Disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

Carcinogenicity

Description of key information

Carcinogenicity Oral

In a Combined repeated dose & carcinogenicity study, Charles River CD male and female rats were exposed to the test chemical. The studies consisted of an in utero and an F1 phase. In the former, the chemical was administered to five groups of the F 0 generation rats (60 of each sex/group) at levels of 0.0, 0.0, 0.1, 0.5 or 1.0% ('original study') and 0.0 or 4.0% ('high-dose study').In male rats, thyroid cystic follicular cell hyperplasia, follicular cysts, follicular adenomas and carcinomas were observed in 4.0 % treated rats of original study and chronic study.When treated with 0.5 %, thyroid cystic follicular cell hyperplasia was observed in original study rats.In female rats, when treated with 0.5 and 4.0 %, follicular adenoma was observed.When treated with 1.0 % in female rats, follicular adenomas and carcinomas were observed. The observed changes in female rat are not statistically significant as compared control. NOAEL was considered to be 0.5 % (251 mg/kg/day) for male rats and 1.0 % (641 mg/kg/day) for female rats when treated with the test chemical.

Carcinogenicity Dermal

Skin painting studies were carried out on mice to evaluate the carcinogenic potential of the test chemical. ICR (Swiss Webster derived) white male and female were exposed to the test chemical in the concentration of 0 and 1428 mg/kg/day and 10 mg of 3, 4-benzpyrene as positive control.

Reticulum cell sarcoma and Mammary gland adeno carcinoma were observed in female mice  but the effect was not significant as compared to control.  The test chemical failed to increase the incidence of neoplasia.Hence, the NOAEL can be considered to be 1428 mg/kg/day when applied twice weekly to the skin of ICR mice for 18 months.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from peer reviewed journals

Qualifier:

according to guideline

Guideline:

other:

Principles of method if other than guideline:

Carcinogenicity study investigating the effect of the test chemical in Charles River CD rats when administered orally for 30 months.

GLP compliance:

not specified

Species:

rat

Strain:

other: Charles River CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Age at study initiation: 63-70 days old
- Weight at study initiation: no data available
- Fasting period before study: no data available
- Housing: They were housed individually in stainless-steel cages except during the mating, lactation and post-weaning periods of the in utero phase.
- Diet (e.g. ad libitum): Purina Rodent Chow ad lib
- Water (e.g. ad libitum): No data available
- Acclimation period: No data available
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21°C
- Humidity (%):40-60%
- Air changes (per hr):No data available
- Photoperiod (hrs dark / hrs light): 12-hr light/day cycle

IN-LIFE DATES: From: To: No data available

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly

- Mixing appropriate amounts with (Type of food): Purina Rodent Chow

- Storage temperature of food: The test compound was stored in sealed containers in a locked closet. The basal diet was stored in an environmentally controlled room with limited access.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data available
- Concentration in vehicle: No data available
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All batches of the basic feed were analysed for heavy metals, chlorinated hydrocarbons and aflatoxin and were found to contain acceptably low levels of these contaminants.

Duration of treatment / exposure:

Original study: F1 males and females received for 129 and 128 wk, respectively
High dose study: 1 males and females received for 125 and 122 wk, respectively

Frequency of treatment:

Daily

Post exposure period:

No data available

Remarks:

Doses / Concentrations:
0.0, 0.0, 0.1, 0.5 or 1.0% ('original study')0, 0,50,250,500 mg/kg/day
Basis:
no data

Remarks:

Doses / Concentrations:
0.0 or 4.0% ('high-dose study')0, 2000 mg/kg/day (in study 2464 mg/kg/day mention for 4%)
Basis:
no data

No. of animals per sex per dose:

70 males and 70 females

Control animals:

yes

Details on study design:

- Dose selection rationale:
For the original study: These dose levels
were selected on the basis of previous subchronic and chronic studies that indicated that 1.0% was the maximum tolerated dose in the rat

