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3.0 Findings

Forensic Identification Services Chemical Carcinogenicity Evaluation

Of the 66 initial chemicals identified for assessment, one, "xylene substitutes", was excluded from further analysis as the identity of the chemical that "substituted" for xylene was not known or stated. Another chemical, Hungarian red, while an identifiable entity, was not associated with any toxicological information. Also, since the chemical structure of Hungarian red could not be identified, a SAR analysis and review of potential structural alerts could not be conducted. As a result this chemical, while "reviewed" per se, was classified as "No data, no conclusions".

Based on our systemic review of the data, the opinions of authoritative bodies such as IARC, NTP, CCOHS, U.S. EPA, etc., of the 64 chemicals which could be ascribed a carcinogen classification category, none were considered to be "Known to be carcinogenic to humans", while 10 were classified as "Reported to produce tumours in experimental animals, but for which human evidence is either lacking or inconclusive".

The remaining 54 chemicals were not found to show evidence of carcinogenic activity in humans or animals. A further analysis of these chemicals on the basis of genetic toxicity data, structural alerts, and similarity to chemicals with known carcinogenic action, revealed that 6 were considered as category C1 or "Not reported to have carcinogenic potential, but may have theoretical risk". The other 48 chemicals were categorized in C2 or "Not reported to have carcinogenic potential, and without theoretical risk".

3.1 Chemicals "Reported to Produce Tumours in Experimental Animals, but for Which Human Evidence is Either Lacking or Inconclusive"

A tabular summary of the classification of each of the chemicals and exposure limits, where available, for chemicals considered to be "Reported to produce tumours in experimental animals, but for which human evidence is either lacking or inconclusive" is presented in Table 2. Target organs (increased incidence of tumours) that have been identified from experimental animal studies are shown in the far right column.

Table 2 - Summary of Chemicals Classified as "Reported to Produce Tumours in Experimental Animals, but for Which Human Evidence is Either lacking or Inconclusive"
Chemical CAS# Use level Exposure Limit Target Organ (Animal Species)
Chloroform 67-66-3 Seldom

20 μg/day (oral)
40 μg/day (inhalation) (CalEPA, 2009)

Kidney, liver
(rats and mice)
Methanol 67-56-1 Often 500 μg/kg bw/day
(US EPA, 2009a)
testes, head/neck
(rats)
Phenol 108-95-2 Rare 300 μg/kg bw/day
(US EPA, 2009b)
adrenal gland, blood
(rats)
Coomassie brilliant blue 6104-59-2 In use 30 µg/day (CalEPA, 2009) (based on Coomassie violet) mammary gland, skin
(rats)
Petroleum ether 8032-32-4 Until 2000, rare 300 ppm in air TWA* (ACGIH, 2009) skin
(mice)
Hydrogen peroxide 7722-84-1 Not stated 1 ppm in air (ACGIH, 2009) intestine
(mice)
Trichloroacetic acid 76-03-9 Rare 1 ppm in air TWA* (ACGIH, 2009) liver
(mice)
Crystal/Gentian violet 548-62-9 In use Not established liver, blood
(rats and mice)
Rhodamine 6G 989-38-8 Seldom Not established adrenal gland, integument
(rats)
Leucomalachite Green 129-73-7 Seldom Not established thyroid, testes, liver
(rats)

*TWA - time-weighted average

A brief assessment of each of the chemicals follows. These reviews are not intended to be comprehensive, but to show the key data involved in regulatory classifications and the underpinning of the category ascribed by Cantox.

