High dose levels are not necessary in rodent studies to detect human carcinogens

CANCER LETTERS Cancer Letters 75 (1993) 183- I94

High dose levels are not necessary in rodent studies to detect human carcinogens Alastair

Monro,

Thomas

S. Davies

(Received 18 August 1993; accepted 8 October 1993)

Abstract Guidelines for the conduct of rodent carcinogenicity studies stipulate that when the test substance is administered via the diet, its concentration need not exceed 5% of the diet. Since it is now apparent that human carcinogens are amongst the most potent of rodent carcinogens, it should be possible to detect accurately potential human carcinogens by using only relatively low dose levels in rodent studies. Our analysis of the potency of human carcinogens in rodent studies leads to the conclusion that, even after applying a safety factor of IO, there is no purpose in using dose levels higher than 500 mg/kg body weight or 1% in the diet. Key words: Carcinogenic

potency;

Rodents;

Humans:

1. Introduction The regulatory rec,uirement that the carcinogenic potential of chmemicals be assessed by two year studies in mice and the rats (the rodent bioassay) is coming under increasing criticism [2,31, 34,631. The common lhrust to the criticism is that the original objective -- the assessment of carcinogenic risk to humans -- has become subservient to an apparent obsessional desire to maximize the incidence of positives in the rodent bioassay. In striving after this latter experimental objective, investigators have increasingly sacrificed specificity for sensitivity. About 50% of chemicals tested

* Corresponding author.

Dose level limit

(probably well over 1000) in the bioassay, as it is presently conducted, have produced a positive result [5,29,45]. This contrasts with relatively few confirmed human carcinogenic agents i.e., just over 50, of which only 37 discrete chemicals or mixtures could be subjected to evaluation in the rodent bioassay [73]. This discrepancy in incidence between rodents and humans has potentially serious societal repercussions. The classification of a compound as a (rodent) carcinogen often triggers restrictions on its production and use which, if based on poor science, may lead to the unjustified loss of a economically valuable chemical or a useful human or veterinary medicine, or may trigger unnecessary and costly operations to control environmental pollution, or clean up chemical waste sites.

0304-38351931SO6.000 15’93Elsevier Scientitic Pubbshers Ireland Ltd. All rights reserved SSDI 0304-3835(93)03206-K

184

The possible reasons for this discrepancy are several and they have been extensively discussed in the literature. First, it is possible that more human carcinogens will be identified. However, given the dominant role of tobacco, hormones, diet and lifestyle factors [20.21,41], it seems unlikely that epidemiological methods will uncover more than a few chemicals which make a significant additional contribution to human cancer. Second, the condition of the bioassay may mean that rodents are ‘over-exposed’ to the test chemical, in comparison to the conditions under which humans are exposed. Exposure encompasses both intensity (daily dose) and duration (as a proportion of a lifetime). Any attempt to make interspecies comparisons of exposure as a basis for risk assessment is. of course, predicated on the assumption that each species responds in a similar manner to a given exposure. This leads us to the third and farreaching reason for the rodent-human discrepancy i.e., species and tissue differences in response to a given exposure. The fundamental steps in the stochastic process of carcinogenesis - fixation of two or more successive deleterious gene mutations and a proliferation of the key cell populations which outweighs apoptosis - are probably essentially similar in each species. However, there is a striking lack of concordance between the site distribution of cancer in humans and the tissues in rodents in which tumors occur spontaneously or which are the commonest targets for purported chemical carcinogens. Furthermore, the quite different patterns of ‘spontaneous’ tumors in various strains of rodent, and in the species and strain susceptibility to chemical carcinogens, suggest that there are genetically-determined differences in the mechanisms that control one or more of the steps in the process. While genotoxic carcinogens are the most likely to exhibit trans-species activity [5], nongenotoxic rodent carcinogens often are characterized by a singular tissue, sex, species or strain specificity in their target organ [ 12,13,15,32,64]. Some of the mechanisms involved (e.g., hormonal, immunosuppressive) may have relevance for human risk assessment; others (e.g.. chronic irritation associated with an exposure intolerable to humans. prolactin-mediated en-

