Toxicological evaluation of certain veterinary drug ...

WHO FOOD ADDITIVES SERIES 45

Toxicological evaluation of certain veterinary drug residues in food Prepared by the FIFTY-FOURTH MEETING OF THE JOINT FAO/WHO EXPERT COMMITIEE ON FOOD ADDITIVES (JECFA)

IPCS lnternational Programme on Chemical Safety World Health Organization, Geneva

This publication is a contribulion to the International Programme on Chemical Safety.

The Internatlonal Programme on Chamlcal Safety (IPCS), established in 1980, is a joint venture of the United Nations Environment Programme (UNEP), the International Labour Organísation (ILO), and the World Health Organization (WHO). The overall objectives of the IPCS are to establish the scientific basis for assessing the risk to human health and the environment from exposure to chemicals, through internalional peer-review processes, as a prerequisite for the promotion of chemical safety, and to provide technical assistance in strengthening national capacities for the sound management of chemjcals. The Intar-Organlzatlon Programme for the Sound Management of Chemlcals (IOMC) was eslablished in 1995 by UNEP, ILO, the Food and Agriculture Organization of the United Nations, WHO, the United Nations Industrial Development Organization, and the Organisation for Economic Co-operation and Developmenl (Participating Organizations), following recommendations made by the 1992 United Nations Conference on Environment and Development to strengthen cooperatíon and increase coordination in the field 01 chemical safety. The purpose 01the IOMC is to promote coordination 01 the policias and actívities pursued by the Participating Organizations, jointly or separately, to achieve the sound management o, chamicals in relation to human health and the environment.

Health & Environment International, Ud, by agreement with WHO, performed independent literature searches on some 01the substances on which data are summarized in this document in order to ensure lhat all relevant toxicological and related in10rmation was reviewed.

ISBN 92 4 166045 7

L1NCOMYCIN First draft prepareo by Kevin J. Greenlees Center for Veterinary Medicine, Food and Drug Administratian, Maryland, USA

RockvilJe,

Arturo Anadon Depanment

o( Taxicology, Faculty af Veterinary

Camplutense

Medicine, de Madrid, Madrid, Spain

Universidad

and Carl Cerniglia National Center for Toxicolagical Research, Food and Drug Administratíon, Little Rack, Arkansas,

USA

Explanation Biological dala . Biochemical aspecls oo..... ......•..............•.......•......• Absorption, dislribulíon, and excrelíon Biolranslormation Toxicological sludies Acute toxicity .,. Short-term studies of toxicity Long-term studles ef toxicity and carcinogenicity Genotoxicity Reproductive toxicity Multigeneration studies Developmental texicity Special studies Immune responses Otetexicity Microbiological effects Observalions in humans Comments . Evaluation . Aererences .

1.

3 4 4 4 5 6 6 8 10 12 12 12 14 15 15 15 15 21 21 31 31

EXPLANATlON

Líncomycin, Iike pinimycin and clindamycin, belongs lO a class of antibiotics known as Iincosaminides. Lincomycin is active mainly againsl Gram-positive bacteria. It exerts ils antíbiotic action by inhibitin ANA-dependenl protein synthesis through action on the 50S subunit of the ribosome. Lincomycin is used alone or in combination with other antimicrobial agents such as spectinomycin, neomycin, sulfadiazine, and sultadimidine. Hcan be given orally in feed or drinkín -water, by intramuscular injection, or as an lntramammary infusion. The recommended doses are: 0.5 mglkg bw in feed and 3-50 mglkg bw in drinking-water lor poultrY; 0.2-13 mglkg bw in feed, 5-10 mgfKg in drinkingwater, and 5-10 mglkg bw intramusculany in pigs; 5 mglkg bw intramuscularly in calves and sheep; and 200-330 mglquarter of the udder as an intramammary inrusion, three limes after each milking, in dairy cows.

~ -,,-

•

INCOM e/IV The Committee

has not previously evalualed

2.

BJOLOGICA DATA

2.1

Biochemlcal aspects

2. 1. 1

Absorption, distribution, and excretion

Iincomycin.