3.1.1 Chloroform

Chloroform is a common solvent and chemical intermediate. There is no consistent evidence to indicate that chloroform is carcinogenic to humans, as case-control evaluations have had several limitations, such as low statistical power or inconsistent results (IARC, 1999a). A number of studies conducted in rats and mice have shown chloroform to cause kidney and liver tumours by both inhalation and oral exposures (IARC, 1999a). Chloroform has been subject to numerous studies that have evaluated its potential to damage DNA (i.e., to be mutagenic or genotoxic). The results of these studies have either been negative (i.e., show no evidence of DNA-damaging potential), or at most equivocal (CCRIS, 2008a). On the basis of these data, IARC (1999a) has classified chloroform as Group 2B "possibly carcinogenic to humans". In the EU, chloroform is classified as "limited evidence for carcinogenicity" (EU IHCP, 2009a). The ACGIH classified chloroform as "Confirmed animal carcinogen with unknown relevance to humans". In contrast, chloroform is on the California EPA’s List of Chemicals Known to Cause Cancer (CalEPA, 2009). CalEPA has calculated a "No Significant Risk Level" (NSRL), which is a daily, lifetime exposure level at which an increased risk of cancer is not expected (beyond 1 in-100,000 risk), of 20 µg/day (oral exposure) and 40 µg/day (inhalation exposure). The TD50 (the daily dose in mg/kg body weight/day to induce tumours in half of test animals that would have remained tumour-free at zero dose) for liver and kidney tumours is 262 mg/kg body weight/day in rats and 111 mg/kg body weight/day in mice (CPP, 2007a).

3.1.2 Crystal/Gentian Violet

Crystal/gentian violet is a pigment or colorant. In mice and rats dosed orally with crystal/gentian violet, the incidence of tumours of the liver/hematopoietic system/harderian gland were reportedly increased (CCRIS, 1994; CPP, 2007b). The TD50 was 90.5 mg/kg body weight/day (CPP, 2007b). No other exposure limit in relation to carcinogenic activity was identified in the scientific literature reviewed. This pigment was observed to be negative in mammalian (CHO) cells lines and in all strains of Salmonella typhimurium, except one, while it was also positive in an Escherichia coli mutagenicity assay (CCRIS, 1994). This chemical has only been assessed for carcinogenic potential by the EU where it is classified as "limited evidence for carcinogenicity" (EU IHCP, 2009b), dependent on the presence of Michler’s ketone (if present at >0.1%, classified as R45 "may cause cancer"). The exact nature of the gentian violet used is not known, therefore, this chemical was conservatively concluded to be "Reported to produce tumours in experimental animals, but for which human evidence is either lacking or inconclusive".

3.1.3 Rhodamine 6G

Based on oral carcinogenicity studies conducted by NTP (1989), there is equivocal evidence for carcinogenic activity based on marginally increased incidence of integumentary keratoacanthoma) in male rats and, in female rats, marginal increases in the incidence of pheochromocytomas of the adrenal gland. There was no evidence for carcinogenicity in male or female mice. Rhodamine 6G was negative in an Ames assay (a test in bacteria to assess the potential for a chemical to cause mutations in DNA) (NTP, 1989), but positive in a sister chromatid exchange assay (a bench top test to assess a chemicals ability to affect chromosomes) and a chromosome aberration test (a bench top test with cells to assess ability of chemicals to cause breaks and other damage in chromosomes) with metabolic activation, while positive in mouse lymphoma assay (a bench-top test conducted with mouse lymphoma cells to assess ability of chemicals to cause mutations in DNA) without metabolic activation (NTP, 1989, 2009a). On the basis of the available data, IARC (1978a) has classified Rhodamine G as "Not classifiable as to its carcinogenicity to humans". This classification is often invoked when there are no data in humans and only limited evidence in animals, as is this case with Rhodamine G. As a conservative (health protective) measure, Cantox categorized Rhodamine G as "Reported to produce tumours in experimental animals, but for which human evidence is either lacking or inconclusive".

