Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

Carcinogenicity

Description of key information

The substance was not carcinogenic in valid feeding studies with rats and mice at very high doses (Borzelleca et al 1990, IRDC 1981).

No carcinogenicity was observed in a skin painting study in mice at the fairly low dose of 50 mg/kg bw (Carson 1984).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apr 1978 - Apr 1980

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes

Remarks:

Some phases of the study were conducted pre-GLP with no negative affection of the validity and integrity of the study.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Hilton-Davis Chemical Co. Div. of Sterling Drug Inc., Cincinnati, Ohio, USA
- Lot No.of test material: AA 2492
- Purity: not less than 90% pure color

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Mixed weekly with ground basal laboratory diet on a weight/weight basis to provide appropriate dosage levels

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

The Charles River CD-1 mouse has been used extensively to evaluate the toxicity and/or carcinogenicity of new chemicals; considerable control data has been accumulated for this strain of mouse.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laborataries, Portage, MI., USA
- Age at study initiation: 4-weeks old
- Weight at study initiation: 24-31 g (males); 20-26 g (females)
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21 °C
- Humidity (%): 40 - 60%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE PERIOD: FROM 26 April 1978 TO: 28-30 April 1980

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Diets were blended in a twin-shell blender.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: 20-21 °C and a humidity range aof 40-60%.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability were analysed in the prepared diets before study initiation, weekly during the first 13 weeks of study and then monthly thereafter.

Duration of treatment / exposure:

24 months

Frequency of treatment:

daily

Dose / conc.:

661 mg/kg bw/day (actual dose received)

Remarks:

Calculated for males. Equivalent to 0.5% (w/w)

Dose / conc.:

2 064 mg/kg bw/day (actual dose received)

Remarks:

Calculated for males. Equivalent to 1.5% (w/w).

Dose / conc.:

7 354 mg/kg bw/day (actual dose received)

Remarks:

Calculated for males. Equivalent to 5% (w/w).

Dose / conc.:

819 mg/kg bw/day (actual dose received)

Remarks:

Calculated for females. Equivalent to 0.5% (w/w).

Dose / conc.:

2 562 mg/kg bw/day (actual dose received)

Remarks:

Calculated for females. Equivalent to 1.5% (w/w).

Dose / conc.:

8 966 mg/kg bw/day (actual dose received)

Remarks:

Calculated for females. Equivalent to 5% (w/w).

No. of animals per sex per dose:

60

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: indicated to be based on existing studies

Two identical control groups were used to account for random biological variation.

Positive control:

no positive control included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times daily (Mon-Fr) twice daily (weekends and holidays)
- Cage side observations were morbidity, mortality and overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly through the first 14 weeks, biweekly for week 16-26 and monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Determined weekly through the first 14 weeks, biweekly for week 16-26 and monthly thereafter
- Compound consumption were calculated from the diet consumption measurement

FOOD EFFICIENCY:
No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, 12, 18 and 24 months
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 10 per sex and dose group
- Parameters checked: haemoglobin, haematocrit, total eryrhrocyte count, total and differential leucocyte counts

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

- Organ weights: brain, thorax, and abdominal cavities

HISTOPATHOLOGY: Yes (all animals trom the two control groups and from the high-dose (5.0%) group). Further groups were analysed in case of findings at the high dose groups.
Organs examined: adrenal (two), aorta (abdominal), bone and marrow (femur), blood smear, brain (three sections: frontal cortex and basal ganglia. parietal cortex and thalamus. and cerebellum and pons), oesphagus. eye, (two. with optic nerve), gallbladder, gonads (ovaries (2), testes with epididymis (2)), heart (with coronar vesels), intestine (caecum, colon, duodenum and ileum), kidneys (two), liver (2 sections), lung and mainstem bronchi, lymph nodes (mesenteric and mediastinal), mammary gland, mandibular salivary gland, nerve (sciatic), pancreas, pituitary, prostate, seminal vesicles, skeletal muscle (rectus femoris), skin, spinal cord (cervical), spleen, stomach, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus, any tissue with gross changes of an uncertain nature together with an apparently normal section of the same tissue, and any tissue masses or suspected tumours together with regional lymph nodes.

Other examinations:

Test diet analyses: Samples were taken of each batch of basal laboratory feed for IRDC analysis of the pesticide, aflatoxic and heavy metal content.
Test sample analyses: Periodically throughout the study, samples were collected and analyzed for volatiles, pure dye content and HPLC purity. In addition, the test substance was tested for aerobic plate count, mold and yeast count and for the absence of Salmonella and E. Coli.