Dogs A prelíminary study, which did nol comply with good laboralory practice (GLP), conducted in a male and a female dog showed thal Jincomycin is rapidly absorbed after intramuscular injecUon at 20 mg/kg bw (Sokolski, 1962). Peak serum concentrations were reached 30 min after injecUon of 9 mg of the base per m!. In two beagle dogs given a single dose 01 300 mglkg bw by capsule, peak absorption occurred within 1-2 h of dosing (Grady & Treiek, 1961; Gray & Purmalis, 1961 a). In a 90-day study, Iincomyein was determined in Jung, Iiver, kidney, muscle, bile, spinal f1uid, and serum 01 beagles dosed orally at O, 400, or 800 mglkg bw par day. The highest coneentrations were observed in kidney and bile (66 and 680 mglg, respeetively) and the Jowest concenlrations in spinal fluid (below the limit 01 detecUon) from animals al the high dose. One control had a concentralion of 39 mglg in the lung and trace levels in lhe liver; il was speeulated fhat lhe animal had been inadvertently dosed just before sacrifice (Gray & Purmalis, 1964a).

Humans The pharmaeokinetics of Iincomyein in humans has been determined after administration by several roules (Table 1). About 72% is bound to proteins in human serum. Lincomyein is widely dislributed, with a volume of distribution approximating total body water, and it is exereted in the Jaeces. Biliary excretion has also been reported to be an important route of elimination. Significant coneentrations of Iincomycin are aehieved in a number of tissues and r1uids regardless 01 the route of administration. These inelude bile, peritoneal Iluid, pleural fluid, the eye, brain, bone, bone marrow, joint capsules, synovial fluid, and cerebrospinal fluid. It is generally poorty distributed into eerebrospinal fluid except in the presence of inflammadon. Therapeutie concentrations have been achieved in the presence of meningitis (Fass, 1981). . Uncomycín has been shown to cross the placenta, and peak concentrations in amniotic fluid of 0.2-3.8 mglml are sustained for 52 h aftar a single intramuscular injeetion 01600 mg to pregnanf women. Uneomycin has been found to be present in milk post partum (Fass, 1981). In a report written to support the safety 01 the lincosaminide pirlimycin, which aJso addressed the safety 01lincosaminides in general and ollincomycin in particular, it was noted that only a fradion 01 an oral dose 01 lincosaminides

UNCOMYCIN

Table 1. Pharmscokinetics of líncomycín in humsn Roule

Parameter

Dose (mg)

Peak serum concentratian (¡J.glml) AUCt024 (mgJmr'h) AUClo~ (mg/ml'h) lime of peak (h) Elimínalion 11/2 (h) Peak saliva concentration (mglml) Time of peak (h) AUCl024 (mglml'h)

1000 17 120 130 1.2 1.5 4.5 5.3 0.86 1.6 3.7 4.7 5.3 10

2.7 3.9 18

Mean concentration (mg/ml)

300 600 7.7-12

16-21

lntramuscular

Inlravenous, 2h Oral

600 12 82 92

Aeference 1500 22 150 160 0.92

Smílh el al. (1981 )

5.3

Fass (1981)

Adults 500 1000 Fass (1981) Peak serum concentralíon {mglml)a 1.8-5.3 2.5-6.7 Eliminalion 1112 (h) 4.2-5.5 Time of peak (h) 2-6 usually 4 I

Children 22-33 mgJkg bw Peak serum concentration (mglml) 4-9 (maintained above 1.0 mg/ml far 15 h) • Presence of food in the slomach markedly impairs absorption (Kucers & Bennelt, 1979; Fass, 1981). The oral bioavailabtlity is estimaled to be 25-50% alter fasting but only 5% wilh a meal (Homish et 81.,1987). reaches the lower intestine (Kotarski, 1995). While orally administered clindamycin is neany completely absorbed after oral administralion (KapusnikUner el aL, 1996), Iincomycin is poony allhough rapidly absorbed rrom the gaslroinlestinal tract (Goodman & Gilman, 1975). The human bioavailability 01 orally administered Iincomycin is estimated to be 25-50% tor fasting individuals but only 5% after a meal (Homish et al., 1987). About 10% of orally administered clindamycin is excreted unaltered in the urine, and asma" fraetion is found in the faeces (Kapusnik-Uner et al., 1996).

