Mutation Research, 264 (1991) 155-162 © 1991 ElsevierSciencePublishers B.V. All rights reserved 0165-7992/91/$03.50 ADONIS 0165799200000000

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MUTLET 0546

Carcinogenic ranking of aromatic amines and nitro compounds H.C. Crabtree a, D. Hart b, M.C. Thomas c, B.H. Witham c, I.G. McKenzie d and C.P. Smith d alCI Colours and Fine Chemicals, P.O. Box 42, Hexagon House, Blackley, Manchester M9 3DA, blCI Group Occupational Health, Alderley Park, Macclesfield, Ches. SKIO 4TJ, cCiba-Geigy PLC, Hulley Road, Macclesfield, Ches. SKIO 2NX and dHickson and Welch Ltd., Wheldon Road, Castleford, West Yorkshire WFIO2JT (Great Britain)

(Received5 March 1991) (Revision received10 July 1991) (Accepted 22 July 1991)

Keywords: Amines, aromatic; Nitro compounds; Ranking scheme

Summary A scheme is proposed for ranking the carcinogenicity of aromatic amines and nitro compounds based on both qualitative (weight o f evidence) and quantitative (carcinogenic potency, i.e. the TDs0 value) factors. The scheme has been drawn up specifically with a view to linking with workplace hygiene controls. Other essential features are that a reliable database exists for the TD~o values for many compounds and that the scheme is capable of usage by non-toxicologists. Validation o f the scheme using 38 aromatic amines or nitro compounds indicates that the main objectives have been met. Extension to different chemical classes should be possible but has not been attempted in this work. An example of a potential hygiene control scheme for use alongside the carcinogenicity ranking is described.

Industrial handling o f potentially carcinogenic substances requires appropriate workplace hygiene The authors of this article comprised members of a small working group responding to the U.K. Chemical Industries Association (C.I.A.) Dyestuffs Health and Safety Committee chaired by Dr. I.G. Laing (Ciba-Ceigy, PLC).

Correspondence: Dr. D. Hart, 1CI Group Occupational Health, Alderley Park, Maeclesfield, Ches. SK10 4TJ (Great Britain).

measures. To facilitate this, a carcinogenicity ranking system has been developed and tested by application to 38 industrially important aromatic amines and nitro compounds. The primary aim has been a pragmatic scheme suitable for use by nontoxicologists, based on readily available, validated data and capable o f being linked to a limited range o f practical workplace controls. Such a scheme is seen as important in enabling users o f aromatic amines and nitro compounds to

156

fulfil obligations under health and safety law such as the UK COSHH (Control of Substances Hazardous to Health) Regulations. Proposed ranking schemes other than the one described here were considered but none incorporated the requisite ease of use and in particular the ability to arrive at a ranking using relatively easily available data. Most existing proposals for assessing carcinogenic potential utilise either a qualitative approach (weight of evidence indicating potential for causing cancer in humans) or a quantitative approach (some indicator of potency or magnitude of effect). It was felt that, for the present purposes, a method combining both factors was most suitable and the methodology described by Brown et al. (1986) was used as a model. Sowinski et al. (1987) described criteria for identifying and classifying carcinogens; these were found to be clear and relatively easy to use. They were therefore taken with slight modification as the qualitative descriptor in our scheme. This qualitative (weight of evidence) assessment was based upon International Agency for Research on Cancer (IARC) reviews where available, the formal

IARC classification was not used (except for Proven Human carcinogens) as it was feld essential to have flexibility to consider chemicals not classified by IARC and/or to include more recent relevant data. In addition to IARC reviews the conclusions from the bioassay reports published by the U.S. National Cancer Institute/National Toxicology Programme were also considered. The relative potency indicator selected was the TDso value obtained from the Carcinogenic Potency Database of Gold et al. (1984). The definition of the TDs0 value is" 'The chronic dose rate (in mg/kg body weight per day) which would have the actuarially adjusted percentage of tumour free animals at the end of the 'standard' life-span for the species'. The TDs0 value is only one of a number of potency indicators but was selected on the following basis: it has a sound statistical background; a reliable database exists which is updated from time to time; after familiarisation with the database, TDso values can be reproducibly selected from the database by non-experts. Using these qualitative and quantitative parameters a ranking matrix (Table 1) was

TABLE 1 MATRIX FOR RANKING CARCINOGENICITY OF AROMATIC AMINES AND NITRO COMPOUNDS Weight of evidence

TDs0 potency category

Classification

1 Proven human

Any

Proven Human

2 Suspect human Proven animal Suspect animal

< <


100 mg/kg/day 10- 100 mg/kg/day 1- 10 mg/kg/day

-) ~

B

4 Proven animal Suspect animal

100-1000 mg/kg/day 10- 100 mg/kg/day

7

5 Suspect animal

100-1000 mg/kg/day

C D

6 Non-classifiable

alncapable of classification

7 Negative evidence

Negative evidence

aRefer to expert.