A. Monro, T.S. Davies/Cancer

Letl. 75 (1993) 183-194

dometrial tumors in old rats, liver tumors caused by peroxisome proliferators, kidney tumors in male rats via azU-globinopathy) appear to have no relevance to human carcinogenic risk assessment. Despite the widespread recognition of the many flaws in the bioassay there will be an understandable reluctance on the part of regulatory authorities, in the face of public opinion, to reduce the current requirements for and of the bioassay. Even if one accepts the near inevitability of this politically conservative attitude there is, nevertheless, still enormous scope to improve the bioassay. In the longer term, it is possible that genetic engineering will produce transgenic animal models which are of much greater relevance to humans than are the current laboratory rodent models. However, much development and validation of the models is still before us and the realization of this objective must be some years off. On the other hand, by contrast, there is one major improvement which could be effected immediately i.e.. the development of a guideline for dose selection (exposure) in the bioassay which has relevance to the human situation. The dogma that the sensitivity of the bioassay be maximized by use of the ‘maximum tolerated dose’ has generated extensive debate and controversy [2,14,16,30] and it will not be reviewed here. Clearly, however, the use of lower dose levels in the bioassay would lead to a lower incidence of positive results. The question then arises as to whether this lower positivity would also result in human carcinogens being ‘missed’ and we shall discuss this. A fourth reason for the discrepancy may be the well documented species differences in metabolic disposition of xenobiotics. These differences may underlie species differences in response (carcinogenicity, in this case), but a priori it is difficult to understand why such differences should lead to an apparently generally greater susceptibility of rodents than of humans. So, what alternatives are available to improve this situation in which the bioassay, as currently conducted, produces about a 50% positivity rate for substances, most of which appear to carry no or a negligible cancer risk for humans? In simple terms, one can either continue with the present basic procedure and try to improve the risk

A. Monro. T.S. Davies/Cmcer

assessment

185

Lett. 75 (1993) 183-194

process at the end of the study; or, one

can try to change the format of the bioassay with the intention of carryl.ng out an experiment which, from the outset, is designed to generate information more relevant to human risk assessment. In the former category, one finds the various attempts to classify rodent carcinogens in terms of the risk they portend for humans [4,47,55]. Such analyses start with the data which exist, or are still being generated, from the rodent bioassay. All other information of possible relevance, such as genotoxicity data, chronic toxicity, endocrine effects, pharmacodynamics and pharmacokinetics, are considered and a.judgement of carcinogenic risk is based on the weight of the evidence. In a recent paper, Tennant argues that the rodent carcinogens which should command the greatest attention as potential human carcinogens are the trans-species (rat and mouse) multiple-site carcinogens [71]. This category is considered to be the least susceptible to genetic variability across species and the findings would be expected to be more reasonably extrapolable to other species. including humans. This approach, however, is still limited inasmuch as one would expect that the differences in response between humans and rodents to be greater than those between rodent species. It is interesting to note that the top priority category possessed the greatest proportion of mutagens (Sulmonellu) and were also more potent carcinogens than the single species/sex/site rodent carcinogens. This prioritizat,on of the existing bioassay data is important and must continue. Proposals to modify the current protocol for the bioassay would fall Into the latter category. In 1992. the NTP Board Iof Scientific Counselors proposed that the use of the MTD should be reexamined and that mechanistic studies should be juxtaposed with the c#Dnduct of the bioassay [63]. In this paper we examine whether there is an empirical basis for, quite simply, using lower dose levels and whether such a strategy would have implications for human risk assessment. If one starts from the premise that the current bioassay is producing ‘too many’ rodent carcinogens, how does one decide which of this surfeit of carcinogens are of relevance? Is it simply the few most potent ones? At first sight this might appear

