1-(4-methyl-2-nitrophenylazo)-2-naphthol

Carcinogenicity

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Species:

rat

Strain:

other: F344/N

Sex:

male/female

Route of administration:

oral: feed

Duration of treatment / exposure:

2-year feed studies

Remarks:

Doses / Concentrations:
6000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
12500 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
25000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

60

Control animals:

yes, plain diet

Conclusions:

Evaluation by IARC:

The International Agency for Research on Cancer evaluated Pigment Red 3 in 1992 and published the results in 1993 [17]. The data base for this evaluation was the NTP study reviewed above. IARC concluded for Pigment Red 3 that
• there is inadequate evidence in humans for the carcinogenicity of CI Pigment Red 3, and
• there is limited evidence in experimental animals for the carcinogenicity of CI Pigment Red.
Accordingly IARC grouped Pigment Red 3 in Group 3 as it ‘cannot be classified as to its carcinogenicity to humans’.

Overall conclusion:
The available carcinogenicity studies revealed some evidence carcinogenic activity in male and female rats, in male mice but not in female mice. Multiple tissues were affected. Significant increases in tumor response were seen mainly at the highest dose levels exceeding the MTD. Tumors were paralleled by tissue toxicity (liver of female rats and kidneys of male mice) or linked to a high spontaneous tumor incidence (pheochromocytoma in the adrenal medulla of male rats). The biological significance of thyroid adenomas in male mice is unclear. The fact that the carcinogenic effects are observed at relatively high dose levels only (LEL for carcinogenic activity ~ 600 mg/kg) is in agreement with the toxicity observed at high concentrations and indicating that the test-substance, its metabolites and/or its by-products were absorbed at a significant level. No information on the metabolism and tissue disposition of Pigment Red 3 is available. It is concluded that additional information on genotoxicity and on mode of action are useful.
Based on the existing data a classification as Category 3 mutagen is not warranted, but should be re-considered when new data on genotoxicity become available. A classification as Category 3 carcinogen cannot be excluded until there is good evidence that the mechanism of tumor formation can not be extrapolated to man. The fact that evidence of carcinogenic activity was based on tumors observed at high dose levels and that estimated exposure to Pigment Red 3 and/or its metabolites and by-products is considered minimal under normal handling and use conditions support that the chemical cannot be classified as to its carcinogenicity to humans.

Executive summary:

Carcinogenicity

The U.S. National Institutes of Health performed toxicity and carcinogenicity studies on Pigment Red 3 as part of the National Toxicology Program [16]. The purity of the test-article was goven as > 97%. The studies were performed between 1981 and 1985. These included 2 week and 13 week (range-finding) studies as well as 2-year (chronic toxicity/) carcinogenicity studies with interim sacrifice after 15 months in B6C3F1 mice and F344/N rats. The protocols used for these studies are only in partial agreement with the current OECD guidelines for subchronic (OECD 408) and chronic toxicity/carcinogenicity testing (OECD 451/453). Nevertheless, these studies are fully valid (Klimisch 1). The purity of the test-article was > 97%.

Studies in rats

The subacute and subchronic studies were conduced at extremely high dietary dose levels, i.e. up to 100000 ppm in the 2-week (~10000 mg/kg in males, ~ 8000 mg/kg in females) and up to 50000 ppm in the 13 week study (~3200 mg/kg in males, 3800 mg/kg in females). Treatment resulted in a reduced body weight gain particularly in females (-20% at 50000 ppm). Treatment resulted in increased absolute and/or relative liver (all dose levels), kidney (males ≥ 12500 ppm) and lung weights (males ≥ 25000 ppm, females all groups). Anemia characterized by a reduced number of red blood cells and lower hematocrit values was observed in all male groups and in females at ≥ 6000 ppm. The anemia was accompanied by increased numbers of reticulocytes, bone marrow hyperplasia and increased extramedullary hematopoiesis in spleen and liver. As further secondary change to the anemia an increased incidence of (hemosiderin) pigments in liver, spleen and kidneys was observed. In males an increased incidence of casts in renal tubules was observed at dose levels ≥ 12500 ppm. This was interpreted as a possible exacerbation of the progression of chronic nephropathy commonly seen in ageing F344/N rats.

The dose levels selection for the chronic rat study were based on the body weight effects in female rats (decrease of body weight gain at 25000 ppm by 15%). The dose levels selected were 0, 6000, 12500 and 25000 ppm. This resulted in a time weighted average daily dose levels of 272, 567, and 1219 mg/kg b.w. in males and 323, 686, and 1388 mg/kg in females at 6000, 12500 and 25000 ppm, respectively.