157 developed. It should be noted that the proposed ranking scheme covers solely carcinogenic potential. In practical situations, other toxicological and physico-chemical properties must also be considered in drawing up workplace control strategies. Methods

38 aromatic amines and nitro compounds have been assessed by personnel from three companies working under the auspices of the CIA Dyestuffs Health and Safety Committee. Results are encouraging: a carcinogenicity ranking has been achieved which can be used for drawing up occupational hygiene strategies; non-toxicologists (albeit persons with a basic knowledge of 'product safety') are able to operate the system, given an initial familiarisation and working to guidelines on the selection and scrutiny of data used in the assessment; once basic data selectin factors were defined, there was close agreement in ranking obtained by persons working independently; the basic system appeared capable of application to other chemical classes but would require validation for each class prior to use. Where doubt existed on selection of a TD50 value or the weight of evidence, the most rigorous option was taken in every case. Thus 'borderline' substances were allocated to the more severe of any two possible rankings.

Ranking guidelines In order to achieve consistency of ranking by different persons, it was found to be essential to drawup detailed guidelines. These covered both the weight of evidence assessment, the choice of TDso value and the significance of various sites and types of tumours. The last of these was included in the guidelines for the use of non-toxicologists. For the purpose of this paper, only the weight of evidence categories are described in detail below. However some outline comments are included on the selection of TDs0 values.

Weight of evidence categorisation (the qualitative assessmenO The following categories are based on Sowinski et al. (1987).

Proven human carcinogen.

This categorisation would result from the conclusions of an IARC evaluation if consistent with other known, valid data. It would also result from good quality epidemiological studies showing a cause and effect relationship. In epidemiological studies, cohort or case control data are given more weighting than multiple case reports. If both cohort and case control studies exist, then expert advice should be sought with regard to which is most significant. Clear identification of a causative agent is important.

Suspect human. This group comprises proven animal carcinogens together with suggestive epidemiological evidence e.g. several case reports or clusters (if both exist, the former is given more weight than the latter). Consideration should be given to inclusion in this group where suggestive epidemiology exists together with only limited positive animal studies but reinforced by positive in vivo genotoxicity data. Proven animal carcinogen. Indicated by clear, statistically significant increases in malignant tumours; tumour incidence should be apparent in more than one species. Where data exists for one species only, careful consideration of tumour type is necessary before placement in this group. Support from genotoxicity tests is important particularly in the latter instance (with in vivo results given more weighting than in vitro results). Positive in vivo genotoxicity data strongly supports placement of a compound in this category. Evidence of a dose-response relationship supports classification in this group. Suspect animal carcinogen. Indicated by conflicting and/or limited evidence or positive studies of good quality but with results of questionable relevance e.g. where tumours occur only at ex-

158 TABLE 2 RANKING RESULTS IN ORDER OF CARCINOGENIC POTENTIAL Based on evidence available, August 1989. TDso value selected a(Species, sex, tumour site, tumour type)

Weight of evidence

Not applicable Not applicable

Proven human Proven human

8.98 mg/kg (R,f, mgl,car) < 1.78 mg/kg (D,f,ubl,mix) 2.12 mg/kg (D,f,ubl,ptc) 1.34 mg/kg (M,m,sub,hes)

Proven Proven Proven Proven

90-94-8

5.47 mg/kg (R,f,liv,hpc)

Proven animal

137-17-7

6.13 mg/kg (M,f, liv,hpc)

Proven animal

4,4' -Diaminodiphenylmethane

101-77-9

22.3 mg/kg (M,m,liv,hpc)

2-Methoxyaniline 2-Methoxy-5-methylaniline 2-Methoxy-5-nitroaniline

90-04-0 120-71-8 99-59-2

27.8 mg/kg (R,f, ubI,MXA) 44.7 mg/kg (M,m,ubI,MXA) 30.5 mg/kg (R,m,ski,MXA)