reasonable, given the general (though fairly loose) overall concordance between the potencies (in mg/kg/day) of carcinogens in mice, rats and humans [1,19,27,30]. In addition, since (a) genotoxic substances are more likely than non-genotoxic ones to show trans-species carcinogenicity [5], (b) genotoxic rodent carcinogens are more potent than non-genotoxic rodent carcinogens [ 11,581 and (c) most human carcinogens are mutagenic, one would expect human carcinogens also to be potent rodent carcinogens. However, cancer is a complex process and that there are several non-mutagenic human carcinogens e.g., hormones, immunosuppressants, which may act by receptor or other mechanisms which can show marked species differences in sensitivity. We have reviewed the rodent carcinogenicity data for known human carcinogens to determine whether there is a basis for imposing an arbitrary upper limit on the dose levels used in the rodent bioassay. 2. Method We examined the original reports of the rodent bioassays which employed oral or parenteral (but excluding inhalation) administration of the Group I chemicals (confirmed human carcinogens) of the IARC Monographs Programme [47]. We sought the lowest level which was judged by the investigator (or, in a few cases, by us) to provide either clear or some evidence of carcinogenicity in any tissue and these data are summarized in Table 1. We retained the original expression of dose level; this could be mg/kg body weight, ppm in the diet or in the drinking water. No correction was made for 5 days/week dosing, nor for the duration of the study. Table 2 lists the lowest carcinogenic dose for each chemical, the data being derived from Table 1. To facilitate comparisons, in Table 2 dietary concentrations that were expressed in the original publication only in terms of mg/kg body weight have been recalculated to give an equivalent dietary concentration; gavage or injections made only sporadically, for example once or twice per week, have been converted to the corresponding equivalent average daily dose, a practice supportable at least for genotoxic carcinogens for

7, 14, 28, 55”, 110”. 220” ppm 76. 19”. 38”, 75”. 150”. 300” ppm

Dose lowered from 300 to 150 ppm then increased to 250 ppm

Subcutaneous injection Drinking water Drinking water

Diet

Intraperitoneal injection lntraperitoneal injection Gavage Gavage Gavage

Gavage

Rat (M & F)

Mouse (F)

Rat (F)

Mouse (M)

Rat (M)

Rat (F)

Mouse (M)

Mouse (F)

Azathioprine

Mouse (F)

Mouse (M)

Diet

Rat (M)

4-Aminobiphenyl

0.25”, 0.5”. l.Oa ppm

Diet Gavage

0.001. 0.005, 0.015, 0.050”. 0.1 OOa ppm 0.015”. 0.3”, 1.0” ppm

Dose level (ppm or %)

1) and some hormones

Rat (M & F) Rat (M & F)

Group

Diet

Route

(IARC

Rat (M)

B,

carcinogens

Aflatoxin

tests with human

Species

I

Chemical

Table Rodent

x IO days

x 5 days,

15” mg/kg 3x/week months 30” mg/kg 3 x /week months

25”, 50”. 100” mg/kg

25”. 50”. 100” mg/kg

25”. 50”. 100” mg/kg

50”. 100”. 200” mgikg

7.5”. for 6 7.5”. for 6

Mean total dose: 3.6-5.8” g/kg over 250-376 days ( - 15 mg/kg/day)

80 &rat 40” &rat

Dose level (mg or rg per kg or per animal) site or type

Zymbal’s gland, oral cavity, skin Zymbal’s gland oral cavity Zymbal’s gland. preputial gland, Harderian gland. lung, malignant lymphoma Zymbal’s gland, ovary, mammary gland, lung. malignant lymphoma

Lymphosarcoma. lung Lymphosarcoma. lung. uterus

Thymic lymphoma. ear duct

Large intestine. mammary gland Angiosarcoma (multiple sites). urinary bladder Angiosarcoma (multiple sites), liver. urinary bladder

Liver. kidney

Liver Liver

Liver

Tumor

54

54

54

54

77

77

17

67

67

76

24

81 81

82

Ref.

lntraperitoneal injection Intraperitoneal injection

Mouse (F)

Rat (M)

Methyl-CCNU

Diet

Mouse (F)