2. 1.2

Bíotransformation

The comparative metabolism.of lincomycin was reported in rals, COWS, pigs, and chickens. Lincomycin was extensively metabolized in all tissues but not in cow's milk (after inlramammary infusion only). Some 16 metabolites were identified, although there were as many as 26 in pig Jiver. The principal residues were parent lincomycin, N-desmethyl lincomycin, and lincomycin sulfoxide (Homish et aL, 1987; Nappier. 1998). Approximately 5% of an oral dose administered to rats was excreted in urine, where Iincornycin and lincomycin sulfone comprised 97% o, the excreted drug; 95% o, the drug was found in the gastrointestinal traet (Homish et al.,

1987). The principal urinary and fascal metabolite in dogs and humans after oral and inlramuscular administration was unchanged Iincomycin, representing 40%

•

INCOMYCIN

6

of the excreted dose; most of the remainder was unidentified. There was no evidence of glucuronide or sulfate conjugation (Hornish et aL, 1987). Pig excreta contained markedly less unchanged lincomycin than lhat of other species studied. The urine contained 11-21% of an oral dose, and half of this was unehanged parent. Only trace amounts of N-desmethyllincomycin were identified. The contents of the gastrointestinar tract accounted for 79-86 % of the excreted drug. In faecal samples, only 17% 01 the excreted dose was unchanged parent, and the remainder was uncharacterized metabolites (Homish el aL, 1987) 2.2

ToxlcologlcaJstudles

2.2. 1

Acute toxíc;ty

Lincomyein was toxic in miee and rats when administered parenterally and was practically nontoxic after oral administralion. Lincomycin was toxic to rabbils by all routes of administration. Mica The acute LOso in male mice treated orally was determined for USP grade and Premix grade Iincomycin (Buller, 1979). No significant difference between the LOso values of 20 000 and 17 000 mg/kg bw was determined in this nonGLP study. An L050 value of 210 mglkg bw was measured in mice lreated intravenously, and the signs of toxicity in the survívors included severe sedation lasting 1-2 min (Gray & Highstrete, 1963a). Rats The acute toxicity of Iincomycin was determined in a preliminary non-GLP study in newbom and adult rats treated by subcutaneous injection (Gray & Purmalis, 1962a). The LD50 in newbom rats was 780 mglkg bw, while lhat in adults was 10 000 mg/kg bw. An intravenous injeetion was reported to be more toxic, with an LDso of 340 mg/kg bw (Gray & Highstrete, 1963a). The aeute toxicity 01 agricultural-grade lincomycin (Glenn & Garza, 1971) and o, USP-grade lincomycin (Brown, 1977a,b) was determined in a series of non-GLP studies in Sprague-Oawley rals. Lincomycin was adminislered orally to groups of five animals of eaeh sex al doses of 5000-16 000 mg/kg bw, and clinical signs and body weights were monitored for 2 weeks after treatment. Al! doses resultad in clínical signs of toxicity including diarrhoea and ataxia. Depression was observed al doses ~ 8000 mglkg bw, and death, preceded by coma, was observed at dases of 12 500 and 16 000 mglkg bw. While there was no significant effect on body-weight gain, the survivors continued to have diarrhoea tor up to 36 h after treatment. The LDso was determined by probit analysis to be about 15 000 mg/kg bw for USP Iincomycin and 11 000 mglkg bw for the agricultural-grade product. In a separate study, an LOsa 01 16000 mg/kg bw was determined tor a premix grade preparation 01 lincomycin (Nielsen, 1975).