Proven/suspect animal Proven animal Proven animal Proven animal

121-66-4 95-83-0 121-14-2 615-05-4 101-61- 1 95-53-4

90.2 mg/kg (R,m,mul,grl) 212 mg/kg (R,f,ubl,MXA) 31.1 mg/kg (R,m,liv,MXB) 113 mg/kg (R,f,cli,mix) 27.7 mg/kg (R,f, thy,fcc) 131 mg/kg (R,m,ubl,mix)

Suspect animal Proven animal Suspect animal Proven animal Suspect animal Proven animal

117-79-3 82-28-0 119-34-6 62-53-3 95-79-4 102-50-1 99-55-8 94-52-0 80-08-0

268 mg/kg (R,m,liv,hpc) 150 mg/kg (R,m,liv,hpc) 309 mg/kg (R,m,ubl,tcc) 160 mg/kg (R,m,spl,hes) 213 mg/kg (M,m,-,hes) 470 mg/kg (R,m,ubl,tcc) 266 mg/kg (M,m,liv,hpc) 423 mg/kg (M,m,liv,hpc) 167 mg/kg (R,m,spl,fbs)

Suspect animal Suspect animal Suspect animal Suspect animal Suspect animal Suspect animal Suspect animal Suspect animal Suspect animal

615-66-7 95-70-5 119-90-4 119-93-7 134-32-7

No clear evidence of carcinogenicity based on largely inadequate studies No TDso value No TDso value No TDso value

Substance

CAS No.

Proven human

Benzidine 2-Naphthylamine

92-87-5 91-59-8

Class A

2,4-Diaminotoluene 3,3' -Dichlorobenzidine 4,4'-Methylenebis(2-chloroaniline) 2-Methyl-l-nitroanthraquinone (N.B. purity of test article in question) Michler's ketone (N.B. purity of test article in question) 2,4,5-Trimet hylaniline

95-80-7 91-94-1 101-14-4 129-15-7

animal animal animal animal

Class B

Class C

2-Amino-5-nit rot hiazole 4-Chloro- 1,2-phenylenediamine 2,4-Dinitrotoluene 4-Methoxy- 1,3-phenylenediamine 4,4' -Methylenebis(N, N-dimethylanili ne) 2-Methylaniline Class D

2-Aminoanthraquinone l-Amino-2-methylanthraquinone 4-Amino-2-nitrophenol Aniline 5-Chloro-2-methylaniline 4-Methoxy-2-methylaniline 2-Methyl-5-nitroaniline 5-Nitrobenzimidazole 4,4' -Sulphonyldianiline Incapable o f classification

2-Chloro- 1,4-phenylenediamine 2,5-Diaminotoluene 3,3' -Dimethoxybenzidine 3,3' -Dimethylbenzidine 1-Naphthylamine

159 TABLE 2 (continued)

Substance

CAS No.

TDso value selected a(Species, sex, tumour site, turnout type)

Weight of evidence

Negative evidence 2-Aminobenzoic acid 3-Chloro-4-methylaniline 2,6-Diaminotoluene l-Nitronaphthalene 4-Nitro- 1,2-phenylenediamine 1,4-Phenylenediamine

118-92-3 95-74-9 823-40-5 86-57-7 99-56-9 106-50-3

Not Not Not Not Not Not

Negative evidence Negative evidence Negative evidence Negative evidence Negative evidence Negative evidence

applicable applicable applicable applicable applicable applicable

a For explanation of the codes given in brackets, see L.S. Gold et al. (1984).

tremely high doses (i.e. greater than 1000 mg/kg bodyweight) or doses that disrupt the normal function of the target organs.

Non-classifiable. Indicated by animal data of doubtful significance, for example, due to the route of dosing (injection or implantation site tumours, etc.) or effects known to be due to a secondary mechanism. With good quality studies, attribution of excess tumours to a secondary cause must be based on a clear understanding of the mechanism. Doubts over the purity of the chemical or evidence that an atypical material was tested may also result in allocation of a chemical to this group. Negative evidence. Indicated by good quality negative studies.