For high dose study: the dose range was mandated by FDA as a prerequisite for regulatory action

- Rationale for animal assignment (if not random): No data available
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included.Deaths, morbidity and clinical signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

DERMAL IRRITATION (if dermal study): Not applicable
- Time schedule for examinations: Not applicable

BODY WEIGHT: Yes
- Time schedule for examinations: body weights values for the F1 rats were measured weekly for the first 14wk, biweekly for the next 12wk, and monthly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption values for the F1 rats were measured weekly for the first 14wk, biweekly for the next 12wk, and monthly thereafter.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes , Intake of FD & C
Red No. 3 was calculated from body weight,
food consumption and dietary concentration and expressed in mg/kg/day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examinations were conducted on all rats once during the
F 0 generation, and at initiation and months 3, 6, 12, 18 and 24 of the chronic phase
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood: at months 3, 6, 12, 18 and 24 and at termination of the chronic phase
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: Ten males and ten females from each group randomly selected
- Parameters checked in table [No.?] were examined. The haematological parameters examined were haemoglobin, haematocrit, erythrocyte counts and erythrocyte morphology, and total and differential leucocyte counts.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at months 3, 6, 12, 18 and 24 and at termination of the chronic phase
- Animals fasted: No data
- How many animals: Ten males and ten females from each group randomly selected
- Parameters checked in table [No.?] were examined.: The serum chemistry tests performed were for serum glutamic-oxalacetic transaminase (SGOT), serum glutamic-pyruvic transaminase, alkaline phosphatase, blood urea nitrogen, fasting glucose, total protein and creatinine.

URINALYSIS: Yes
- Time schedule for collection of urine: at months 3, 6, 12, 18 and 24 and at termination of the chronic phase
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. Urine was examined for gross and microscopic appearance, specific gravity, pH and the presence of protein, glucose, ketones, bilirubin and occult blood.

NEUROBEHAVIOURAL EXAMINATION: No data - Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: The brain, gonads, kidneys, liver, spleen and thyroid were weighed and relative organ weights were calculated.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, A complete histopathological evaluation was conducted on all rats from the three control groups and the highest dose group from each study (1.0 and 4.0%) and also on ten rats of each sex randomly selected from each group for an interim kill at 12 months.

The adrenals (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), oesophagus, eye (two, with optic nerve), heart (with coronary vessels), intestine (caecum, colon, duodenum and ileum), kidneys (two), liver, lung (inflated with formalin) and mainstem bronchi, lymph nodes (mesenteric and mediastinal), mammary gland (inguinal), nerve (sciatic), ovaries, pancreas, pituitary, prostate, salivary gland (mandibular), seminal vesicles (two), skeletal muscle (biceps femoris), skin, spinal cord (cervical), spleen, stomach, testes with epididymides, thymus, thyroid with parathyroid, trachea, urinary bladder (inflated with formalin) and uterus were examined histologically, together with any tissue showing macroscopic lesions of an uncertain nature (with a section of normal appearance from the same tissue) and any tissue masses (with the regional lymph nodes)002E If a potential effect was consistently noted in a tissue, then that tissue was examined histologically in all the animals. Microscopic evaluation was also conducted on any other rats with gross lesions or masses.

Other examinations:

Female rats in the in utero phase were weighed on gestation days 0, 6, 15 and 21 and on lactation days 0, 4, 14 and 21.

Statistics:

The variances of the two control groups were tested for equality using the F test. If the variances were equal, a standard independent two sample test was used to determine equality of means. If the variances differed, Welch's t test was used to determine equality of means using the Smith-Satterthwaite correction for unequal variances.

Statistical evaluation of equality of means in control and treated groups was made by the appropriate one-way analysis of variance followed by a multiple comparison procedure if indicated. First, Bartlett's test was performed to determine whether groups had equal variance and parametric procedures were used if they did not. The one-way ANOVA was applied using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's test was used to determine which means were significantly different from the control. If a non-parametric procedure for testing equality of means was indicated, the Kruskal-Wallis test was used.