3.1.4 Coomassie Brilliant Blue

This chemical is a pigment used in various lab techniques. There is very little toxicological information available on it. It was found to be negative in one in vitro DNA damage assay (Reifferscheid and Heil, 1996). In contrast, a closely related substance Coomassie Violet has been the subject of several carcinogenicity studies in rats and mice (IARC, 1978b; JECFA, 1977). Coomassie violet is carcinogenic in rats following its oral or subcutaneous administration: it produced mammary carcinomas and squamous-cell carcinomas of the skin after its oral administration to female rats and local fibrosarcomas following its subcutaneous injection in male and female rats. It also increased the incidence of benign mammary tumours in female rats following ambient exposure. Based on these data, IARC (1987a) has classified Coomassie violet as Group 2B substance [The agent (mixture) is possibly carcinogenic to humans]. A NSRL of 30 µg/day has been established by CalEPA (2009). Based on the data for Coomassie violet, Coomassie brilliant blue has been classified as "Reported to produce tumours in experimental animals, but for which human evidence is either lacking or inconclusive".

3.1.5 Leucomalachite Green

Leucomalachite green is a colouring agent. Based on evidence from a 2-year carcinogenicity study conducted by the NTP (2005a; 2009b), there is equivocal evidence for thyroid gland/testes/liver tumours in male and female rats, with no evidence for tumourigenicity in female mice. There were no human data available for assessment. Leucomalachite Green was found to be active (i.e., positive) in a micronucleus assay (a test that evaluates the potential for chemicals to damage chromosomes in whole animals usually rats), based on the same series of NTP studies. Leucomalachite green also shows structural alerts for genotoxic activity (Ashby and Tennant, 1991; Benigni and Bossa, 2008). Leucomalachite green has not been classified with respect to carcinogenic potential by an authoritative body, including IARC, the EU, NTP, U.S. EPA, CalEPA, or ACGIH.

3.1.6 Methanol

Methanol, or methyl alcohol, is a colourless liquid with a strong odour, used as a solvent and cleaner (NJHSFS, 1996a). Limited inhalation studies in mice, rats, and monkeys have not shown carcinogenic effects, and no effects were observed in one dermal carcinogenicity study in mice (CCOHS, 2007b). In one lifetime rat drinking water study (Soffritti et al., 2002), significant increases in carcinomas of the head and neck, and of testicular tumours was reported. However, the veracity of this study has not been independently verified. Despite some equivocal information from a few in vitro studies, the majority of the data indicate that methanol is not mutagenic, especially in in vivo mutagenicity assays (CIR, 2001; CCOHS, 2007b; CCRIS, 2008c). Methanol does not contain structural alerts for either genotoxicity or in carcinogenicity. It is critical to note that methanol was classified as "Reported to produce tumours in experimental animals, but for which human evidence is either lacking or inconclusive" on the basis of a poorly conducted and reported study, the results of which were not replicated in other studies. The weight of evidence would suggest that methanol has no carcinogenic potential.

3.1.7 Petroleum Ether

This is a chemical mixture composed of highly volatile hydrocarbons that is used as a non-polar solvent. It is a petroleum distillate intermediate between naphtha and kerosene. IARC (1989) has categorized petroleum ether as "Not classifiable as to its carcinogenicity to humans". However, in the EU has indicated that petroleum ether "May cause cancer" (EU IHCP, 2009c), while the ACGIH (2009) has classified as it in the category A3 or "Confirmed animal carcinogen with unknown relevance to humans". The animal studies and the in vitro (bench-top studies) have shown ambiguous and/or equivocal results, and the "positive" results (i.e., showing activity) may be likely related to the presence of polycyclic aromatic hydrocarbons (PAH) as impurities in the petroleum ether (IUCLID, 2000a). PAHs are substances that are known to show genotoxic activity. Based on its toxicology profile, petroleum ether was classified as "Reported to produce tumours in experimental animals, but for which human evidence is either lacking or inconclusive". However this classification may be overly conservative for petroleum ether derived from well characterized refining sources and which are free of PAHs.