Statistics:

All statistical analyses were done for each sex seperately with significant differences determined by the probability level p<0.01, two-tailed.
Body weights, food consumption, and clinical studies were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torries using Dunnett's multiple comparison tables to judge significance of differences.
The two control groups were compared against each other. If no significant differences were identified, controls were combined and compared against treated groups, otherwise each control group was compared against each treated individually.
Survival data and time to neoplastic lesions were analyzed by the methods described by Thomas, Breslow, and Gart.
For the histopathologically proved neoplastic lesion incidences the two control groups were compared against each other. If no significant differences were identified, control groups were combined and tested against the high dose group. Otherwise, each control group was compared against the high dose group seperately.
The number of neoplastic lesion-bearing animals were analyzed at study termination using the chi-square test criterion.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental findings noted in similar low numbers of treated and control mice included tremors, labored breathing, localized hair loss, disrended abdomen, masses in the abdominal, inguinal and/or anogenital region, pale exposed skin and ocular changes (pale exes, excessive lacrimation, red and/or swollen eyelids, corneal opacity, dilated pupils, and white areas in the internal eye.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 104 weeks of study, the mean body weight values indicated an apparent compound-related decrease for the treated males and females.
Males: At 1.5% and 5.0%, significance at most intervals analyszed and occasional significance (0.5% dose level) during the first 14 weeks.
Females: Body weight decreases exhibited significance (primarily seen when values were compared to Control 2 or combined controls) at most intervals analyzed throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental findings noted during the study for a few control and treated mice included slight to marked decreases in hemoglobin, hematocrit and total erythrocytes and/or slight to marked increases in total leucocytes.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The hair color and exposed skin of the treated mice were blue. Feces of the treated mice appeared blue when sectioned and smeared. No other changes considered to be related to the test substance were seen.

Details on results:

There was no indication that the substance was carcinogenic in mice. With one exception (hemangioma, splee, female), there were no statistical analyses which were significant at the 0.01 level. This included analysis of all tumors, benign tumors, malignant tumors and all neoplasms with two or more observations in the high dose group. The actual incidence was 0%, 2%, 2%, and 5% in control, low dose, mid dose, and high dose groups, respectively. Hemangioma of the spleen is a common spontaneous tumor for this species and strain. It was unusual that none was observed in the controls and the incidence in the high dose animals was considered to be spurious. The single statistically significant value was not toxicologically significant and the compound was not oncogenic for hemangioma of the spleen.
The slight decrease in body weights after 24 months was considered to be related to the reduced nutritional value of the diet. The tested doses were extremely high.

Dose descriptor:

NOAEL

Effect level:

>= 5 other: % in the diet (> 7000 mg/kg bw)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of carcinogenic effects at the highest tested dose.

Critical effects observed:

no

Results of neoplastic findings in mice treated with the test substance:

Neoplastic summary table
	Control 1		Control 2		0.5%		1.5%		5.0%
	M	F	M	F	M	F	M	F	M		F
Number Animals Examined Microscopically	60	60	60	60	49	57	47	59	60	60
Total tumor bearing animals	20	28	17	29	11	23	10	21	18	23
Total number of tumors	21	33	18	40	12	23	10	22	19	29
Total benign tumor bearing animals	7	12	4	18	3	10	3	11	5	11
Total number of benign tumors	7	15	5	24	3	10	3	12	5	17
Total malignant tumor bearing animals	14	17	13	15	9	13	7	10	14	12
Total number of malignant tumors	14	18	13	16	9	13	7	11	14	14

Conclusions:

No treatment related effects were seen in behavior, survival, food consumption or hematological parameters. No compound related changes were noted in macroscopic or microscopic pathologic examinations. The test subtance was not found to be oncogenic in this study under the conditions chosen. The No-Effect Level is condidered to be 5%.