A statistical test for trend among the dose levels was also performed. In a parametric case (equal variance), standard regression techniques were used with a test for trend and lack of fit. In the non-parametric case, Jonckheere's test for monotonic trend was used.

The statistical analysis of tumour incidence data was performed using contingency table techniques. The table was evaluated by the Fisher exact test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Localized hair loss (due to cage friction) and nasal and ocular discharges occurred in low incidence throughout the study and were similarly distributed in control and treated rats.

High dose study: Hair discoloration ranging from pink to red was noted among treated rats and dark-red faeces were reported in treated rats in the high-dose study. Hair discoloration ranging from pink to red was noted among treated rats and dark-red faeces were reported in treated rats in the high-dose study.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

Necropsies were conducted on all animals dying spontaneously, killed in a moribund condition or killed on schedule.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights were significantly (P <0.01) higher for males and females in the 1% group and for females in the 0.5% group compared to the combined controls during the first 14 wk of the original study, but were similar in the control and all the treated groups (0.1, 0.5 and 1.0%) throughout the rest of that study. Mean body weights of rats in the 4% group were lower than those of the concurrent control group throughout the study, and for the females the differences were statistically significant (P < 0.01) at most intervals and at the end of the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food Consumption: Food consumption of all treated rats was higher than that of the controls. The increases were dose related and were occasionally statistically significant

Compound intake: The average intakes of the colouring were 49, 251,507 and 2464 mg/kg/day by the male rats and 61, 307, 641 and 3029 mg/kg/day by the females fed the
0.1, 0.5, 1.0 and 4.0% diets,

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Ophthalmoscopic examinations revealed focal and diffuse retinopathy, conjunctivitis, uveitis and cataracts in rats of all groups. None of these findings was compound related.

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There was a slight increase in SGOT (serum glutamic-oxalacetic transaminase) values, at 18 months only, in males receiving 0.5 or 1.0% of the test chemical

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Pink or red-coloured urine was excreted by rats receiving 0.5 or 1.0% for the first year; thereafter, the urine colour was comparable to that of the controls ("yellow" or "straw coloured").

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean thyroid weights were increased in males in the 4.0% group and in females in the 0.5% group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No compound-related macroscopic lesions were noted at necropsy in the original study.No compound-related macroscopic lesions were noted at necropsy in the high-dose study.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the ten rats of each sex from each group killed and necropsied after 1 yr on test in both studies, there were no compound-related microscopic changes. No increases in the incidence of non-neoplastic or neoplastic lesions in other tissues were noted in either study.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In male rats, thyroid cystic follicular cell hyperplasia, follicular cysts, follicular adenomas and carcinomas were observed in 4.0 % treated rats of original study and chronic study.When treated with 0.5 %, thyroid cystic follicular cell hyperplasia was observed in original study rats.In female rats, when treated with 0.5 and 4.0 %, follicular adenoma was observed.When treated with 1.0 % in female rats, follicular adenomas and carcinomas were observed. The observed changes in female rat are not statistically significant as compared control.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

251 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Key result

Dose descriptor:

NOAEL

Effect level:

641 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Critical effects observed:

no

Table1. Mean thyroid weights of rats fed the test chemical in the diet for up to 30 months

Dietary level (%)	Male		Female
	Dose of colouring(mg/kg/day)	Thyroid weight (mg)	Dose of colouring(mg/kg/day)	Thyroid weight (mg)
	Original study
0.0	0	47±0.13(16)	0	37±0.04(16)
0.0	0	45±0.05(21)	0	34±0.03(17)
0.1	49	43±0.04(12)	61	41±0.10(19)
0.5	251	40±0.03(15)	307	98±1.67(11)
1	507	54±0.11(15)	641	46±0.24(14)
	High dose study
0.0	0	44±0.04(16)	0	41±0.09(14)
0.4	2464	92±0.42(24)	3029	40±0.08(25)
*Values are means + SEM for the numbers of thyroids indicated in brackets.