8-Methoxypsoralen with UV light

500a ppm

35

35

71

Skin

Skin

Peritoneum, lung, subcutis. breast, brain

0.4” mg/mouse/day

1.5”. 3.0” mgikg 3 x/week for 6 months

A Mouse

69 0.02, 0.1”. 0.4a, l.6a mg/kg; 12 inj in 4 weeks

Lung (Strain Assay)

Intraperitoneal injection

Mouse (M & F)

Melphalan

40 66

o.ol5%u” Daily intake: 1, 4, 16a mg/kg (cont. in ppm not given)

Thymic lymphoma Malignant lymphoma

Diet Diet

Mouse (M) Mouse (M & F)

Cyclosporin

68

Hematopoietic, nervous. and lymphoid systems, urinary bladder

Daily intake; 0.31, 0.63”. 1.25”, 2.5a mg/kg (cont. in ppm not given; drug given in 5 ml water after water deprivation overnight ~ see Note)

Drinking water

Rat (M & F)

Cyclophosphamide

69

Mouse (M & F)

69

7

75

10 36

A Mouse

Intraperitoneal injection

Mouse (M & F)

3mg/kglx,2xa,4.5~“, 9 x Ymonth

Liver, Harderian gland

gland

Lung (Strain Assay)

Gavage

Rat (F)

50a, lOOa, 150” ppm

1.2a, 2.5a, 3.5, 5.0” mg/kg (13 doses in 30 &j;ij

70

70

6.2, 25”. 100” 400” mg/kg; I2 inj in 4 weeks

Diet

Mouse (M & F)

170” ppm

glands to external canal

glands to external canal, liver,

Intraperitoneal injection

Diet Gavage

Rat (F) Rat (F)

I x/week for life

15” m&at

Sebaceous adjacent auditory rectum Sebaceous adjacent auditory Liver Mammary

0.8, 3.1a, 12.5”. 35.0” mgkg; 12 inj in 4 weeks

Subcutaneous injection

Rat (F)

1x/week for life

15a mg/kg

Nervous, hematopoietic and lymphatic systems. mammary gland, ext. ear canal Lung (Strain A Mouse Assay)

Subcutaneous injection

Rat (M)

Chlornaphazine

Chlorambucil

Benzidine

Vinyl chloride

300” mg/kg

300” mg/kg

46. 139*, 424= ppm

46. 139a, 424” ppm

Diet

Diet

Diet

Diet

Gavage

Gavage

Diet

Rat (M & F)

Mouse (M & F)

Rat (M)

Rat (F)

Rat (M)

Rat (F)

Rat (M & F)

0.49, 4.49. 44.1” ppm

Daily intake: 44a mgikg (talc. from weekly dose of 310 mg/kg; cont. in ppm not given) Daily intake: 23” mgikg (talc. from weekly dose of 160 mgkg; cont. in ppm not given)

Gavage

Rat (F) 51 weeks

0.1-3, 10, 30” mg/rat 5 days/week x 1 year 300a mg/kg; 1 x /week for

Gavage

Rat (M & F)

2-Naphthylamine

9 Liver

Liver. angiosarcoma, (liver. lung and abdomen); Zymbal’s gland Angiosarcoma (liver, lung and abdomen): Zymbal’s gland Liver (neoplastic nodule, angiosarcoma. .. *

Liver, angiosarcoma, (liver and lung); Zymbal’s gland; neoplastic nodules in liver were increased at all doses levels Liver, angiosarcoma (liver, lung and abdomen); neoplastic nodules in liver were increased at all doses levels

9

72

25

25

25

25

44

38

18

42

43

Ref.

Liver (only I rat with a liver tumor) Urinary bladder, kidney, ureter Urinary bladder

lymphoma.