Executive summary:

The test substance was offered in the diet to Charles River CD-1 mice at dose levels of 0.5, 1.5, and 5.0% in a 24 -month long-term feeding study. Sixty mice/sex were randomly assigned to each of two control groups and each of three treatment groups. The mice were observed daily for signs of toxicity, moribundity and mortality. Detailed observations were recorded weekly. Individual body weights and food consumption values were recorded weekly during weeks 1 through 14, biweekly weeks 16 through 26, and once every 4 weeks thereafter. Hematologic studies were condcuted at 3, 6, 12, 18 and 24 months of study. The hair color and exposed skin of the treated mice were blue. The feces of treated mice appeared blue when sectioned and smeared. No other changes in general behavior and appearance or pharmatoxic signs, considered to be related to compound, were seen. Slightly lower mean body weight values were noted for the treated groups when compared to the controls. Food consumption values were similar for control and treated mice. All other parameters were within the expected range for this species at this age. There were no macroscopic or microscopic changes which could be attribited to the test compound. Statistical analysis indicated that the test compound was not oncogenic for animals of this species and strain under the conditions of this study.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun 1977 - Dec 1979

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes

Remarks:

Some phases of the study were conducted pre-GLP with no negative affection of the validity and integrity of the study.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Hilton-Davis Chemical Co. Div. of Sterling Drug Inc., Cincinnati, Ohio, USA
- Lot No.of test material: AA 2492
- Purity: not less than 90% pure color

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Mixed weekly with ground basal laboratory diet on a weight/weight basis to provide appropriate dosage levels

Species:

rat

Strain:

other: Charles River CD

Details on species / strain selection:

The Charles River derived rat has been used extensively to evaluate the toxicity and/or carcinogenicity of new chemicals; considerable control data have been accumulated for this strain of rat.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories,Wilmington, MA, USA
- Age at study initiation: weanlings
- Weight at study initiation: 108-155 g (males); 98-141 g (females); F1 generation started exposure at weaning
- Housing: individually (except during mating and lactation period); mating: in pairs
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: approximately two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21 °C
- Humidity (%): 40 - 60%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Other: For F1-generation a maximum of two rats per sex from each litter were selected.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Diets were blended in a twin-shell blender.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: 20-21 °C and a humidity range aof 40-60%.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability were analysed in the prepared diets before study initiation, weekly during the first 13 weeks of study and then monthly thereafter.

Duration of treatment / exposure:

30 months (plus in-utero phase) (with 10 animals per sex and dose for interim sacrifice after 12 months)

Frequency of treatment:

daily

Dose / conc.:

0.1 other: % (w/w)

Remarks:

equivalent to 50 mg/kg bw for males and 62 mg/kg bw for females (calculated, actual ingested).

Dose / conc.:

1 other: % (w/w)

Remarks:

equivalent to 514 mg/kg bw for males and 631 mg/kg bw for females (calculated, actual ingested).

Dose / conc.:

2 other: % (w/w)

Remarks:

equivalent to 1072 mg/kg bw for males and 1319 mg/kg bw for females (calculated, actual ingested).

No. of animals per sex per dose:

F0: 60 for treated groups and 120 for control group until week 11. After that large control group was split into two groups of 60 rats/sex
F1: 70

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: indicated to be based on existing studies
Two identical control groups were used to account for random biological variation.

F0 rats were mated to produce the F1 litters. Females were vaginally smeared daily until sperm or a copulatory plug was observed (= gestation day 0). The day all pups of a litter were delivered was defined as lactation day 0. F1 pups were counted, sexed and weighed. F1 males and Fa females were randomly selected and assigned to control or treatment group.

Positive control:

no positive control included.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were morbidity, mortality and overt signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly. In addition, F0 females weighed on gestation day 0, 6, 15, and 21 and on lactation day 0, 4, 14, and 21. Individual body weights were recorded weekly through the first 14 weeks, biweekly for week 16-26 and monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Determined weekly through the first 14 weeks, biweekly for week 16-26 and monthly thereafter
- Compound consumption were calculated from the diet consumption measurement

FOOD EFFICIENCY:
No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: After the selection of the F1 rats was completed, and at 3, 6, 12, 18 and 24 months of study and prior to study termination weeks 111 (females) and 116 (males).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, 12, 18 and 24 months and before termination weeks 111 (females) and 116 (males)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes / No / No data
- How many animals: 10 per sex and dose group
- Parameters checked: haemoglobin, haematocrit, total eryrhrocyte count, total and differential leucocyte counts. and erythrocyte morphology

CLINICAL CHEMISTRY: Yes
- Time schedule: At 3, 6, 12, 18 and 24 months and before termination weeks 111 (females) and 116 (males)
- Animals chosen: 20 per sex from control and 10 per sex from treated groups
- Parameters analyzed: blood urea nitrogen, fasting blood glucose, total serum protein and creatinine, serum glutamic pyruvic transaminase activity (alanine aminotransferase), serum glutamic oxaloacetic transaminase activity (aspartate aminotransferase), serum alkaline phosphatase activity