3.1.8 Hydrogen Peroxide

Hydrogen peroxide is a colorless, odourless liquid and is usually in a water solution, being used as a common oxidizing and bleaching agent and in deodorants, water and sewage treatment, and rocket fuels, as a disinfectant, and in making other chemicals (NJHSFS, 1998c). Hydrogen peroxide has induced intestinal tumours in highly susceptible strains of mice administered high-doses in drinking water (Munro et al., 2006; CPP, 2007c). However, in a standard 2-year carcinogenicity study in which rats were administered hydrogen peroxide in the drinking water at a concentration of up tom 0.6%, no evidence of carcinogenicity was reported (Munro et al., 2006). With respect to genotoxic effects, equivocal results have been obtained in bacterial mutagenicity assays, but positive results have been obtained in numerous other in vitro assays including, mouse lymphoma and micronucleus tests (CCRIS, 2009). However negative results have been obtained in other in vivo studies, since in vivo systems contain detoxification mechanisms not present in vitro (CCOHS, 1998; Munro et al., 2006). IARC (1999d) has classified hydrogen peroxide as Group 3 (not classifiable as to its carcinogenicity to humans) while ACGIH (2009) considers it an A3 substance (confirmed animal carcinogen with unknown relevance to humans) (ACGIH, 2009) and (IARC, 1999d). Based on the results of non-standard studies conducted in mice, hydrogen peroxide was classified as "Reported to produce tumours in experimental animals, but for which human evidence is either lacking or inconclusive". Given that the in vitro genetic toxicity data and the results of studies conducted in special strains of mice are of limited or no relevance to humans, hydrogen peroxide is likely to be of no carcinogenic concerns to humans.

3.1.9 Trichloroacetic Acid

Trichloroacetic acid is a colourless, crystalline solid, which is used in liquid solutions in the manufacture of pharmaceuticals and pesticides (NJHSFS, 1997a). It has been classified by ACGIH (2009) as A3 or "animal carcinogen with unknown relevance to humans" (ACGIH, 2009) and Group 3 or "not classifiable as to its carcinogenicity to humans" by IARC (1995). These classifications are based on the results of drinking water studies in which mice were administered trichloroacetic acid for 52 or 61 weeks. Treatment was associated with an increased incidence of liver tumours in males only. In contrast, in a 2-year feeding study in which rats were exposed at 250, 630, 1,600, or 10,000 mg/kg feed and in a 2-year drinking water study, also conducted in rats, showed no evidence of carcinogenicity, though some liver toxicity was observed (OECD SIDS, 2005). It is suspected that the liver tumours noted in mice treated with high doses of trichloroacetic acid, is likely a species-specific effect and not indicative of human carcinogenic risk. Both positive and negative results in in vitro and in vivo mutagenicity studies of have been reported (OECD SIDS, 2005), however, the positive results may have largely been due to the pH lowering effect of the acid. Given the drinking water studies conducted in mice, trichloroacetic acid was classified as "Reported to produce tumours in experimental animals, but for which human evidence is either lacking or inconclusive" although the mechanisms by which the tumours appeared to arise may not have relevance for human carcinogenic risk.

3.1.10 Phenol

Phenol is a colorless to white crystalline solid or a pink to red liquid in aqueous solution if not in pure form, and is used in the manufacture of plywood, pharmaceuticals, adhesives, plastics, and rubber (NJHSFS, 1995d).

Phenol has been tested extensively for genotoxicity in a variety of in vivo and in vitro tests (ATSDR, 2008). The results of these assays have generally been mixed. Statistically significant increases in adrenal gland tumours and leukemia or lymphomas were observed in male rats exposed to low doses of phenol in a feeding study, but not at higher doses, or in female rats or in mice (CCOHS, 1999). There is also some evidence that phenol is a tumour promoter, based on oral and dermal carcinogenicity studies with known carcinogens (CCOHS, 1999). Overall, the evidence is considered inconclusive and thus phenol has been classified by the ACGIH (2009) as A4 (not classifiable as a human carcinogen) (2009) while IARC (1999e) has considered it as a Group 3 substance (not classifiable as to its carcinogenicity to humans). This classification also echoes that of the U.S. EPA.