Survival of rats was not adversely affected by treatment. Survival of low dose males was significantly higher than that of control males. Body weight development was slightly impaired in high dose males and in all female groups. Greater than 10% difference in body weight were observed for high dose males from week 82 onwards and for females from weeks 82, 66, and 42 onwards at 6000, 12500, and 25000 ppm. Food consumption was not affected by treatment.

The 15 months interim evaluation revealed the same principal findings as observed in the subchronic study, i.e. indication of anemia with secondary changes in liver and spleen (extramedullary hematopoiesis and hemosiderin deposition) as well as in bone marrow (hyperplasia). An increased incidence and/or severity of chronic nephropathy was observed in high dose males and in all treated female groups. Additional histopathological findings to those observed in the 13-week study consisted of a dose related increase in the incidence of biliary tract proliferation as well as of hepatic foci of cellular change which in males was accompanied cystic degeneration. The incidence of neoplastic findings was generally low (with the exception of testicular interstitial cell adenoma) and not indicative of a treatment-related effect.

Treatment-related histopathological changes were observed in various organs. In the liver an increased incidence of hepatocellular adenoma was observed in female rats at the high dose level (Table 7; p = 0.001). The incidence of hepatocellular adenoma in males was within the historical control range and did not display a dose-response relationship. As already observed at the interim sacrifice the incidence eosinophilic and mixed cell foci of cellular change was increased in both sexes at all dose levels. Females also had an increased incidence of eosino- philic and mixed cell foci. Many of these foci were large and occupied several hepatic lobules but exhibited limited or no compression of the adjacent parenchymal tissue. The eosinophilic foci contained large hepatocytes resembling those described for hepatocellular adenoma. Mixed cell foci usually had a predominance of cells similar to those in the eosinophilic foci, but also included admixtures of cells that exhibited clear, vacuolated, or amphophilic cytoplasm. The foci in males were generally smaller than those in females with little evidence of compression of the surrounding parenchyma. Treated males displayed an increased incidences of cystic degeneration and angiectasis. These conditions were often associated with foci of hepatocellular alteration or hepatocellular adenoma. Cystic degeneration occurs spontaneously at low incidences in aging rats.

The incidence of benign pheochromocytomas of the adrenal medulla was increased in males. The trend test was significant and a pairwise comparison between control and mid and high dose groups was statistically significant. There was no apparent increase of malignant pheochromocytoma but there was a positive trend for the combined incidence. The incidence of benign and combined pheochromocytoma was outside the historical control for all treated male groups. It is interesting to note that the control incidence was at the upper end of the historical control range.

The incidence of skin squamous cell papilloma was (significantly) increased in males and outside the historical control range. When benign and malignant squamous cell tumors were combined the trend test remained marginally significant only due to the presence of one skin carcinoma in a control male.

An increase of Zymbal gland carcinoma was observed in high dose males. The incidence exceeded the historical control range marginally. The NTP report mentioned that Zymbal gland neoplasms infrequently occur spontaneously but are induced by a variety of carcinogens, particularly the aromatic amines. Therefore, it was suspected that the marginal increase in carcinomas in this study may have been associated with chemical administration.

The incidence tumors of the preputial and clitoral gland, of mammary gland fibroadenoma in females and of mononuclear cell leukemia in both sexes was decreased in treated groups. The latter was previously observed in studies with aniline and aniline-related compounds.

In addition there were a number of changes of non-neoplastic histopathologic changes including an increased severity of chronic nephropathy in all treated male and female groups, an increased incidence renal tubule hyperplasia, kidney cysts and hyperplasia of the papillary transitional epithelium. Secondary to the kidney lesions, an increased incidence of parathyroid hyperplasia, fibrous osteodystrophy and glandular stomach mineralization was observed in male mainly at the mid and high dose level. In addition, a dose-dependent, treatment-related increase of focal and multifocal pancreatic acinus atrophy was observed in both sexes Based on the above data, i.e. the increased incidence of benign pheochromocytoma of the adrenal gland in males and of hepatocellular adenoma in females there was ‘some evidence of carcinogenic activity’ of Pigment Red 3. The NTP considered the marginal increased incidences of skin squamous cell papilloma and Zymbal gland carcinoma as possibly related to PR3 administration. It has to be considered that at the top dose level the body weight development was impaired in both sexes by more than 10% and therefore the MTD was slightly exceeded.

Evaluation by IARC

The International Agency for Research on Cancer evaluated Pigment Red 3 in 1992 and published the results in 1993 [17]. The data base for this evaluation was the NTP study reviewed above. IARC concluded for Pigment Red 3 that

• there is inadequate evidence in humans for the carcinogenicity of CI Pigment Red 3, and

• there is limited evidence in experimental animals for the carcinogenicity of CI Pigment Red.