URINALYSIS: Yes
- Time schedule: At 3, 6, 12, 18 and 24 months and before termination weeks 111 (females) and 116 (males)
- Parameters analyzed: specific gravity, pH and presence of protein, glucose, ketones, bilirubin and occult blood, appearance (gross and microscopic), microscopic examination of the sediment, urobilinogen and nitrite

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

- Organ weights: brain, gonads, kidneys, liver, spleen, testes, ovary and thyroid/parathyroid

HISTOPATHOLOGY: Yes (all animals trom the two control groups and from the high-dose (5.0%) group). Further groups were analysed in case of findings at the high dose groups.
Organs examined: adrenals (two), aorta (abdominal), bone and marrow (femur), blood smear (if anemia, enlarged thymus, lymphadenopathy, or hepatosplenomegaly was present), brain (three sections: frontal cortex and basal ganglia. parietal cortex and thalamus. and cerebellum and pons), oesphagus, eye, heart (with coronar vesels), intestine (caecum, colon, duodenum and ileum), kidneys (two), liver (two sections), lung and mainstem bronchi, lymph nodes (mesenteric and mediastinal), mammary gland, nerve (sciatic), ovaries, pancreas, pituitary, prostate, salivar gland (mandibular), seminal vesicles, skeletal muscle (rectus femoris), skin, spinal cord (cervical), spleen, stomach, testes with epididymides (two), thymus, thyroid with parathyraid, trachea, urinary bladder, uterus, any tissue with gross changes of an uncertain nature together with an apparentdy normal section of thc same tissue, and any tissue masses or suspect tumours rogether with regional lymph nodes.

Other examinations:

Test diet analyses: Samples were taken of each batch of basal laboratory feed for IRDC analysis of the pesticide, aflatoxic and heavy metal content.
Test sample analyses: Periodically throughout the study, samples were collected and analyzed for volatiles, pure dye content and HPLC purity. In addition, the test substance was tested for aerobic plate count, mold and yeast count and for the absence of Salmonella and E. Coli.

Statistics:

All statistical analyses were done for each sex seperately with significant differences determined by the probability level p<0.01, two-tailed.
Body weights, food consumption, clinical studies, and absolute and relative organ weights were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie using Dunnett's multiple comparison tables to judge significance of differences.
The two control groups were compared against each other. If no significant differences were identified, controls were combined and compared against treated groups, otherwise each control group was compared against each treated individually.
Survival data and time to neoplastic lesions were analyzed by the methods described by Thomas, Breslow, and Gart.
For the histopathologically proved neoplastic lesion incidences the two control groups were compared against each other. If no significant differences were identified, control groups were combined and tested against the high dose group. Otherwise, each control group was compared against the high dose group seperately.
The number of neoplastic lesion-bearing animals were analyzed at study termination using the chi-square test criterion.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental findings noted in similar numbers for control and treated rats included rales, red material around the eyes, nose or anogenital region, redness around the eyes, white areas in the internal eyes, corneal opacities, dilated pupils, increased distance between the pupil and the cornea and palpable masses primarily in the abdominal, thoracic or anogenital regions.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Four parental rats died during the study, one female in the 0.1% group, one male and one female in the 1.0% group and one male in the 2.0% group. These deaths were not attributed to treatment. Based on the mortality of the high-dose females, all females were sacrificed at 111 weeks. The males were sacrificed at week 116 when the mid-dose group mortality reached 80%.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Occasional statistically significant differences were considered unrelated to the compound. After week 90, mean body weights of the 2% females began a downward trend. No statistically significant differences between the control and 2% group mean body weights were found at the intervals tested, and the difference was not considered biologically significant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was slightly greater for the males and females at the 2.0% dosage level than for the control males and females. These differences were statistically significant for the males between weeks 27 and 66 and for the females between weeks 27 and 38 and between weeks 79 and 90 and can in all probability be attributed in part to the non-nutrient value of the compound at this dosage level.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Chorioretinal degeneration (seen at 3 and 18 months), chorioidal hypoplasia (at 6 months), and chorioretinal hypoplasia (at 12 months). Conjunctivitis, keratitis, cataract were the most frequent findings. All changes in ophthalmic findigs during the study period were considered related to age rather than to compound administration.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Leucocyte mean values at the 2.0% level were slightly and very slightly increased for males and females, respectively, when compared to the combined control groups 1 and 2. The slight increase noted for the males involved slight-to-moderate elevations (i.e. 16.40 to 21.37x10³/cmm) for five of the ten individual values. The increase in the mean was statistically significant. The very slight increase noted for the females is due primarily to a moderately elevated increase for only one rat (21.54x10³ cmm).
Review of the hematologic profiles obtained during the conduct of the study did not indicate any changes which could be attributed to the test article. The statistically significant differences noted occurred at random and were not considered of biological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental findings (for 3 and 6 months) included slightly elevated blood urea nitrogen and creatinine levels for one rat at the 1.0% dosage level and one rat at the 2.0% dosage level at 6 months of the study. Mean SGPT value for the low-dose females was slightly higher than control means, but the increase was not considered to be physiologically significant. Elevated BUN values (one low-dose and two mid-dose males), slightly elevated glucose values (four control, three low-dose, two mid-dose and one high-dose male(s), two control, one low-dose and two-mid-dose females). No dosage relationship or definite compound relationship was evident in these changes.
Review of the biochemical values obtained during the conduct of this study did not indicate any changes whi'ch could be attributed to the test article, Some statistically significant differences were noted for a few biochemical parameters at some intervals, however, no biological significance was interpreted because there were no trends or consistent differences between control and treated groups.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The color of the urine samples collected from the treated rats was noted as light blue or green to dark blue or green in color in all mid- and high-dose treated rats and most low-dose treated rats. No other consistens differences were noted between the control and treated groups. Review of the urinalyses did not indicate any other changes which could be attributed to the test substance.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no toxicological significant variations in organ weights. There were no morphological correlations for the several statistcally significant variations.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Blue green discoloration of the stomach, small intestine and large intestine was observed in many of the compound treated animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no lesions which could be attributed to the compound. The lesions observed were those of spontaneous disease and they were not unusual in type or incidence for rats of this strain. There were no hyperplastic or neoplastic lesions which could be attributed to the compound.