Thymic ovary

12a mg/kg; 4 inj in 8 wks

Intravenous injection

Mouse (F)

Myerlan

Mouse (M & F)

Subcutis (injection site) Lung (Strain A Mouse Assay)

O.OSa ml of 0.05% solution per mouse 1 x/week for 6 weeks 0.25” ml of 0.006-0.007% solution per mouse; 4 inj in 8 days

site or type

Subcutaneous injection Intravenous injection

Mouse (M & F)

gas

Mustard

Dose level (ppm or %) Tumor

Route Dose level (mg or gg per kg or per animal)

Species

Chemical

Mouse (F)

Norethynodrel mix withimestran01 (50: 1)

Testosterone

“Dose level at which tumors were observed. Note: We consider this to be more similar to once daily gavage

Mouse (F) (neonatal)

dose than to continuous

gland

administration

via the drinking

water.

53

Mammary

5”. 20” &mouse

x 5 days

74

Uterus

Subcutaneous implants Subcutaneous injection

56

Prostate

61

60

65

65

48

28

53

78

0. 10, 20”. 30” m&at; replaced every 6-8 weeks for 6 months or more (pellets contain approx 10 mg testosterone each) I-2“ mgmouse 2 x/week for life

Mouse (F)

Rat (M)

Prostate

80-100” mg/rat for 6 months (implants renewed at 3 months)

Subcutaneous implant Subcutaneous implant

gland

Mammary

Rat (M)

gland

gland,

Mammary pituitary

Mammary

gland

Mammary

gland

gland

Mammary

Mammary

gland.

Mammary pituitary

Pituitary

every 2 months

x 5 days

28

gland,

Mammary pituitary 33

80 26

49

Liver, pituitary Mammary gland

Liver (angiosarcoma)

7”. 70” &mouse

40” mg/mouse for 1 year

5”. 20” &mouse

IO” mg/kg

0.03, 0.3, 1.0. 3.33, 16.65”. 50.0” mg/kg

Gavage

1.1” mg/kg

Daily intake:

Diet

Mouse (M)

Norethynodrel

75” &kg

Mouse (M)

Mestranol

Daily intake:

Subcutaneous implant

Mouse (F)

Medroxyprogesterone

Daily intake: 0.07”, 0.7” mg/kg (cont. in ppm not given)

0.5” mg/l

0.0062Sa, 0.0125. 0.025, 0.05”. O.l”, 0.5”, 1.0” ppm Daily intake: 0.02a, 0.2a mgikg (cont. in ppm not given) 0.640” ppm

Diet

Diet

Rat (M & F)

Estrogen (conjugated)

Diet

Mouse (F)

Mouse (F) (neonatal)

Diet

Rat (M & F)

Mouse (F)

Gavage Diet

Rat (F) Mouse (F)

Gavage

Drinking water Subcutaneous injection

17/3-Estradiol

DES

Hormones

Rat (M & F)

5

A. Mom-o. T.S. Davies/ Cancer Left. 75 (1993)

190

Table 2 Lowest dose level at which human

Aflatoxin B 4-Aminobiphenyl Azathioprlne

carcinogens

and some hormones

Species

Diet/drinking

Rat Rat Mouse Rat

0.015c

Benzidine

Chlorambucil Chlornaphazine Cyclophosphamide Cyclosporin Melphalan 8-Methoxypsoralen with UV Methyl-CCNU Mustard gas

Vinyl chloride

GavageIinjection

(mg/kg)b

0.6’ 15C

P.0 s.c

3.2c 25’ 25c 8.6c 2.0c

i.p. p.0. p.0. s.c p.0.

0.2 1.33 10.7 0.63

p.0. i.p. i.p.

0.043 12.oc

i.p. i.p.

5oc

P.0

I 5oc I12 5ooc

0.64c

Rat Mouse Mouse Mouse Rat Mouse Rat

Myleran 2-Naphthylamine

water (ppm)”

in rodents

7c 150-3ooc

Rat Mouse Rat Rat Mouse Rat Mouse Mouse Rat Mouse Mouse Mouse Mouse

Benzene

have been detected

183-194

161’ 44

O.lC 0.27c 0.86C 43

i.p. S.C. i.v. i.v. p.0.

16.7

p.0.

