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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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:

DEVELOPMENTAL TOXICITY AND PSYCHOTOXICITY OF TEST CHEMICAL IN RATS

Author:

CHARLES V. VORHEES et.al.

Year:

1983

Bibliographic source:

Toxicology, 1983

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: developmental and neurotoxicity test

Principles of method if other than guideline:

The test chemical was evaluated for for its developmental toxicity and psychotoxicity in 2 different experiments

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:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Laboratory Supply Co., Indianapolis, Indiana, USA
- Age at study initiation:90-110 days of age of the offspring
- Weight at study initiation:Male (200-220 g) and female (200-220 g)
- Fasting period before study:No data available
- Housing:No data available
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period:5 days

Administration / exposure

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: powdered Purina rat chow

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For male : fed diets containing the dye for 2 weeks.
For female: The dye was fed to each treatment group for 2 weeks prior to breeding, 1-14 days during breeding (males and females housed together in pairs), then to the females during gestation and lactation.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: [artificial insemination / purchased timed pregnant / cohoused]:cohoused
- If cohoused:
- M/F ratio per cage:1:1
- Length of cohabitation:Till the confirmation of breeding
- Further matings after two unsuccessful attempts: [no / yes (explain)]:No data
- Verification of same strain and source of both sexes: [yes / no (explain)]:No data
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:The presence of sperm in the daily vaginal lavage of the females and designated as day zero of gestation.
- Any other deviations from standard protocol:No data

Duration of treatment / exposure:

The dye was fed to each treatment group for 2 weeks prior to breeding, 1-14 days during breeding (males and females housed together in pairs), then to the females during gestation and lactation, the males being discarded after confirmation of breeding (determined by the presence of sperm in the daily vaginal lavage of the females and designated as day zero of gestation).

Frequency of treatment:

Daily

Duration of test:

From parent to offspring generation

Doses / concentrations

Remarks:

0% in the diet, 0.25% , 0.5%, 1.0% in diet

No. of animals per sex per dose:

19 – 22 animals/sex/dose

Control animals:

yes

Details on study design:

no data available

Examinations

Maternal examinations:

BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
OTHER:females for reproductive success.

Ovaries and uterine content:

No data available

Fetal examinations:

Other: Cincinnati Psychoteratogenicity Screening Test Battery, plus weight, food consumption, physical landmarks of development, and brain weight.

Statistics:

Analysis of variance for unequal group sizes, with split-plot designs where repeated measures were made, were performed on the majority of the data. Duncan a posteriori multiple range comparison tests for unequal group sizes (Kramer 1956) were used on those measures on which significant F-ratios occurred. Frequency data (e.g., mortality) were analyzed by Fisher's test for uncorrelated proportions (Guilford 1965). On preweaning tests, data on individual subjects were averaged for the entire litter or within sex and the litter was, therefore, the unit of analysis. On postweaning tests, only one male and one female was used from each litter on any given test,therefore, litter was again the unit of analysis, but no litter averaging was required. Because of the large number of tests done, and therefore, the number of statistical tests required, effects were only accepted as significant if the overall test (i.e., usually an F-test) occurred at a level ofp < 0.01 or beyond. Follow-up a posteriori individual group comparisons, however, were less stringent (p < 0.05) under the assumption that the F-test provided an adequate protection level when used at p < 0.01.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A statistically significant treatment x days interaction was found for prebreeding and lactation body weights (both p <0.01), but not for gestation weights. Although these effects were statistically
significant they were very small (< 5%) and group comparisons revealed no significant differences between any treatment group and the control group (R3 0.0), indicating that these differences were not reliable treatment-related effects.In the second Red-3 experiment, no significant reductions were found in food consumption or body weight of the parental animals during any of the exposure periods. There were statistically significant increases found in food consumption during all three parental phases that were associated with all three dye groups.
These effects were not, however, reflected in any significant changes in body weights.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences found among the groups in the first experiment for food consumption prior to breeding or during gestation or lactation in the parental animals.
In the second Red-3 experiment, no significant reductions were found in food consumption or body weight of the parental animals during any of the exposure periods. There were statistically significant increases found in food consumption during all three parental phases that were associated with all three dye groups.

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:

no effects observed

Description (incidence and severity):

No significant reductions in day 90 brain weights were observed in either experiment. In the second experiment there was a significant treatment main-effect on cerebellar weights (p < 0.01), which, by individual group comparisons, was found to be the result of a significant increase in cerebellar weight in the R3 0.5 group (7.5%) and in the R3 0.25 group (11%) compared to the R3 0.0 control group. As before, these isolated, non-dose-dependent' increases are not thought to be treatment-related.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

No significant effects of any of the measures of reproductive performance were found in either experiment.