LlNCOMYCIN

Rabbits Aabbits have been shown to be quite sensitive to orally administered Iincomycin (Gray et aL, 1965a). After a single intravenous injection 01 0.5 mglkg bw, 5 out 01 10 rabbits either died or were killed 10r humane reasons within 2 weeks 01 dosing, and 7 out ot 10 rabbits had died by 1.5 months. In two studies that did not comply with GLP, in which groups of three rabbits were given lincomycin, only the lowest dose of 0.5 mglkg bw was not lethal. Al! the other dosas (5. 50, 100, and 150 mg/kg bw) caused death, such that by 4 weeks 9 out of 15 and 12 out of 15 rabbits had died. Histological examination revealed gastrointestinal stasis and, in those animals that died, haemorrhagic suffusion of the serosal surlace of the caecum. Attempts to modify the toxicity by supplementation with Lactobacillus culture or intubalion with 1resh (rabbit) caecal contents were not success1ul. The observed toxicity was considered to result from gastrointestinal Gram-positive floral imbalance. The irritability of Iincomycin to tissues was investigated in rabbits in a series ot studies that did not comply with GLP. Doses 01 up to 300 mg/kg bw were injected into the lumbar muscle at pH 4 (Gray & Purmalis, 1962b) or pH 7.4 (Gray & Purmalis, 1962c). No difference was seen in the minimal to mild muscular irritation after slaughter up to 7 days after treatment. Injection 01 up to 150 mg of lincomycin into the sUfle joint 01 New Zealand white rabbits caused no trealment-related effécts, such as intra-articular irmatíon (Gray & Highstrete,

1965). Dogs A series 01 studies that did not comply with GLP were conduc1ed in dogs. In one study of single intrathecal injections of lincomycin, two dogs received 15 mg in 1 mi 01solution, eight dogs received 50 mg in 1 mi of solution, and 10 dogs received the vehícle (isotonic saline and benzyl alcohol at 9 mg/ml). No treatment-relatad clinical effects were reportad. Cerebrospinal fluid samples taken up to 48 h after injeclion were c10udy and had increased cell counts consisting predomínantly of Iymphocytes. Gross and microscopic examinalion 24-72 h after injection did nol revea! treatment-related effects, such as meningeal irrilation (Gray et aL, 1965b). When íntravenous or inlramuscular injectians af 150 mg/kg bw per day were given for 5 and 3 days, respectively (Gray & Purmalis, 1962d), no treatment-related eHects were reparted. Lincomycin was injected subcutaneously for 14 days ¡nta four pups fram each of three Iitters wilhin 24 h of birth at a dose of 0,30,60, ar 90 mglkg bw per day. No significant treatment-related effects were reparted (Gray et aL,

1962). In a preliminary sludy,líncomycin was adminislered at a dose of 4000 mglkg bw orally by gavage for 5 days to two remale beagles. While both animals

INCOMYCIN

vomited for 1-2 h after gavage, no treatment-related effects (such as diarmoea) wara raportad. In the same study, a third dog received an intravenous dose of 940 mglkg bw in a volume of 230 mi as two injections. Transianl prostration and slightly incraased activilies of alenine and aspartate aminotransaminases were seen, suggesting some hepatic toxicity. Tha heart rate and respiration remained normal, and no histopathological alterations ware found in a liver biopsy sample 5 days later. The dog appeared clinically normal during the subsequant 2 weeks of observation, and no other treatment-related effects were reporte (Gray & Purmalis, 1963a).

2.2.2

Short-termstudies 01 toxicity Mics

Lincomycin of pramix grade was administered for 90 days to groups of 15 B6C3F1 mice ot each sex al concentrations of O, 70, 200, 700, 2000, or 20000 mglkg in the reed,'squivalent todaily dosas of 0,10,30, 100,300, and 3000 mglkg bw par day. While lhe study was completad in 1979, bato re initiation of GLP requirements, a quality assurance statement was provided which addressed deviations from GLP. The highest dose resulted in a significant supprassion of body weight, increased food consumption and intestinal weight (with pancreas), a decreased sarum glucose concentration, and, in females, increased serum corticosterone concentration, decreased serum globulin concentralion, and decreased mea n 'hymus weighL While 'he mean organ weights of animals at the highest dose were the lowest for heart, liver, spleen, and kidney (males only), the differences were not statistically significantly differenl 1rom controls. Histolog~lIy, the lumina of the large and small intestine were found to be distended and dilatad. The next highest dose of 300 mglkg bw per day also íncreasad intestinal weight (with pancreas) and increased the incidence of luminal distention and dilatation 01 the small and large intestines. The serum glucose values were also depressed. The NOEL was 100 mglkg bw per day (Platte & 5eaman, 1981).

