39) ug Ni/l Air sampling: Day 1, Post Shift: 21.5 +/- 17.4 (5.0-67.5) breathing zone ug Ni/l samples (Ni and Co in Day 2, Pre Shift: 20.1 +/- 13.1 (6.3-39.4) total airborne dust ug Ni/l were analysed) Day 2, Post Shift: 20.9 +/- 16.7 (4.7-52.9) Urine sampling and ug Ni/l analysis for each - A good correlation was found between worker (2 urine samples per day for 2 Co and Ni in post-shift urine. consecutive days) Statistics: Student’s paired t-test and regression analysis Study type: biological Nickel levels in Khimprom workers exposure monitoring depending on occupational contract with in the Khimprom nickel [N; mean nickel concentrations (+/chemical plant, SEM) for no contact workers, exposed; pNovocheboksarsk city, value]: central Russia. hair (ug/g): 262; 0.360 (+/- 0.028); 0.459

Remarks restrictions)

Reference Johyama Y, Kunitani Y, Test material Michitsuji H, (common name) Yamada S nickel hydroxide (2007)* (CAS number): 12054-48-7

mixture of metallic cobalt, cobalt, oxyhydroxide and nickel hydroxide dust.

Type of population: occupational

(+/- 0.057); < 0.1

2 (reliable with restrictions) Test material (Common name):

Grabeklis AR, Skalny AV, Nechiporenko SP, Lakarova EV (2011)

nickel

whole blood (mg/L): 246; 0.052 (+/0.046); 0.006 (+/- 0.002); > 0.1

Details on study blood plasma (mg/L): 244; 0.009 (+/design: Whole blood, 0.001); 0.012 (+/- 0.004); > 0.1 plasma, urine and hair Supporting publications 2015:EN-478

246



Method Results Remarks were collected from urine (mg/L): 248; 0.005 (+/- 0.000); 263 male workers (25- 0.006 (+/- 0.001); < 0.05 58 years old) Among the biosubstances investigated, All the samples were hair was found to be the most sensitive to analysed by combined most of toxic trace metals. ICP-OES/ICP-MS method. Occupational contact with nickel was 208 workers with occupational contact reflected in a pronounced increase of their with Na, Pb, Mn, Ni, content in hair. Cr, Be, Si, B, Zn. The levels of nickel were not reflected in 55 workers, without occupational contact the blood mineral profile and trends were used as control group. generally the same in the plasma samples.

Reference

Multielement analysis of urine detected an Statistics: Student's t- increased level of nickel in those workers who had occupational contact with this test, one-way metal. ANOVA, Spearman rank order correlation Cncentration of nickel in the urine was generally low and the difference was not very pronounced. Nickel levels in blood and nickel levels in plasma were positively correlated (Spearman R: 0.23, p