Accordingly IARC grouped Pigment Red 3 in Group 3 as it ‘cannot be classified as to its carcinogenicity to humans’.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

other: Rat and Mice

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: The unusual study design, 78 week testament followed by a 20 week treatment free 'recovery period', the exclusion of animals which died pre-term as well as deficits in reporting make it impossible to assess the validity of the study results

Qualifier:

no guideline followed

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: feed

Duration of treatment / exposure:

78 weeks

Post exposure period:

20 weeks treatment free "recovery period"

Remarks:

Doses / Concentrations:
1000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
2000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Conclusions:

The results of the present study thus demonstrated that D&C Red No. 36 at the concentrations of 1,000 ppm and 2,000 ppm in the diet is not carcinogenic either to male or female Wistar rats.

Executive summary:

In this study groups of 50 male and 50 female Wistar rats were administered D&C Red No. 36 (Warner Jenkinson Company, NJ, USA; Purity > 95%) at dietary dose levels of 0, 1000, and 2000 ppm for 78 weeks (estimated daily dose: 50 and 100 mg/kg b.w.). At week 98 all surviving animals were killed and various organs were sampled and stored in formalin. The organs sampled included at least lung, liver, spleen, kidney, urinary bladder, mammary gland and thyroid gland. Survival of rats was not affected by treatment. The number of survivors at termination (week 98) was 50, 48 and 48 in males and 50, 47, and 50 in females at 0, 1000, and 2000 ppm, respectively. The 7 animals which died were excluded from evaluation since they died before week 40. Body weight development was not affected in males but slightly, but statistically significant lower in treated females when compared to the control. Besides body weight data no information is available to assess the systemic toxicity caused by PR4 and thus obtain an indication on the bioavailability of the pigment.

Tumor incidences were reported for liver, thyroid, adrenals, urinary bladder, and mammary gland (Table 4). The incidence of benign tumors was low (£4%) in most tissues and not indicative of a treatment-related effect. Higher tumor incidences were only observed in the liver and mammary gland. The mammary tumor incidence in females and of liver tumors in males was not indicative of an adverse effect. In females the incidence of “liver tumors' was dose-dependently increased (Please note: the incidences reported also include hyperplastic nodules!). This increase was however not statistically significant. The incidence of malignant tumors was likewise low and did not indicate a relation to treatment.

 

Based on the data presented the authors conclusion was: “The results of the present study thus demonstrated that D&C Red No. 36 at the concentrations of 1,000 ppm and 2,000 ppm in the diet is not carcinogenic either to male or female Wistar rats. While the occurrence of benign liver tumors in female rats may be related to dye treatment, the lack of any apparent dose-dependence or any statistically significant difference from the control group (P = 0.06) suggests that this is unlikely.”

A treatment-related increase of her tumors in females is questionable since a) various types of her tumors and pre-neoplastic lesions were lumped together and b) the historical background of liver tumors in the strain of rats used in that laboratory is not known.

Furthermore, the unusual study design, 78 week testament followed by a 20 week treatment free 'recovery period', the exclusion of animals which died pre-term as well as deficits in reporting make it impossible to assess the validity of the study results.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

rat

Justification for classification or non-classification

The International Agency for Research on Cancer evaluated Pigment Red 3 in 1992 and published the results in 1993. The data base for this evaluation was the NTP study reviewed above. IARC concluded for Pigment Red 3 that

• there is inadequate evidence in humans for the carcinogenicity of CI Pigment Red 3, and

• there is limited evidence in experimental animals for the carcinogenicity of CI Pigment Red 3.

Accordingly IARC grouped Pigment Red 3 in Group 3 as it ‘cannot be classified as to its carcinogenicity to humans’.

Additional information

Pigment Red 3 is one of the most widely used red pigments for colouring of paints, inks, plastics, rubber and textiles. The pigment was tested for carcinogenicity in rats and mice. In those species only limited evidence for carcinogenicity was established. An overall evaluation of the pigment, carried out by IARC, stated that it cannot be classified as to its carcinogenicity to humans (IARC, 1993).

Justification for selection of carcinogenicity via oral route endpoint:
Evaluation by IARC
The International Agency for Research on Cancer evaluated Pigment Red 3 in 1992 and published the results in 1993 [17]. The data base for this evaluation was the NTP study reviewed above. IARC concluded for Pigment Red 3 that
• there is inadequate evidence in humans for the carcinogenicity of CI Pigment Red 3, and
• there is limited evidence in experimental animals for the carcinogenicity of CI Pigment Red 3.
Accordingly IARC grouped Pigment Red 3 in Group 3 as it ‘cannot be classified as to its carcinogenicity to humans’.