Rats In a study that did not con10rm to GLP, groups 01 five Wistar rats of each sex were given lincomycin at a dose of O, 30. 100, or 300 mg/kg bw per day by oral gavage for 30 days. Effects on body weight, food consumption, organ weights, haema'ological values, and pathological findings were reported, but no significant treatment-effects were found at any dose. The NOEL was 300 mg/kg bw par day, the highest dose tested (Gray & Purmalis, 1961 b). In an extension of this study to 3.5 months, Iincomycin was administered orally by gavage at a dosa 01O, 30, 100, or 300 mglkg bw per day to groups of 10 Upjohn Wistar rats 01 each sexo No drug-relaled effects were observed on body-weight gain, 1000 consumption. or pathological findings. The NOEL was 300 mglkg bw per day, the highest dose testad (Gray & Purmalis, 1962e). When the sludy was repeated in groups of 20 rats 01each sex at a dose 01 600 or 1000 mglkg bw par day tor 3 months, the average weight of the intestinal

UNCOMYC/

I

tracts of all treated animals was greater than that of comrols, but it was not clear whether thi was due to the tissue or the content, as no changes were observed in the intestinal wall or mucosa on gross or microscopic examination. The NOEL was 1000 mg!kg bw per day, the highest dose tested (Gray & purmalis, 1964b).

Dogs In a study that did not conform to GLP, groups of lhree beagles received lincomycin by intramuscular ¡njection at a dose of O, 15, 30, or 60 mglkg bw per day adminislered twice daily tor 4 weeks. Two o( the dogs-one control and one at 60 mg!k.gbw par day-showed mild Iymphocytic infiltralion of lhe thyroid. Since this effeet occurred in only two animals and equally in treated and control groups, it was not aseribed to lincomycin. Aside 1rom mild inflammatory reactions ~t the ¡njecUon site observed at necropsy in all groups, including controls, no treatmenHelated eHeds were reported. The NOEL was 60 mglkg bw per day, the highest dose test~d (Gray & Purmalis, 1962f). In a further study that did not comply with GLP, lincomyein was given to groups of three beagles by capsule three times a day for 30 days at a dose of O, 30, 100, or 300 mglkg bw per day. Daily examinalions tor body weight, haematological and urinary analyses, and gross and histopathological examination revealed no treatment-related effects (Gray & Purmalis, 1962g). In another study that did not comply with GLP, groups of four beagles of each sex were given Iincomycin at a dose of 0,400, or 800 mg/kg bw par day in gelalin capsules adminislered three times a day for 90 days. Transient increases in serum alanine aminolransferase activity were observed during the first month 01lreatment at 800 mg/kg bw per day and in ene animal at the low dose, but lhe level had retumed to normal by Ihe end of the study. Bilateral Iymphocytic thyroiditis was observad in three controls, two animals at 400 mg/kg bw per day and two at 800 mg/kg bw per day. This condition had also been observad in other beagle colonies. Mild Iymphocytic infiltration was reported in other organs as wall. These lesions did not appear to be treatment·relaled. The NOEL was BOO mglkg bw per day, the highest dose lested (Gray & Purmalis, 1964a). In a study that did not comply wilh GLP, lincomycin was administered in capsules to groups 01 twe beagles of each sex al a dosa of O, 30, 100, or 300 mglkg bw per day for 6 menths. No treatment-related effects we re reportad on body weight, organ weights, or haematological, clinical chemical, er urinary end-points. The summary and conclusions of lhe report state that further histopathelogical analysis revealed Iymphocytic lhyroiditis in the male and female at the high dose and similar infillration 01 the kidney in one 01 the animals (Grey et al., 1963b). Lesions of lhis type were observed at all doses in a later, 90-day study (Gray & Purmalis, 1964a). The European Medicines Evaluation Agency (1998) identilied a NOEL of 100 mg/kg bw per day in lhe study of Gray et al. (1963b) on the basis of an ¡ncrease in adrenal weight at the high dese. However, while a paired ttest showed a significant difference

