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

Endpoint summary

Description of key information

Repeated Dose Toxicity 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').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.

Repeated Dose Inhalation Toxicity

A short-term toxicity study need not be conducted as exposure of humans via inhalation route during production/use is highly unlikely based on thorough and rigorous exposure assessment provided. Taking into account the low vapour pressure of the substance (7.572711605e-21 Pascal), the exposure to the test chemical via inhalation is highly unlikely. Hence this endpoint can be considered for waiver.

Repeated Dose Dermal Toxicity

A short-term toxicity study need not be conducted as exposure of humans via dermal route during production/use is highly unlikely based on thorough and rigorous exposure assessment provided. The acute dermal LD50 (section 7.2.3) was greater than 2000 mg/kg. Hence, this study can be considered for waiver

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

combined repeated dose and carcinogenicity

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: combined repeated dose/carcinogenicity

Principles of method if other than guideline:

A combined repeated dose and carcinogenicity study investigating the effect of the test chemical in Charles River CD rats when administered orally

GLP compliance:

not specified

Limit test:

no

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. Animals were identified by re-tagged and/or toe-clipped.
- Diet (e.g. ad libitum): Food was available ad lib(Purina Rodent Chow)
- 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

Vehicle:

other: Purina Rodent Chow

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dose was prepared by adding 0.1, 0.5, 1.0 and 4.0 % FD & C Red No. 3 (Erythrosine) in Purina Rodent Chow. Feed were blended in a twin-shell blender with test compound.

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): Purina Rodent Chow were used.
- Concentration in vehicle: 0.0, 0.1, 0.5, 1.0 and 4.0 %
- 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:

Doses were analysis for homogeneity and stability, and 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:

For 30 months or until survival decreased to ten rats of one sex in any group, at which time all the animals of that sex in the study were to be killed

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
Original study: 0.0, 0.0, 0.1, 0.5 and 1.0 (0, 0, 49, 251 and 507 mg/kg/dy for Male and 0,61, 307 and 641 mg/kg/day for female )
Basis:
no data

Remarks:

Doses / Concentrations:
High-dose study: 0.0 and 4.0 % (0, 2464 mg/kg/day for male and 0, 3029 mg/kg/day for female)
Basis:
no data

No. of animals per sex per dose:

Total : 1820
For utero phase
Original study:
0.0% : 60 male, 60 female
0.0% : 60 male, 60 female
0.1% : 60 male, 60 female
0.5% : 60 male, 60 female
1.0% : 60 male, 60 female

High-dose study:
0.0% : 60 male, 60 female
4.0% : 60 male, 60 female

For chronic phase
Original study:
0.0% : 70 male, 70 female
0.0% : 60 male, 60 female
0.1% : 70 male, 70 female
0.5% : 70 male, 70 female
1.0% : 70 male, 70 female

High-dose study:
0.0% : 70 male, 70 female
4.0% : 70 male, 70 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of previous sub chronic and chronic studies indicated that 1.0% was the maximum tolerated dose in the rat.
- Rationale for animal assignment (if not random): For original study, 60 male and 60 female. Maximum of two rats of each sex from each litter were randomly selected for the chronic phase following completion of the in utero phase.

For chronic study, 70 male and 70 female animals were selected.
- 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: During the mating, lactation and post-weaning periods of the in utero phase and twice daily throghout study.
- Cage side observations checked in table [No.?] were included.Mortality and morbidity were examined.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily (with at least 5 hr between observations).

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

BODY WEIGHT: Yes
- Time schedule for examinations: For utero phase female on gestation days 0, 6, 15 and 21 and on lactation days 0, 4, 14 and 21.
For chronic phase weekly for the first 14 week, biweekly for the next 12 week, and monthly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):Yes,
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, For utero phase weekly for the first 14 week, biweekly for the next 12 week and monthly thereafter.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, dietary concentration of compound was calculated.

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:Once during the utero phase at generation, and at initiation and months 3, 6, 12, 18 and 24 of the chronic phase.
- Dose groups that were examined: All 1820 animals were examined.