IOC

P.0

Hormones DES 17&Estradiol Estrogens (conjugated) Medroxyprogesterone Mestranol Norethynodrel Testosterone

0.4c 0.0062’ o.5c 1.4c

Rat Mouse Mouse Rat Mouse Mouse Mouse Rat Mouse

o.15c

S.C.

2OC

S.C.

1.6 17c

SC.

0.52’ 7.7’ S.C.

“Dietary dose levels expressed in the original as mgikg body weight were multiplied by 7 for mice and 20 for rats to convert to ppm of diet. bDoses given in Table I as per rat or per mouse were multiplied by 4- I4 (depending on the weight of the rat) and 30. respectively to give an approximation to mgikg. Doses given originally as several doses over d days were cumulated and divided by d to give a daily dose. CLowest dose tested; a tumorigenic response could be present at a lower dose.

which cumulative dose is important [22,79]. Doses expressed per animal have been converted to mg/kg body weight. 3. Results and discussion We .decided

to treat

separately

the data

from

gavage, dietary and drinking water studies. The different rates of input of a given dose can have pharmacokinetic-pharmacodynamic implications for the pattern of chronic toxicity and potency of a carcinogenic response [52,62]. Thus, the hepatocarcinogenic potency of chloroform in mice depends on whether it is administered by gavage or

A. Monro. T.S. Davies/Comer

Let!. 7S (1993)

191

183-194

in the drinking water. In an analysis of 379 bioassays from the NTP data base, it transpired that chemicals administered (at the MTD) by gavage are more likely (41% vs. 26%) to be carcinogenic than chemicals administered in the diet [39]. The same analysis also indicated that the inverse correlation between the MTD and the number of positive experiments (sex/species) applied primarily to gavage and not to feeding studies. Another reason for separating dietary from gavage studies is that in the former there is a marked reduction in food intake per unit of body weight over the first few months of life, with a concomitant reduction in the daily dose (in mg/kg body weight). Without knowing the significance for carcinogenicity of the dose rate early in life versus that late in life, it may not be justified to use an averaging factor for diet-gavage conversion of dose rate. In our analysis (Table 2), it should be noted that for most substances tumorigenicity was observed at the lowest dose *iested. Therefore, the true lowest effect level could be even lower than the doses listed in the Table. For non-hormonal chemicals administered by gavage or injection the minimum daily dose to produce a carcinogenic effect ranged. in mice, from 0.04-25 mg/kg; in rats, the range was from 0.2-43 mg/kg. For chemicals administered via the diet, the minimum dose for mice was from 7-500 ppm; for rats, the range was from 0.015-880 ppm. The extraordinary potency of the hormones highlights the known marked species differences in sensitivity to hormonal effects (for sex hormones see, for example, 1371). Thus, overall, human carcinogens are amongst the most potent of rodent carcinogens. This observation is consistent with the oft-noted correlation between trans-species (mouse-rat) carcinogenicity and toxicity [6,8,11,29,39]. The lower the MTD, the more likely a chemical is to be a trans-species carcinogen. For example, in the Brown and Ashby analysis [l l] (in which diet and gavage studies were confounded), the proportions of mouse or rat carcinogens positive in the other species were about 70% in the dose range up to 300 mg/kg, but only 41% at dose levels over 800 mgikg. Our findings are also consistent with another recent analysis, in which the author applied a dif-