Selection of TDso values (the quantitative assessment) Before a TDso value is selected it is necessary to become familiar with the contents of a typical record. This has been described in detail by Gold et al. (1984). In selecting a TDso value, our principle was to look for clear increases in the incidence of malignant tumours, with most weighting being given to the malignant turnout type showing the highest

numerical increase compared to the control group. This is recommended as most likely to ensure that the potential for carcinogenesis in multiple species including man is evaluated. Where there are no values based solely on malignant tumours then combinations of benign and malignant tumours may be used but in this event the tumours should be derived from the same cell type. Combinations of malignant tumour types associated with the same cell types (e.g. several tissues all of which contain sebaceous glands) may he selected in the absence of other more suitable TD~o values. In the validation exercise we utilised the lowest TDso (most potent carcinogenic effect) meeting our selection criteria. Subsequent to the validation exercise Gold et al. (1989) published a review of their TD~o data for 492 rodent carcinogens. They selected single TDso values for each of the two main rodent species (but not for other pertinent species e.g. the dog); the selection method is described in Gold et al. (1989) and differs in detail from that described in this article, i.e. in basing the choice on statistical significance of the dose-response where available. Where this was not available, they selected any positive site. In neither case was the relevance of the site to multispecies carcinogenicity considered. It follows that values selected by the Gold et al. (1989) procedure will not necessarily lead to identical TDs0 values to those from our scheme. In such cases, it would be prudent to either

160 select the lower of the two values or seek expert advice. Results The results from the validation exercise with the 38 aromatic amines and nitro compounds are shown in Table 2. To help demonstrate the process by which these were obtained, two worked examples are described below.

Worked examples (a) 2-Methoxy-5-nitroaniline dine) CAS Registry No. 99-59-2

(5-nitro-o-anisi-

The 'qualitative assessment' was based on the IARC review in Monograph 27. Data from the mouse study suggested an increased incidence o f hepatocellular carcinoma in males; however there was no clear dose-response and the group showing the highest incidence were from a different shipment o f animals than the controls. In contrast the rats showed a very high incidence of carcinoma of the skin in males, and o f the zymbal gland or skin of the ear in both sexes. There was also a clear increase in clitoral gland adenoma or carcinoma and probably an increase in mammary gland adenocarcinoma in females. Based on clear evidence in the rats and some carcinogenicity in mice, this chemical was classified as a 'proven animal carcinogen'. The 'quantitative assessment' involved identifying the most marked treatment-related effects on tumours (the majority o f which were malignant). The associated TDs0 value was 30.5 m g / k g for a combination o f skin tumours in the rat (this was also the lowest quoted TDso value excluding those that our guidelines state should not be used). Combination of 'proven animal' classification and a TDso value of 30.5 mg/kg resulted in an overall classification o f 'B'.

(b) 4-Methoxy-2-methylaniline CAS Registry No. 102-50-1

(m-cresidine)

Data used for the 'qualitative assessment' were obtained from the IARC review in Monograph 27

based on two National Cancer Institute bioassays. Some problems were evident with the data from the mouse study due to excessive mortality in the males; lack o f any apparent increase in tumour incidence may have been influenced by the poor survival rate. An apparent small increase in hepatocellular carcinoma in female mice was well within historical background control incidence and, therefore, could not be conclusively attributed to treatment. In the rat study, there was also significantly increased mortality in both males and females at the highest dose level. In the high dose males there was an increased incidence of urinary bladder transitional cell carcinoma. With the females there was a possible increase in the same tumour type but this was probably not biologically significant. A high incidence of renal hyperplasia was also evident. No information was given about the occurrence of bladder calculi in the rats with bladder tumours. As the presence o f such calculi can induce these tumours, it was difficult to fully assess the significance of these findings. Applying our guidelines for the 'qualitative assessment' and taking into account the lack of any clear carcinogenic effects in mice and female rats led to a classification of 'suspect animal carcinogen'. For the quantitative assessment, the only effect definitely associated with feeding the chemical was the transitional cell carcinoma of the bladder in the male rats. The TDso value for this was 470 mg/kg. Combination o f the 'suspect animal' classification and a TDso value of 470 mg/kg gave an overall classification o f 'D'. Discussion The proposed ranking scheme offers a mechanism for ranking aromatic amines/nitro compounds using readily available data from the literature and presents the ranking in a format which can be linked to workplace hygiene control strategies as outlined in Table 3. Proven human carcinogens would be handled as if they were classified A. However in the selection of a hygiene

161 TABLE 3 EXAMPLES OF POTENTIAL CONTROL MEASURES/HYGIENE STRATEGIES Classification Negative

Classification C/D

Classification B

Classification A

Required GOHP a

Required GOHP a Extraction system. Up-graded operating instructions/procedures. Reduction of skin contact.

Required GOHP a High efficiency extract systems. Special operating instructions and training. Minimisation of skin contact. Monitoring to demonstrate containment (air and poss. biological). High reliability chemical plant. Detoxification and special disposal of containers.