r

1

LlNCOMYCIN

in adrenal weights, the rela1ive weights were not significantly altered and no significant difference was found in an unpaired ttest The NOEL was 300 mglk bw per day, the highest dose tested. Lincomycin was administered orally as 1he premix grade al a dose 010, 0.38,0.75, or 1.5 mglkg bw per day or as the US grade at 1.5 mg/kg bw per day in gelatin capsules for 1 year to groups of fíve beagles 01 each sex in a study thal did not conform to GLP. The doses were chosen to support a proposed tolerance 01 lincomycin of 1 mg/kg in the edible tissues 01 poultry, pork, beef, lamb, and dairy products, and were based on muUiples of 25,50, and 100 times the maximum theoretical human dietary intake. The measured end-points ¡ncluded c1ínical and ophthalmic parameters, food consumption, body weights, clinical pathological, chemical, and urinary parameters, organ weights, and gross and histological appearance. No differences were observed between animals receiving the premix and USP grades, and no treatmentrelated eftecls were reported. The NOEL was 1.5 mg/kg bw per day of each grade 01 lincomycin, the híghest doses tested (Goyings et aL, 1979a). 2.2.3

Long-term studies al toxicity and carcinogenicity

Rats Groups 01 10 rats of each sex received Iincomycin at a dose of O, 30, 100, or 300 mg/kg bw per day by oral gavage for 1 year. The study did not comply with GLP.Al! rats were necropsied, four of each sex per group were examined histologically, and haematological parameters, body-weight gain, organ weights, and pathological find~r')gswere reported. No treatment-related effects were found. While there was a significant diHerence (p 0.019, two-tailed t test) in the weight ot the liver between controls (19 ± 2.3 g) and rats at the high dose (24 ± 4.9 g), the weights relative lo body weight were not significantly different. rhe NOEL was 300 mg/kg bw per day, the highest dose tested (Gray et aL, 1963a).

=

In a study that díd comply with GLP, groups of pregnant Sprague-Dawley rats and groups of 60 of the resulting offspring of each sex received fieldgrade premix lincomycin orally al a dose of 0,0.38,0.75, or 1.5 mglkg bw par day or USP~grade Iincomycin at a dose of 1.5 or 100 mg/kg bw per day. Treatment of the offspring was continued for 26 monlhs. Food consumption per cage (two rats per cage) was monitored weekly; body weights were monitored weekly through week 56 and during altemate weeks thereafter. Serum chemistry was evaluated at 6 and 12 months and at termination, and haematological parameters were assessed betore treatment, al 3, 6, and 12 months, and al termination. Organ weights and urinary parameters were measurad at the ínterim kills and al termination. Al! rats that died or were killed were examined grossly and histologically; a full histopathological examination was pertormed on animals in the control and the two high-dose-gróups. The percenlage survival, clínical and ophthalmological end-points, food consumption, organ weights, and haematological, serum chemicaJ,and urinary parameters were unaffected by treatment. A statistically significant eftect on

