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

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

LIFETIME TOXICITY/CARCINOGENICITY STUDY OF FD & C RED NO. 3 (ERYTHROSINE) IN RATS

Author:

J. F. BORZELLECA, C. C. CAPEN, C. C. CAPEN

Year:

1987

Bibliographic source:

Food and Chemical Toxicology Vol. 25, No. 10, pp. 723-733, 1987

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

240-474-8

EC Name:

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

Cas Number:

16423-68-0

Molecular formula:

C20H8I4O5.2Na

IUPAC Name:

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

Details on test material:

- Name of test material (as cited in study report):
D&C Red 3
- Molecular formula (if other than submission substance): C20-H6-I4-O5.2Na
- Molecular weight (if other than submission substance): 879.8424 g/mol
- Substance type: Organic
- Physical state: Solid

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

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

Doses / concentrations 2

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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

1

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

2

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

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

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.