Required Best industrial hygiene practices. High efficiency extract systems. Frequent maintenance checks. Comprehensive plant operational procedures. Comprehensive measures to reduce skin contact to negligible levels. Monitoring to demonstrate fully effective containment (air, surface and poss. biological). High reliability chemical plant. Thorough detoxification; special cleaning and disposal of containers. Segregated operations. Very careful selection and use of PPE (inc. cleaning). Regular operator traning and updating

Consider Extraction system

Consider Air monitoring and possibly biomonitoring. High reliability chemical plant. Segregated operations where possible. Special care in disposal of containers.

Consider Segregated operations. Mechanical handling. Increased frequency maintenance checks. Integral plant detoxification. Special PPE changing/laundering procedures. Health surveillance.

Consider Mechanical handling. Integral plant detoxification. Pre-campalgn briefing. Automatic, totally contained plant. Bulk or semi-bulk transfer, on-line sampling and process controls.

a 'Good occupational hygiene practice' applicable in handling all chemicals.

strategy appropriate additional measures from those listed under 'Consider' in Table 3 are likely to be required. Predictably, some care has to be exercised in operation but problems encountered were limited and became obvious in any specific assessment. We recommend that, under the following circumstances, the compound under assessment should be considered to have demonstrated potential for multispecies carcinogenicity: good quality positive data from well validated in vivo genotoxicity studies are available (e.g. the mouse micronucleus and liver unscheduled DNA synthesis tests); administration of the compound must have been by a physiologically normal route, such as

oral, dermal or inhalation; protocols used are to acceptable current standards. In such circumstances, our recommendation would be to seek expert advice or to raise the ranking category if the compound is not already placed in Category A or B. Our scheme allows the possibility of a 'suspect human carcinogen' being classified as low as Category 'B'. It would be prudent to seek expert advice on the strength of the epidemiological evidence before deciding upon the ranking of such compounds. In the absence of this, a classification of 'A' is recommended. We believe we have achieved our main objective in developing a ranking system based on available

162

data, for linking to hygiene control strategies. The scheme is designed to conform to accepted toxicological principles whilst, in most instances, avoiding the need for deliberation by experienced toxicologists. Some familiarity with materialssafety is, however, necessary in operating it. The authors would be pleased to discuss practical application of the scheme with interested parties.

Disclaimer The opinions expressed in this article are those of the authors. They do not necessarily represent those of the companies employing them or those of the CIA.

Acknowledgements Thanks are due to Mr. M. Rackham (ICI Pharmaceuticals) and Mr. C. Money (ICI Fine Chemicals Manufacturing Organisation) for support on hygiene matters.

References Brown, H.S., D.R. Bishop and C. Rowan West (1986) A methodology for assessing carcinogenic hazards of chemicals, Toxicol. Ind. Health, 2(3), 205-218.

Gold, L.S., C.B. Sawyer, R. Magaw, G. M. Backman, M. de Veciana, R. Levinson, N.K. Hooper, W.R. Havender, L. Bernstein, R. Peto, M.C. Pike and B.N. Ames (1984) A carcinogenic potency database of the standardised results of animal bioassays, Environ. Health Perspect., 58, 9-319. Gold, L.S., M. de Veciana, G.M. Backman, R. Magaw, P. Loprieno, M. Smith, M. Blumenthal, R. Levinson, L. Bernstein and B.N. Ames (1986) Chronological supplement to the carcinogenic potency database: Standardised results for animal bioassays published through December 1982, Environ. Health Perspect., 67, 161-200. Gold, L.S., Sloane T.H., G.M. Backman, R. Magaw, M. Da Costa, P. Loprieno, M. Biumenthal and B.N. Ames (1987) Second chronological supplement to the carcinogenic potency database: Standardised results of animal bioassays published through December 1984 and by National toxicological program through May 1986, Environ. Health Perspect., 74, 237-329. Gold, L.S., T.H. Slone and L. Bernstein (1989) Summary of carcinogenic potency and positivity for 492 rodent carcinogens in the carcinogenic potency database, Environ. Health, Perspect., 79, 259-272. Sowinski, E.J., B. Broecker et al. (1987) Criteria for identifying and classifying carcinogens, mutagens and teratogens, Regul. Toxicol. Pharmacol., 7, 1-20. Communicated by K. Sankaranarayanan

Carcinogenic ranking of aromatic amines and nitro compounds.

A scheme is proposed for ranking the carcinogenicity of aromatic amines and nitro compounds based on both qualitative (weight of evidence) and quantit...
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