Table 2: Incidence of thyroid histopathology in rats fed the test chemical in the diet

Lesion		Incidencet for groups in:
	Dietary level (%)	0.0	0.0	0.1	0.5	1.0	0.0	4.0
	Males
	Dose (mg/kg/day)	0	0	49	251	507	0	2464
	No. examined‡	70	69	67	68	69	69	69
Cystic follicular cell hyperplasia§		2(2.9)	1(1.5)	8(12)	11* (16.2)	5(7.3)	0(0)	16* (23.2)
Follicular cysts§		7(10.0)	10(14.5)	6(9.0)	8(11.8)	8(11.6)	2(2.9)	10* (14.5)
Diffuse or focal follicular cell hyperplasia§		1(1.4)	0(0)	5(7.5)	5(7.4)	18*(26.1)	4(5.8)	60*(87.0)
Follicular cell adenoma§		0(0)	0(0)	0(0)	2(2.9)	1(1.5)	1(1.5)	15*(21.8)
Follicular cell carcinoma		0(0)	0(0)	3(4.5)	1(1.5)	3(4.4)	2(2.9)	3(4.4)
	Female
	Dose (mg/kg/day)	0	0	61	307	641	0	3029
	No. examined‡	70	69	67	68	69	69	69
Cystic follicular cell hyperplasia§		1(1.4)	1(1.5)	3(4.5)	5(7.4)	4II(5.8)	4(5.8)	5(7.2)
Follicular cysts§		15(21.4)	12(17.4)	14(20.9)	12(17.6)	21(30.4)	7(10.1)	7(10.1)
Follicular cell adenoma§		1(1.4)	0(0)	1(1.5)	3(4.4)	5(7.2)	0(0)	5(7.2)
Follicular cell carcinoma		0(0)	0(0)	0(0)	0(0)	1II(1.5)	0(0)	0(0)
†No. of thyroids affected, expressed in brackets as a percentage of the total examined. ‡No. of thyroid glands examined from a total of 70 rats/group (except for the high-dose study female control group of 69). A small number of thyroids could not be evaluated because of inadequate tissue or autolysis. §Some rats exhibited one or more types of thyroid pathology. II One female rat had both hyperplasia and a carcinoma. Values marked with an asterisk differ significantly from the control value (*P < 0,01).

Conclusions:

In male rats, thyroid cystic follicular cell hyperplasia, follicular cysts, follicular adenomas and carcinomas were observed in 4.0 % treated rats of original study and chronic study.When treated with 0.5 %, thyroid cystic follicular cell hyperplasia was observed in original study rats.In female rats, when treated with 0.5 and 4.0 %, follicular adenoma was observed.When treated with 1.0 % in female rats, follicular adenomas and carcinomas were observed. The observed changes in female rat are not statistically significant as compared control. NOAEL was considered to be 0.5 % (251 mg/kg/day) for male rats and 1.0 % (641 mg/kg/day) for female rats when treated with the test chemical.

Executive summary:

In a Combined repeated dose & carcinogenicity study, Charles River CD male and female rats were exposed to the test chemical. The studies consisted of an in utero and an F1 phase. In the former, the chemical was administered to five groups of the F 0 generation rats (60 of each sex/group) at levels of 0.0, 0.0, 0.1, 0.5 or 1.0% ('original study') and 0.0 or 4.0% ('high-dose study'). The concurrent control groups received the basal diet. After random selection of the F1 animals, the long-term phase was initiated using the. same dietary levels and 70 rats of each sex/group, including the three control groups. Rats were exposed for a maximum of 30 months. Dietary concentrations of the compound were determined weekly during the first 13 wk of the study and monthly i thereafter to demonstrate that the diets were prepared properly and were stable.Deaths, morbidity and clinical signs of toxicity were recorded twice daily (with at least 5 hr between observations). Individual body weights and food consumption values for the F1 rats were measured weekly for the first 14wk, biweekly for the next 12wk, and monthly thereafter. Intake of the test chemical was calculated from body weight, food consumption and dietary concentration and expressed in mg/kg/day. Necropsies were conducted on all animals dying spontaneously, killed in a moribund condition or killed on schedule. The brain, gonads, kidneys, liver, spleen and thyroid were weighed and relative organ weights were calculated. When treated with 4.0 % in rats, Hair discoloration ranging from pink to red and dark-red faeces was observed. Hair discoloration ranging from pink to red were also observed in other treated rats. Palpable masses (primarily in the abdominal, thoracic and anogenital regions), localized hair loss (due to cage friction) and nasal and ocular discharges in low incidence occurred in treated and control groups.Observed effect were not due to test compound. Mean body weights of the female F1 rats on 4.0% test chemical (3029 mg/kg/body weight/day) were significantly lower than those of controls (P < 0.01) throughout the study. Food consumption increased in all treated groups in a dose-related manner. In male rats, thyroid cystic follicular cell hyperplasia, follicular cysts, follicular adenomas and carcinomas were observed in 4.0 % treated rats of original study and chronic study.When treated with 0.5 %, thyroid cystic follicular cell hyperplasia was observed in original study rats.In female rats, when treated with 0.5 and 4.0 %, follicular adenoma was observed.When treated with 1.0 % in female rats, follicular adenomas and carcinomas were observed. The observed changes in female rat are not statistically significant as compared control. NOAEL was considered to be 0.5 % (251 mg/kg/day) for male rats and 1.0 % (641 mg/kg/day) for female rats when treated with the test chemical.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

651 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Klimisch Rating 2

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from peer reviewed journals

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

Skin painting studies were carried out on mice to evaluate the carcinogenic potential of the test chemical

GLP compliance:

not specified

Species:

mouse

Strain:

ICR

Remarks:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Housing: Animals were housed individually in a wire cage and identify by identification number.
- Diet (e.g. ad libitum): Food, ad libitum
- Water (e.g. ad libitum): Water, ad libitum.

Route of administration:

dermal

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Details on exposure:

Details on dermal exposure
PREPARATION OF DOSING SOLUTIONS: Dosing solution was prepared in water at a concentration of 1 %, calculated on the pure dye content of each color. To achieve homogeneous preparations, a Waring blender was utilized, and the materials were kept on a magnetic stirrer during treatment. All materials were prepared fresh weekly.


TEST SITE
- Area of exposure: Dorsal area of skin
- Time intervals for shavings or clipplings: Subsequent periodic clipping was performed according to the rate of hair growth free of lubricating oil.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml dose containing 1 mg of FD & C Red 3.
- Concentration (if solution): 1 mg
- Constant volume or concentration used: Yes, 0.1 ml
- For solids, paste formed: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Concentration (if solution): 1 mg

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

18 months

Frequency of treatment:

twice a week

Remarks:

0.1 ml dose containing 1 mg of dye (1428.5 mg/kg)

No. of animals per sex per dose:

100 mice/dose/sex

Control animals:

yes, concurrent vehicle

Details on study design:

no data available

Positive control:

10 mg of 3, 4-benzpyrene were used as positive control.

Observations and examinations performed and frequency:

Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table [No.?] were included: Mortality and morbidity was observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice weekly

BODY WEIGHT: No data available
- Time schedule for examinations: No data available

Sacrifice and pathology:

Sacrifice and pathology
GROSS PATHOLOGY: Yes,
All mice that died, those sacrificed moribund, and those surviving the 18-month experimental period were necropsied.Skin and any grossly abnormal organs and tissues of all treated animals, 50 vehicle control animals (approximately equal number of males and females); complete pathology on five males and five females randomly selected vehicle control animals that survived 18-month experimental period; and complete pathology on five males and five females randomly selected vehicle control animals that survived 18 month experimental period; and complete pathology of five male and five female animals from the positive control group that included induced skin lesions. Tissues were preserved in 10% formalin and tissues selected for histopathology, sectioned, stained with hematoxylin and eosin and examined.