UNCOMYCJN

growth promotion was observed up to day 57 of the study in animals given 0.75 mglk bw per day of premíx, but not thereafter. An increased incidence of Ron-neoplastic microscopic lesions of the prostate and seminal vesicles (acute prostatilís and seminal vesiculitis) was reporte in males at 1.5 mg/kg bw per day of premix and at 100 mglkg bw per day of USP grade. The frequency of prostalis was 21/59 in controls, 1/35 in rats at 0.38 mg/kg bw per day, 5/45 in rals at 0.75 rnglkg bw par day, 40/60 in those at 1.5 rnglkg bw per day of premix, 3/40 in rats at 1.5 mg/kg bw per day of USP grada, and 31/59 in those at 100 mglkg bw per day of USP grade. At the 1-year interim slaughter, the frequency of prostatis was 4/10 in controls, 2/10 in rats at 0.75 mg/kg bw per day of premix, and 2/10 for those at 100 mg/kg bw per day of USP grade. A :reviewof the data for individual animals showed no dose-response relationship, and there was no increase in lhe relative severity of the lesions. The prostatis was therefore considered to be unrelated to treatment with Iincomycin. The numbers of benign, malignant, and total tumours in each treated group were not statistically significantly different from those in the concurrent vehicle contra' group (Table 2). A statistically significant increase in the number ot males with subcutaneous fibromas was observed at the high dose of USPgrade material when comparad with concurrent controls, but the total number of fibromas was not significantly different. A statistically significant ¡ncrease in the incídence of Iymphosarcoma was observad in females at 1.5 (G/52) and 100 mg/kg bw per day (7/60) o, USP grade when compared with control females (1/59). A trend analysis of these incidences did not, howevar, show a significant linear component, and it was concluded that the Iymphosarcomas were not related to treatment. No increase in the incidence of Iymphosarcomas was seen in males. The incidenca of mammary adenomas and cystadenomas in females at 1.5 mglkg bw per day of USP material (10/52) was greater than !hat in concurrent control females (4/59, p= 0.083) bul there was no difference in the total number of benign marnmary neoplasms. Similarly, the incidence of mammary adenocarcinomas and carcinomas in females at 1.5 mglkg bw par day of USP material (9/52) exceedad that in concurrent remale controls (3/59, p 0.063). Howaver, 'he 5.1% incidanca rate 01 mammary carcinomas in the concurrent con'rol females was well below the 12% incidence rate (23/196) reported for historical female controls. While a larga number 01 pituitary adenomas and mammary fibroadenomas were observed, thesa lesions are common in Upj:TUC(SD) rats and were not related to treatment.

=

TBble 2. Numbers of benign, malignant, containing lincomycin 01 two grades Sex

Males

Tumour

Malignant

Benign Total Females

Malignant Benign Total

Vehlcle

9 39 43 12 43

47

Bnd total tumours

in rBts fed dlets

Pramix (mg/kg bw par day) USP (mg/kg bw par day)

0.38

0.75

1.5

1.5

11

13 33

10

22

37

29

38

9 35 38

15

25

33

40

12 39 44

15

11

18

15

43

44 49

40 49

47 51

47

100

12

INCOMYCI1t

Neither premix nor USP·grade lincomycin was carcinogenic under the conditions of the assay, but the low maximum dose used and the poor survival preelude a definitive conélusion. The NOEL for non-neoplastic eHeets was 100 mg/kg bw per day, the highest dose tested. 2.2.4

1I

Genotoxicity

A battary 01tests that complied with GLP were conducted to address the genatic toxicity of lineomycin (Table 3). The onty positive resull was obtained in an assay for unscheduled ONA synthesis in rat primary hepatocytes (Harbarch & Aaron. 1987). While only an abstraet of this study was available, the positive resutt was duplicated in another assay at the relativaly low dose 010.17 ¡.Lglml.In these assays, scoring could not be done at doses > 0.17 ¡.Lglml because of the cytotoxicity 01Iincomycin. In addition, while the full report with raw data is available, it was nol made available to the Committee. The positive results were addressed in a subsequent report to the US Food and Drug Administration (Aaron, 1988b), whieh refers to a similar assay in whíeh negative ar equivocar results were obtained (5eaman, 1982) after use of an improved procedure for microscope slide preparation. The report also noted that negative results were obtained in a study that díd not eonform to GLP in whieh the same lot 01 lincomyein was used as that in the assay with positive results. In the second assay, the taxicity of Iincornycin was much lower (~ 300 J.Lglml), allowing scoring al doses as high as 1000 Jlglrnl. The lower toxicity is consistent with tha! in other assays with negative results (5eaman, 1982; Aaron. 198aa). The weight of the evidenee suggests that lineomyein is not genotoxic. 2.2.5

Reproductive toxicity (a)