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


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:
Organ weight were recorded.
Absolut and relative weight of brain, gonads, kidneys, liver, spleen and thyroid were recoreded.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross pathology were performed on all animals dying spontaneously, killed in a moribund condition or killed on schedule.
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. All excised tissues not completely used for histological examinations were preserved.

HISTOPATHOLOGY: Yes
A complete histopathological evaluation was conducted on all rats from the 3 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.
Organ examined: 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, 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).

Other examinations:

No data available

Statistics:

For parameters showing no statistically significant differences between the two control groups of the original study, the control data were combined and the treated groups from that study were compared with the pooled controls. When the two control groups were significantly different, the treated groups were compared with each control group separately.
The variances of the two control groups were analyzed for equality by 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 by using the Smith-Satterthwaite correction for unequal variances. All tests were conducted at the 1.0% two-sided risk level. The 4% group was compared only to its concurrent control group.
Statistical analysis of tumour incidence data was performed by using contingency table techniques and evaluated by Fisher exact test. Tests were conducted at the 5.0 and 1.0% two-sided levels.
Neoplastic lesions and the survival, Total benign neoplastic lesions, total malignant neoplastic lesions and animals bearing benign and/or malignant neoplastic lesions were also analyzed.

Clinical signs:

no effects observed

Description (incidence and severity):

For chronic study:
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 gropus.Observed effect were not due to test compound

Mortality:

no mortality observed

Description (incidence):

No significant effect on survival of all treated male and female rats were observed as compared to controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Original study:
When treated with 1.0 % in male and female, significant increased were observed on body weight as compared to combined controls.In female when treated with 0.5 %, significant increase were observed during first 14 week of study.
High-dose study: Significant decreased were observed in body weight of female rat were observed as compared to control.
For chronic phase: Slightly reduction in mean body weight was observed but the effect was not significant as compared to control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The average intakes of the colouring for original study is 49, 251 and 507 mg/kg/dy for Male and 0,61, 307 and 641 mg/kg/day for female.
For high-dose study is 2464 mg/kg/day for male and 3029 mg/kg/day for female.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

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:

no effects observed

Description (incidence and severity):

No significant difference was observed in haematologicl parameters of treated rat as compared to control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In male, when treated with 0.5 and 1.0 % slight increase in SGOT (serum glutamic-oxalacetic transaminase) level were observed at 18 monthes as compared to control.

Urinalysis findings:

no effects observed

Description (incidence and severity):

When treated with 4.0 %, pink or red coloured urine were observed in rat throughout the study. When treated with 0.5 and 1.0 %, Pink or red-coloured urine were observed for 1 year then 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:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic lesions were observed in treated rat of original or chronic study as compared to control.

Neuropathological findings:

not specified

Histopathological findings: non-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.

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 biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

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 biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Conclusions:

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 gropus.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

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

waiver

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Quality of whole database:

waiver

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

waiver

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

waiver

Additional information

Repeated dose toxicity oral

Various studies have been reviewed to evaluate the repeated oral toxicity of the test chemical. These include in vivo experimental studies performed on rats, mice, dogs as well as pigs for the test chemical. 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 result is supported by another 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. Statistically significant decreases in body weights were noted for mice at the 3.0% dosage level during several weeks of the study when compared to either control group means. 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. There were no significant changes for total tumours, benign tumours or malignant tumours. A NOAEL of 3.0 % 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 also supported by a chronic repeated dose toxicity study performed on rats to assess the toxicity potential of the test chemical. Carworth Farm E strain SPF male and female rat were exposed to the test chemical at dose concentration of 0.0, 125, 250, 500 and 1000 mg/kg/day (0.0, 0.25, 0.5, 1.0 and 2.0 %) orally for 90 days. Two additional groups, comprising ten rats of each sex, were given a dietary level of 0 or 2% for periods of up to 40 wk for use in supplementary haematological studies. Body weight and food consumption in the main groups were recorded weekly. At autopsy haematological examinations involved the determinations of haemoglobin and methaemoglobin concentrations, packed cell volume and counts of total erythrocytes, reticulocytes, total and differential leucocytes and erythrocytes containing Heinz bodies. In the two additional groups (0 and 2% test chemical), haemoglobin concentration was measured at wk 20 and 40 while the packed cell volume and erythrocyte count were determined at wk 40.weights of the brain, heart, liver, spleen, kidneys, adrenals, gonads, pituitary and caecum( plusc ontents) were recorded. The thyroids were weighed after fixation. Paraffin-wax sections of these and a wide range of other organs were stained with haematoxylin and eosin for histopathological examination. Sections of the livers of the control rats and of those given 2% test chemical were stained by the periodic acid-Schiff (PAS) reaction to demonstrate glycogen. Fur and faeces of all the treated groups were coloured red; the intensity of color was dose dependent. Although statistically significant reductions were seen in the haemoglobin levels of males at 05 and 2.0% test chemical for 13 wk, this effect did not follow the feeding of the higher dietary level to either sex for 20 or 40 wk. Moreover, no similar effect was seen at any level in the females. This finding was therefore considered to be a chance observation, which may have been produced by an unusually high value for the control male rats.When treated with 0.5 and 2.0 % in male rats, significant decreased was observed in Hemoglobin of treated rat as compared to control in 13 weeks. This decrease was not observed in 40 week treated rats of all animals as compared to control. When treated with 0.25, 0.5, 1.0 and 2.0 % in male and female rat, significantly significant increase were observed in Protein-bound Total and True iodine and non -Protein-bound iodine as compared to control. When treated with 2.0 % in male and female rat, significant increase were observed in absolute and relative weight of caecum and relative weight of thyroid in treated rat as compared to control. When treated with 1.0 % in male and female rat, significant increase was observed in relative weight of caecum.When treated with 0.5 % in male and female, significant increase in relative weight of caecum and in male significant decrease in relative weight of kidney was observed. When treated with 0.25 % in male, significant increase in relative weight of caecum and in female, significant decrease in relative weight of kidney was observed as compared to control. When treated with 2.0 % in male and female, orange pigment in kidney tubules and in female single adenoma of an adrenal gland was observed. When treated with 0.5 and 1.0% in male rat, orange pigment in kidney tubules were observed as compared to control. NOAEL was considered to be 125 mg/kg/day (0.25 %) when Carworth Farm E strain SPF male and female rat were treated with the test chemical.

The above results are further supported by a long term feeding study carried out in Beagle dogs to observe the toxic effects caused by the test chemical. Twenty-four young beagle dogs, divided into four groups of three males and three females, were fed laboratory chow diets containing the test chemical at levels of 0, 0.5, 1.0 or 2.0 % for 2 years. Periodic haematological examinations, including haemoglobin and haematocrit determinations and white cell counts, were performed before and during the test period, and records of both body weights and food consumption were kept throughout the test period. At the conclusion of the test, the dogs were killed and autopsied. Sections of liver, kidney, heart, lung, gall bladder, spleen, pancreas, adrenal, thyroid, parathyroid, stomach, small and large intestines, brain (four levels), rib bone and marrow, testis, prostate, ovary, uterus, submaxillary salivary gland, lymph node and skeletal muscle from the six dogs given the 2.0% test chemical diet and from the six control dogs were examined microscopically. Although these 12 dogs received the more thorough microscopic examination, tissues from other dogs were also examined. In addition, from about half of the dogs, paraffin sections of Zenker-fixed liver and kidneys stained with haematoxylin-eosin and frozen sections of formalin-fixed liver and kidney stained with Oil RedO were examined Approximate myeloid-erythroid cell ratios were determined on Wright-Giemsa-stained bone-marrow smears from 23 dogs.No haematological effects attributable to ingestion of test chemical were noted in the periodic examinations.No compound-related gross pathology was noted. A control male had a congenital absence of the right kidney and emaciation was observed in one dog in each of the groups given the 1.0 and 0.5 % diets, the dog fed the 0.5 % diet being the more emaciated of the two. Obesity was also seen in one female in each of these groups. No intestinal worms were found in these 24 dogs. There were no compound-related histological effects, and only minor incidental abnormalities were seen. Slight chronic thyroiditis was noted in one male and one female given the 2.0% test chemical diet and the male also showed a slight decrease in the myeloiderythroid ratio in the bone marrow. Another male in the group given the 2% test chemical diet had slight testicular atrophy and the third male in this group had cystic mucoid glands in the gall bladder. One control female had slight cystitis of the urinary bladder. No other gross or microscopic pathology was seen. NOAEL was considered to be 500 mg/kg body weight /day (2.0 %) when beagle male and female dogs were treated with the test chemical.