HISTOPATHOLOGY: Yes

Organ examined: Brain, pituitary, thyroid, thymus, liver, spleen, kidney, adrenal, stomach, small intestines, large intestines, urinary bladder, axillary lymph nodes, testes, ovary, and skin from area of treatment, any tissue masses, grossly abnormal organs, or tissues were examined.

Statistics:

no data available

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant variations that could be attributed to application of the test compound.

Dermal irritation (if dermal study):

no effects observed

Description (incidence and severity):

There were no significant variations that could be attributed to application of the test compound.

Mortality:

no mortality observed

Description (incidence):

No effect were observed on survival of treated male and female.

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Swelling in left ear, Swelling in neck and Nodule in liver was observed in treated female mice but the effect is not significant as compared to control.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Inflammatory cyst lined by fibrous tissue capsule, Reticulum cell sarcoma and Mammary gland adeno carcinoma were observed in female mice but the effect is not significant as compared to control.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

The test chemical failed to increase the incidence of neoplasia

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

1 428 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

dermal irritation

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

Critical effects observed:

no

Conclusions:

Inflammatory cyst lined by fibrous tissue capsule, Reticulum cell sarcoma and Mammary gland adeno carcinoma were observed in female mice but the effect is not significant as compared to control. The test chemical failed to increase the incidence of neoplasia.Hence, the NOAEL can be considered to be 1428 mg/kg/day when applied twice weekly to the skin of ICR mice for 18 months.

Executive summary:

Skin painting studies were carried out on mice to evaluate the carcinogenic potential of the test chemical. ICR (Swiss Webster derived) white male and female were exposed toFD & C Red No. 3 (Erythrosine)intheconcentration of 0 and 1 mg/kg/day and 10 mg of 3, 4-benzpyrene as positive control. All mice that died, those sacrificed moribund, and those surviving the 18-month experimental period were necropsied.Skin and any grossly abnormal organs and tissues of all treated animals, 50 vehicle control animals (approximately equal number of males and females); complete pathology on five males and five females randomly selected vehicle control animals that survived 18-month experimental period; and complete pathology on five males and five females randomly selected vehicle control animals that survived 18 month experimental period; and complete pathology of five male and five female animals from the positive control group that included induced skin lesions. Tissues were preserved in 10% formalin and tissues selected for histopathology, sectioned, stained with hematoxylin and eosin and examined.The results show that the test chemical was not toxic, no toxic changes were observed on survival and clinical signs. In addition, gross pathological changes such as swelling in left ear, swelling in neck and nodule in liver was observed in treated female mice. Inflammatory cyst lined by fibrous tissue capsule, Reticulum cell sarcoma and Mammary gland adeno carcinoma were observed in female mice  but the effect was not significant as compared to control.  The test chemical failed to increase the incidence of neoplasia.Hence, the NOAEL can be considered to be 1428 mg/kg/day when applied twice weekly to the skin of ICR mice for 18 months.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 428 ng/kg bw/day

Study duration:

chronic

Species:

mouse

Quality of whole database:

Klimisch Rating 2

Justification for classification or non-classification

Hence, the non-carcinogenic NOAEL for the test chemical can be considered to be NOAEL > 500 mg/kg/day. Therefore the test chemical can be classified under the category “NOT CLASSIFIED” as per CLP Regulation.