Multigeneration studies

In a three-generation study of reproduetive toxicity that did not eomply with GLP, 30 male and 60 female Fo and 10 male and 20 female F2 and F3 Sprague·Oawley rats were treated with Iincomycin premix grade at O, 0.38, 0.75, or 1.5 mg/kg bw per day or USP grade at 1.5 or 100 mglkg bw per day in the diet, beginning with Fo weanling rats, through successive breeding of the Fo, F,. and F2 progeny, to weaning of the F3a Iitters. The dases of premix grade were based on multiples of O, 25. 50, 1DO, and 7000 times the maximum antieipated human dietary intake. given a tolerance in edible tissues 011 rnglkg. No treatment·related effeets were reported on elinical status, fe rtility, or maintenanee of pregnancy in the adults. AII other variables were only summarized, but the report indicated that litter parameters sueh as pup viability,. growth rate. sex ratio, survival rates, clínical status, and gross and histological appearance were also unaffected. The NOEL was 1.5 mglkg bw par day of premix-grade Iincomycin and 100 rng/kg bw per day of USP·grade lincomyein, the highest dases tested (Goyings et al., 1979b). In another study that did not eonform to GLP, groups of 24 pregnant rals were given premix-grade lincomyein by gastric gavage at a dose of O, 10, 30, or 100 mglkg bw per day on days 6-15 of gestation. The dams were killed and

-13

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Tsb/e 3. Results End-point

In vitro Ravarse mutation

o,

o,

tests for the genotoxicíty

líncomycín

Concentration

S. typhimurium

120-1000

J,lglplate Negalivga.b Mazurek & Swenson (1981)

620-5000

J,lglplate Negallve"·t; Aaron & Mazurek (1967)

TAga, TAl 00,

RBSUI1

Reterence

Test objecl

TA1535, TA1537, TA1536 Raverse mutalion

S. typhimurium TAga, TA 100, TA102. TA1535. TA1537

Forward mutation

Chinese hamster 3(}-3000 J,lglml V79 lung fibroblasls, hprt locus

NegativeG

Forward mutation

Chinese hamster 10(}-3000 J.Lglrnl V79 lung fibroblasls, hprt locus

Negative"" Harbach el al. (1982b)

ONA damage (alkalina

Chinesa hamsler V79 lung fíbroblasts

1~ 1300 J.Lglml

Negallve"·1l Petzold (1981)

Unschadulad ONA synthesis

Primary rat hepatocytes

10-2500 J.lglmlh

NegativeJ

5eaman

Unscheduled ONA synthesis

Primary ral hepatocytes

0.17-17

Positiva

Harbarch & Aaron (1987)~

ONA repalr

Human peripheral Iymphocytes

2800-5000

J.lglml

Negalive".l Aaron (1991a)

Cytogenicity

Aat bona marrow

1500-3000 bW"

mg/kg

NegatiVa"

Cytoganicily

Mouse bona rnarrow 150-600

Harbach el al. (1982a)

elulion)

In vivo

5ex·linked Drosophíla recessive lathal me/anogaster rnutation

J.lglm~

mg/kg bw NegativeG

25 000 and 50 000 Negative ¡.tglml

(1982)

Trzos & Swenson (1981) Aaron (1991 b) Aaron (1988a)k

• With Bnd without rat I¡ver microsomal fracUon (S9) 2-Acetylaminofluorene (TA98, TA100, TA1538), cyclophosphBmida (TA1535), and 9aminoacridine (TA 1537) used as positive controls e 2-Aminoanthracene (all strains with 59), 2-nitrofluorane (TA98, TA100 without 59), sodium azida (TA1535 without S9), 9-aminoacridine (TA1437 without S9), and cumene hydroperoxide (TA102 without S9) used as positiva conlrols rI Without S9

. b

• With S9 7,12-Dímethylbenz[a]an!hracene usad as positiva control N-Methyl-N-nitro-N-nitrosoguanjdine and epichlorohydrin usad as complete carcinogen positive controls Bnd benzo[aJpyrene, 4-nitroguinoline-1-oxide, and 2acetylaminofluorene as procarcinogen positive cantrols Concentrations of 5000 and 10 000 ~glml were also testad but wera lethal to the cell culturas. Toxicily WBSobservad at dosas as low as 50 ~glml. 2-Aminoathracene used as positiva control Concentrations > 16.7 ¡..to/mlware lethal lo the cel! culturas. Only abstract provided; fuI! report available from tha sponsor upon request Cyclophosphamide (with S9) and 4-nitroquinoline-1-oxide (wilhout 89) usad as positive controls m One-half the dose WBSadminlsterad at O and 24 h. Administration ot a single dose of 3000 mglkg (one-half of B 6000 mg/l