Furthermore, a repeated dose study was performed to investigate the effect of the test chemical in Osborne-Mendel rats when administered orally for 2 yrs.Five groups of 24 litter-mate Osborne-Mendel rats, evenly divided by sex, were fed test chemical at 0.0, 0.5, l.0,2.0 or 5.0 % dietary levels for 2 years. The rats were housed individually and were allowed food and water ad libitum. Weekly records were kept of animal weights, mortalities and clinical observations. At approximately 3, 6, 14 and 21 months, blood samples were collected from the tail vein of 10 rats in’each group, and haemoglobin, haematocrit and total and differential leucocyte counts were determined. At the end of 2 yr, the survivors were killed by decapitation and autopsied and the weights of heart, liver, spleen, kidneys and testes were recorded. Rats that died before the end of the experiment were autopsied but organ weights were not recorded. Of the 120 rats started on the experiment, 109 were submitted for pathological examination, the rest being discarded chiefly because of advanced post-mortem autolysis. Sixty one rats were sectioned microscopically, 21 in detail and 40 with sections limited to kidney, liver, testes, tumours (if present) and other organs with abnormalities. The haematoxylin eosin stained paraffin sections from the rats studied in detail included sections of lung, heart, liver, spleen, pancreas, stomach, small intestine, colon, caecum, kidney, adrenal, thyroid, testis and prostate (or ovary and uterus), urinary bladder, leg bones with voluntary muscle and marrow, and any tumour or other tissue appearing abnormal. The parathyroid was often included with the thyroid. No effect was observed on growth of treated rat as compared to control. No effects (to P > 0.05) on mortality were observed in rats given test chemical at dietary levels up to 5.0%. No effects weights of liver, heart, kidney or testes were observed in rats given test chemical at dietary levels up to 5% but there were significant reductions in spleen weights. The average spleen-to-body weight ratios (g/kg) for the control males and for the males given the 5.0, 2.0 and 0.5 % dietary levels of the test chemical were 2.51, 1.64 (P<0.02), 1.54 (P<0.01) and 1.71 (P <0.05), respectively. while the ratios for the control females and for females given the 5.0% dietary level were 244 and 1.78 g/kg (P<0.02) respectively. On an absolute organ-weight basis, only the spleen weights of the males given the 5.0 and 2.0% levels of test chemical were significantly lower than those of the controls (P <0.01 and P< 0.05, respectively). When treated with 5.0 %, Moderate and slight distension of the caecum was observed in treated rat as compared to control. Treatment with 1.0 % and 2.0 % in rat, slight distension of the caecum was observed as compared to control. In male rat treatment with 5.0 %, splenic reticulum cell sarcoma and thyroid colloid adenoma were observed.Treatment with 2.0 %, fibrosarcoma, squamous cell carcinoma and adrenal cortical carcinoma was observed.  In female rat treatment with 1.0 %, ovarian thecal cell tumor was observed.  Treatment with 0.5 %, endometrial sarcoma was observed. Observed changes are due to change in variation. The effects observed in the feeding study in rats were limited to the dietary levels of 1.0 % and higher, and involved only growth depression and caecal enlargement. Hence, the LOAEL can be considered to be 1% in diet i.e 1000 mg/kg/day.