Additional information

Carcinogenicity Oral

Various studies have been reviewed to evaluate the carcinogenic potential of the test chemical. These include in vivo oral studies for the test chemical in rats as well as mice. The results are mentioned below:

In a Combined repeated dose & carcinogenicity study, Charles River CD male and female rats were exposed to the test chemical. The studies consisted of an in utero and an F1 phase. In the former, the chemical was administered to five groups of the F 0 generation rats (60 of each sex/group) at levels of 0.0, 0.0, 0.1, 0.5 or 1.0% ('original study') and 0.0 or 4.0% ('high-dose study'). The concurrent control groups received the basal diet. After random selection of the F1 animals, the long-term phase was initiated using the. same dietary levels and 70 rats of each sex/group, including the three control groups. Rats were exposed for a maximum of 30 months. Dietary concentrations of the compound were determined weekly during the first 13 wk of the study and monthly i thereafter to demonstrate that the diets were prepared properly and were stable.Deaths, morbidity and clinical signs of toxicity were recorded twice daily (with at least 5 hr between observations). Individual body weights and food consumption values for the F1 rats were measured weekly for the first 14wk, biweekly for the next 12wk, and monthly thereafter. Intake of the test chemical was calculated from body weight, food consumption and dietary concentration and expressed in mg/kg/day. Necropsies were conducted on all animals dying spontaneously, killed in a moribund condition or killed on schedule. The brain, gonads, kidneys, liver, spleen and thyroid were weighed and relative organ weights were calculated. When treated with 4.0 % in rats, Hair discoloration ranging from pink to red and dark-red faeces was observed. Hair discoloration ranging from pink to red were also observed in other treated rats. Palpable masses (primarily in the abdominal, thoracic and anogenital regions), localized hair loss (due to cage friction) and nasal and ocular discharges in low incidence occurred in treated and control groups.Observed effect were not due to test compound. Mean body weights of the female F1 rats on 4.0% test chemical (3029 mg/kg/body weight/day) were significantly lower than those of controls (P < 0.01) throughout the study. Food consumption increased in all treated groups in a dose-related manner. In male rats, thyroid cystic follicular cell hyperplasia, follicular cysts, follicular adenomas and carcinomas were observed in 4.0 % treated rats of original study and chronic study.When treated with 0.5 %, thyroid cystic follicular cell hyperplasia was observed in original study rats.In female rats, when treated with 0.5 and 4.0 %, follicular adenoma was observed.When treated with 1.0 % in female rats, follicular adenomas and carcinomas were observed. The observed changes in female rat are not statistically significant as compared control. NOAEL was considered to be 0.5 % (251 mg/kg/day) for male rats and 1.0 % (641 mg/kg/day) for female rats when treated with the test chemical.

This is supported by a similar combined repeated dose & carcinogenicity study, where Charles River CD-1 COBS male and female mice were exposed to the test chemical. The test chemical was administered to groups of 60 Charles-River CD-1 mice/sex/dose via diet. Animals were randomly assigned to treatment and control groups. Dietary levels of 0, 0.3 %, 1 % and 3.0 % corresponded to average food consumptions of 0, 424, 1474, and 4759 mg/kg bw/d in male animals and 0, 507, 1834 or 5779 mg/kg bw/d in female animals. The mice were observed 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 weeks 1 through 14, biweekly during weeks 16 through 26, and once every 4 weeks thereafter. Haematological studies were conducted after 3, 6, 12, 18 and 24 months of the study. Pink hair coloration was noted for treated mice. The exposed skin areas of the treated mice were noted as light pink, bright pink and pink (0.3, 1.0 and 3.0% dosage levels, respectively). The urines of male mice at the 1.0% dosage level were light orange in colour. The urines of male and female mice at the 3.0% dosage level were orange and light orange, respectively. Faeces of the treated mice were red in colour when sectioned and smeared.Haematological values were similar for control and treated mice after 3, 6, 12 and 18 months of the study. At 24 months of study, three females at the 1.0 % dosage level and one male at the 3.0% dosage level showed marked increases in total leucocytes and decreases in haemoglobin, haematocrit and erythrocytes. For two of these females at the 1.0 % dosage level, the leucocytes were predominantly lymphoblastic. Statistically significant decreases were seen in lymphocyte values (24 months) for female mice at the 1.0% dosage level.Haematological values were similar for control and treated mice after 3, 6, 12 and 18 months of the study. At 24 months of study, three females at the 1.0 % dosage level and one male at the 3.0% dosage level showed marked increases in total leucocytes and decreases in haemoglobin, haematocrit and erythrocytes. For two of these females at the 1.0 % dosage level, the leucocytes were predominantly lymphoblastic. Statistically significant decreases were seen in lymphocyte values (24 months) for female mice at the 1.0% dosage level. No significant differences in the numbers of mice with other types of tumours among control and treated groups were observed. Varieties of other lesions were observed at the microscopic level with similar incidences in control and treated mice. Hence, the NOAEL of 3.0 % of the test chemical in the diet (corresponding to 4579 mg/kg bw/d) was derived for male animals and a NOAEL of 1 % in the diet (corresponding to 1834 mg/kg bw/d) was derived for female animals.