Other effects:

no effects observed

Description (incidence and severity):

Male and female fertility indices, gestation length, pup survival indices through weaning, number of live and stillborn pups and pup weights at birth and weaning were similar for control and treated rats.
Rats at all three dosage levels had changes in the color of the hair, exposed skin and feces. Rats at 0.1% dosage level experienced light blue hair and skin returning to normal by week 4 and week 28, respectively, of the long-term phase of the study. Feces from these rats appeared green when sectioned and smeared. Rats fed at 1.0% and 2.0% had a blue hair and skin color at the initiation of the study which continued to termination. Feces collected from these rats appeared blue in color when sectioned and smeared.

Details on results:

However, some of the statistical analyses of life table curves and of the adjusted incidence were significant at the 0.01 level for total tumors and for all benign tumors in females. These analyses indicate that the tumors of high dose females occurred earlier in life than in controls although there was no increase in total numbers of tumors. There were no toxicologically significant analyses for any individual tumor type.

Relevance of carcinogenic effects / potential:

In summary, the analyses did not indicate an increase in the unadjusted incidence of tumors. In fact, a negative trend occurred.
Analyses of the adjusted incidence for total tumors in the female suggested that the neoplasms occurred earlier in the high dose animals.
The compound was not considered to be oncogenic for any of the individual tumor types and the statistical analyses of adjusted incidence for total tumors in the female were equivocal.

Dose descriptor:

NOAEL

Effect level:

>= 2 other: % in the diet (> 7000 mg/kg bw)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of carcinogenic effects at the highest tested dose

Dose descriptor:

NOEL

Effect level:

1 other: % (w/w) in the diet

Based on:

test mat.

Sex:

female

Basis for effect level:

other: adverse effects on body weights starting week 102 at 2%. No effects for males

Critical effects observed:

no

Results of tumor incidences in rats:

Neoplastic summary table
	Control 1	0.1%	1.0%	2.0%
All tumors, female	84%	77%	77%	80%
All benign tumors, female	79%	71%	74%	70%
All tumors, male	57%	37%	46%	53%
All benign tumors, male	50%	24%	31%	47%

Moratlity data:

	Survival/number initiated*
Treatment	Male**	Female***
Control 1	18/60	28/60
Control 2	13/60	24/60
0.1%	12/60	23/60
1.0%	12/60	28/60
2.0%	18/60	10/60

 

*Less interim sacrifice

**Survival at week 116

***Survival at week 116

Food consumption data:

Average basal food consumption (Control 1 & 2) and average food with compound consumption (g/rat/day)			Average compound consumption (mg/kg/day)
Treatment	Male*	Female**	Male*	Female**
Control 1	27.3	20.7	-	-
Control 2	27.1	21.0	-	-
0.1%	26.5	21.2	50	62
1.0%	28.0	21.7	514	632
2.0%	28.1	22.2	1073	1318

* week 114

** week 110

Conclusions:

The feeding of the test substance to rats at dose levels of 0.1, 1.0, and 2.0% beginning with the parents prior to mating and the F1 generation for 26 months, produced slight body weight decreases for treated rats. Although changes were seen in color of hair, skin and feces of rats, these signs were not considered toxicologically significant because they were due directly to the color of the dye. No treatment related effects were seen in behavior, survival, food consumption, hematologic, biochemical or urinalyses parameters. No compound related changes were noted in macroscopic or microscopic pathologic examinations. On visual inspection of the neoplastic incidence data, the test substance did not appear to be oncogenic. Statistical analyses did not indicate an increase in the unadjusted incidence of tumors. In fact, a negative trend was occurred. Analyses of the adjusted incidence for total tumors in the females suggested that the neoplasms occurred earlier in the high dose animals. The compound was not considered to be oncogenic for any of the individual tumor types and the statistical analyses of adjusted incidence for total tumors in the female were equivocal. While there is a slight possibility on statistical grounds that the test substance could be oncogenic, the only evidence is earlier appearance of tumors in the female and no definite relationship has been established in this study. The no-effect level of the test substance in this study was considered to be 2.0%.

Executive summary:

The test substance was offered at dosage levels of 0.1, 1.0, and 2.0% throughout a long-term feeding study in rats exposed in utero (F1 generation). Sixty animals/sex F0 rats and 70 animals/sex F1 rats were initiated at each dosage level and in each of two control groups. During the in utero portion of the study, the corresponding parental (F0) generation rats were fed identical dosage levels of the test substance. Rats were observed twice daily for signs of overt toxicity, moribundity and mortality. Detailed observations were recorded weekly. Individual body weights and food consumption values were recorded weekly during the F0 phase. These values were recorded weekly for the first 14 weeks, biweekly for the next 12 weeks, and once every 4 weeks thereafter during the F1 phase. Ophthalmoscopic examination of all rats were conducted at 3, 6, 13, 18, and 24 months and prior to termination at 25 and 26 months. Blood and urine samples were obtained from 10 males and 1o females randomly selected from each group at 3, 6, 12, 18, and 24 months and prior to termination at weeks 116 (males) and 111 (females) for hematologic and biochemical studies and urinalyses. Blue coloration of the hair and exposed skin was apparent for treated rats at the 1.0% and 2.0% levels throughout the study. Sectioned feces were green in color for rats at the 0.1% dosage level and blue in color for rats at the 1.0% and 2.0% dosage levels. Four parental rats died during the in utero phase. One hundred fifty seven (157) control and 257 treated F1 rats died or were sacrificed in extremis during 116 weeks of study for the males, or 111 weeks of study for the females. No specific compound-related pattern of mortality developed. Other than an indication of a partially-compensated anemia at 24 months (of unknown significance) and blue or green urine coloration, review of the clinical pathology data did not indicate any differences between the treated and control animals that were possibly attributable to the test article.

No compound-related gross or microscopic changes were observed. The inflammatory, degenerative and/or proliferative changes described were regarded of spontaneous nature, not uncommon among rats of this age and strain and unrelated to compound administration. On visual inspection of the neoplastic incidence data, the test substance did not appear to be oncogenic. Statistical analyses did not indicate an increase in the unadjusted incidence of tumors. In fact, a negative trend occurred. Analyses of the neoplams occurred earliert in the high dose animals. The compound was not considered to be oncogenic for any of the individual tumor types and the statistical analyses of adjusted incidence for total tumors in the female were equivocal. While there is a slight possibility on statistical grounds that the test sustance could be oncogenic, the only evidence is earliert appearance of tumors in the female and no definite relationship has been established in this study. The no-effect level of the test substance in this study was considered to be 2.0%.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Species:

mouse

Quality of whole database:

The tested doses were low; the dose was chosen for assessment of use in lipsticks.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. No carcinogenic effects were observed at dose levels exceeding the limit dose in life-long feeding studies in rats and mice. As a result the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.