ferent measure of carcinogenic potency, the TD,,*, to chemicals in IARC groups I, IIA, IIB, and III [50]. It was found that all group I chemicals (human carcinogens) had a TDjo of < 100 mg/kg for both mice and rats, while for group IIA (probable human carcinogens) the proportion with a TDso of < 100 mg/kg was 93% for rats and 91% for mice. For groups IIB and III, a 90% rate was only achieved at TD,, values near 1000 mg/kg. This means that any strategy to use lower dose levels in the bioassay could not be criticized for its failure to encompass ‘probable’ human carcinogens. An other perspective, and which is not without interest in the present context, is provided by a different analysis of the dose levels required in rodent studies to detect human carcinogens. Without commenting on the absolute values, the author observed that in no case was the use of the MTD necessary to detect human carcinogens [3]. 4. Conclusion and proposal As the Guidelines for the conduct of the bioassay have evolved there has appeared the apocryphal statement to the effect that the highest dose level should not exceed 5% (50 000 ppm) of the diet [46,5 1,571. One can find vague speculations that to exceed this limit might have deleterious consequences for the nutrition of the animals. It is ironic to realize today that if the fear was based on a possible reduction in food intake (in animals feeding ad lib), the consequence might have actually been beneficial to the model! However, in the frenzy to maximize the exposure of the animals no consideration appears to have ‘been given to whether the administration of such high dose levels would produce results of relevance to human risk assessment. Our analysis shows that all human carcinogens to which humans are exposed by the oral route can be detected in rddent bioassays at dose levels at or below 43 mg/kg body weight or 880 ppm in the diet. We propose, therefore, that there is a strong empirical basis for reducing the current upper dose limits in the

*Defined as the chronic dose rate (mgikglday) which would halve the probability of animals remaining tumor-free [5?].

192

A. Monro. T.S. Davies/Cancer

guidelines to levels which are more relevant to the objective of the bioassay. The uncertainties, already discussed, of comparing does levels given by gavage and by diet (based on factors which may be compoundor mechanism-dependent, and strain-, diet-or husbandrydependent) leads us to make separate recommendations for each method of administration. We propose that the maximum dose levels that need be administered to either rats or mice should be:

genotoxic chemicals among animal and human carcinogens evaluated in the IARC Monograph series, Cell Biol. Toxicol.. 5, 115-127. Berger, M.R., Habs. M. and Schmahl, D. (1985) Comparative carcinogenic activity of prednimustine. chlorambucil. prednisolone and chlorambucil plus prednisolone in Sprague-Dawley rats. Arch. Geschwulstforsch. 55.

I

8

9

Gavage:

500 mg/kg

Diet: 10 000 ppm (1%)

These dose levels offer arbitrary safety margins of slightly more than 10 (500143 or 10 000/880) over the highest levels shown to be necessary to detect the human carcinogens identified so far. For interest, application of the widely-used factors for conversion of gavage doses to dietary concentrations (about 7-10 for mice and lo-20 for rats, depending on their age), indicates that the proposed gavage limit of 500 mg/kg corresponds to 3500-10 000 ppm (0.35-l%) diet. So there is consistency in the dual proposal. One division of the Environmental Protection Agency of the United States already recommends an upper limit dose of 1000 mgikg body weight for carcinogenicity testing [23].

10

11

12

I3

14

I5

5. References 16 Allen. B.C., Crump, K.S. and Shipp, A.M. (1988) Correlation between carcinogenic potency of chemicals in animals and humans, Risk. Anal., 8, 531-544. Ames, B.N. and Gold, L.W. (1990) Chemical carcinogenesis: too many carcinogens. Proc. Nat. Acad. Sci., 81. 1112-1116. Apostolou, A. (1990) Relevance of maximum tolerated dose to human carcinogenic risk. Regul. Toxicol. Pharmacol.. 11. 68-80. Ashby, J., Doerrer. N.G., Flamm. F.G.. Harris. J.E.. Hughes. D.H.. Johannsen, F.R., Lewis, SC., Krivanek. N.D., McCarthy, J.F., Moolenaar. R.J., Raabe, G.K.. Reynolds, R.C., Smith, J.M., Stevens, J.T., Teta, M.J. and Wilson, J.D. (1990) A scheme for classifying carcinogens, Regul. Toxicol. Pharmacol., 12. 270-295. Ashby, J. and Tennant, R.W. (1991) Definitive relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP. Mutat. Res.. 257, 229-306. Bartsch, H. and Malaveille, C. (1989) Prevalence of

I7

18

19

20 21

4299442. Bernstein, Hoel, D.G. parison of dam. Appt.

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