All of the available results are supported by a short-term toxicity study performed in pigs to evaluate the toxic potential of the test chemical. Pigs of both sexes of the Large White strain, weighing initially approximately 20 kg and obtained from a minimal-disease herd were used for the study. The test chemical was given to groups of three male and three female pigs by mouth for 14 wk in doses of 0 (control), 167,500 or 1500 mg/kg body weight/day. The test chemical was dissolved in water to give a dose volume of 4.0 ml/kg. This was added to part of the daily ration of food given before the main feed. In this way, the dose was eaten immediately and its accurate administration was ensured. The pigs were weighed initially the thereafter twice weekly. Haematological investigations were carried out on blood taken from an ear vein at wk 4 and 8 and from the superior vena cava immediately prior to autopsy at wk 14. Measurements were made of the haemoglobin concentration and packed cell volume together with count of erythrocytes and reticulocytes as well as total and differential leucocyte counts. Methaemoglobin concentrations were determined in the blood taken at autopsy. All the organs were preserved in 10% buffered formalin. Paraffin -wax sections of these tissues were stained with haematoxylin and eosin for histopathological examination. The pigs appeared healthy throughout the experiment. at no levels of treatment did the body weights of the treated animals differ significantly from those of the controls, although there was a slight reduction at the highest level of treatment, the mean gains at wk 14 being 26.7 and 26.5 kg in the males and females, respectively, of this group compared with 28.4 and 30.1 kg in control males and females. Comparisons between litter mates showed that the gains at wk 14 were lower than the controls in two of the males and all three females.When treated with 500 and 1500 mg/kg body weight/day in female rat significant increase were observed in relative thyroid weight as compared to control. When treated with 1500 mg/kg body weight/day in male and female, deceased in hearts and lungs weight of treated rat but these differences were not statistically significant when the weights were expressed relative to body weight. Flattened acini in thyroid glands, In the lungs, small areas of alveolar collapse and peribronchiolar cuffing were observed and, in the kidney, and liver there were occasional areas of cytoplasmic vacuolation and cellular infiltration were observed in 1500 mg/kg body weight/day treated rat. The slightly lowered rates of body-weight gain at the 1500 mg/kg bw/day level of treatment and the reductions in the heart weights were inconsistent with a depressed thyroid function and there were no other indications of such a depression. There was no indication from the present study of the cause of the slight reductions in body-weight gain. Because the thyroid gland was enlarged and the serum-thyroxine level was reduced in pigs fed the 167 mg/kg bw/day of the test chemical, it was not possible to establish a no-untoward-effect level from this experiment.LOAEL was considered to be 167 mg/kg body weight/day when Large White male and female pig was treated with the test chemical.

Based on the available results, the NOAEL value of the test chemical after repeated oral exposure can be considered to greater than 500 mg/kg/day. Hence, the test chemical can be considered to be comparatively non-toxic and classified under the category “Not Classified” as per CLP Regulation.

Repeated Dose Inhalation Toxicity

A short-term toxicity study need not be conducted as exposure of humans via inhalation route during production/use is highly unlikely based on thorough and rigorous exposure assessment provided. Taking into account the low vapour pressure of the substance (7.572711605e-21 Pascal), the exposure to the test chemical via inhalation is highly unlikely. Hence this endpoint can be considered for waiver.

Repeated Dose Dermal Toxicity

A short-term toxicity study need not be conducted as exposure of humans via dermal route during production/use is highly unlikely based on thorough and rigorous exposure assessment provided. The acute dermal LD50 (section 7.2.3) was greater than 2000 mg/kg. Hence, this study can be considered for waiver

Justification for classification or non-classification

Based on the available results the test chemical can be considered to comparatively non-toxic when exposed repeatedly via oral, dermal, inhalation route of exposure. Hence, the test chemical can be classified under the category "Not Classified" as per CLP Regulation.