These results are further supported by another l ifetime chronic oral toxicity study performed to evaluate the toxic potential of the test chemical. Two groups of 50 7-week old ICR mice/sex were fed diets containing the test chemical at dose levels of 1.25 % and 2.5% corresponding to approximately 1875 and 3750 mg/kg/day for 18 months.The mice received test chemical in cube diet for the first 20 weeks, and thereafter the test chemical was mixed with the basic powder diet. All animals in the experimental groups were fed the basic diet free of the test chemical for an additional 6 months, after which they were sacrificed and autopsied.Mortality was greater among animals exposed to the test chemical than among the controls (approximately 61% of the animals died in the 2.5% group, 59% in the 1.25% group, and 36% in the control group). Animals in both experimental groups exhibited a high incidence of lymphocytic leukaemia, and sporadic cases of pulmonary adenomas were observed. The frequency of both lesions was in the range spontaneously-occurring in this strain of mice. Hence, the NOAEL for male and female ICR mice dosed orally with the test chemical was considered to be 3750 mg/kg/day.

Hence, the non-carcinogenic NOAEL for the test chemical can be considered to be NOAEL > 500 mg/kg/day. Therefore the test chemical can be classified under the category “NOT CLASSIFIED” as per CLP Regulation.

Carcinogenicity Dermal

Skin painting studies were carried out on mice to evaluate the carcinogenic potential of the test chemical. ICR (Swiss Webster derived) white male and female were exposed to the test chemical in the concentration of 0 and 1428 mg/kg/day and 10 mg of 3, 4-benzpyrene as positive control. All mice that died, those sacrificed moribund, and those surviving the 18-month experimental period were necropsied. Skin and any grossly abnormal organs and tissues of all treated animals, 50 vehicle control animals (approximately equal number of males and females); complete pathology on five males and five females randomly selected vehicle control animals that survived 18-month experimental period; and complete pathology on five males and five females randomly selected vehicle control animals that survived 18 month experimental period; and complete pathology of five male and five female animals from the positive control group that included induced skin lesions. Tissues were preserved in 10% formalin and tissues selected for histopathology, sectioned, stained with hematoxylin and eosin and examined.The results show that the test chemical was not toxic, no toxic changes were observed on survival and clinical signs. In addition, gross pathological changes such as swelling in left ear, swelling in neck and nodule in liver was observed in treated female mice. Inflammatory cyst lined by fibrous tissue capsule, Reticulum cell sarcoma and Mammary gland adeno carcinoma were observed in female mice  but the effect was not significant as compared to control.  The test chemical failed to increase the incidence of neoplasia.Hence, the NOAEL can be considered to be 1428 mg/kg/day when applied twice weekly to the skin of ICR mice for 18 months.