Maternal developmental toxicity

Number of abortions:

not specified

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No significant reductions were found in offspring food consumption or body weight in either experiment from birth through day 90 of postnatal life. Significant increases in postweaning food consumption in the dye groups were found in both experiments (both p < 0.01), but were not reflected in altered body weights in the second experiment and were primarily in the low-dose (R3
0.25) group in the first experiment. The latter effect had disappeared by 90 days of postnatal life

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The R3 1.0, R3 0.5, and R3 HU groups all showed significant (all p < 0.01) increases in pre-weaning mortality in the first experiment. No changes were found during later phases. The increase in
pre-weaning mortality in the dye groups was not replicated in the second experiment. In fact, the R3 1.0 group showed a significant reduction in pre-weaning mortality. It is noteworthy that pre-weaning mortality rates were significantly higher (p < 0.01) in the controls (R3 0.0) in the second experiment compared to the first. This increased level of background mortality was not due to any detectable infection in the animal colony and appears to merely represent normal variability in animal viability as they come from the supplier.

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Description (incidence and severity):

No malformations were seen in the offspring based upon external examinations the day after birth

Skeletal malformations:

not specified

Visceral malformations:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Behaviorally, there were no significant effects found on the tests of surface righting, pivoting, cliff avoidance, negative geotaxis, auditory startle, pre-weaning open-field, rotorod, or active avoidance in either experiment. There was no significant effect on appetitive operant discrimination learning tested only in the first experiment. There were no significant differences on tests of incisor
eruption, eye opening, testicular appearance, olfactory orientation, or water M-maze behavior tested only in the second experiment.Significant treatment effects were found in both experiments on swimming development. In the first experiment, the significant effects only occurred on the measure termed swimming-angle. On this measure both the main-effect and the treatment x days interaction were significant (both p <0.01). Individual group comparisons showed that these differences were most pronounced on day 10 and were reflected as accelerated swimming-angle development in the R3 1.0 and R3 0.25 groups.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in postnatal survival

external malformations

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

The treatment groups for Experiment 1 were test chemical as 0.0, 0.25, 0.5, or 1.0% of the diet ,and a positive control group treated with the toxin hydroxyurea on days 2-10 of life (50 mg/kg/day, s.c.); Experiment 2 was a replication of Experiment 1 with the same dose groups, but without the positive control group.Male were discarded after breeding confirmed.

Offspring mortality expressed as a % of those alive at the beginning of each phase

	Treatment	Days of age			No. Of liveoffspring after culling on day 1
		1-21	22-24	25-90
Experiment 1	R3 1.0	9.3^a	0.0	0.7	161
	R3 0.5	10.2^a	0.0	1.5	147
	R3 0.25	1.5	0.0	0.0	128
	R3 0.0	3.2	0.0	0.8	412
	R3 HU	8.6^a	0.0	1.5	219
Experiment 2	R3 1.0	9.9^a	0.0	6.9	161
	R3 0.5	15.9^a	0.0	3.4	157
	R3 0.25	23.3	0.0	1.0	193
	R3 0.0	22.5	0.0	0.0	129
^aSignificantly different from the 0% dye exposure group, p < 0.01

Swimming development ratings (mean _+ SE)

	Treatment	n^a	Swimming-direction day 6	n^a	Swimming-angle day 10
Experiment 1	R3 1.0	14	1.9±0.1	12	2.4±0.2*
	R3 0.5	13	1.6±0.1	13	2.1±0.1
	R3 0.25	12	1.8±0.1	11	2.4±0.3*
	R3 0.0	35	1.7±0.1	35	1.8±0.1
	R3 HU	19	1.7±0.1	19	2.1±0.2
Experiment 2	R3 1.0	14	2.2±0.2**	14	1.9±0.1
	R3 0.5	13	2.6±0.2	13	2.1±0.2*
	R3 0.25	14	2.6±0.1	14	2.3±0.2**
	R3 0.0	11	2.6±0.2	11	1.9±0.2
* F-test p < 0.01, individual group comparison to R3 0.0 group, p < 0.05 ** F-test p < 0.01, individual group comparison to R3 0.0 group, p < 0.01 an represents the number of litters tested, n varies slightly due to missing data points n = 35 in the R3 0.0 group because of controls from a previous similar experiment

Applicant's summary and conclusion

Conclusions:

No adverse effects were found in the present experiments on the test chemical on parental weight, food consumption, or reproductive performance. The offspring were evaluated for growth and behavioral development. There was an increase in pre-weaning mortality in the high and mid-dose groups in the first experiment, but this effect was not replicated in the second experiment. No adverse effects of the test chemical on offspring growth or adult regional brain weight were observed. Thus, it was concluded that no evidence was obtained in these two experiments to suggest that the test chemical was a developmental psychotoxin in rats at doses of up to 1.0% of the diet. Hence, the NOAEL can be considered to be 1% (1000 mg/kg/day) in diet.

Executive summary:

The test chemical was evaluated for for its developmental toxicity and psychotoxicity in 2 different experiments. Sprague-Dawley male and female rats were exposed to the test chemical administered at dietary dose levels of 0, 0.25, 0.5 and 1.0 % (corresponding to approximately 250, 500 and 1000 mg/kg/day) mixed in powdered Purina rat chow. Two separate experiments were conducted using the test chemical. The two experiments were separated by 2 years, and the second experiment was intended as a replication to resolve some of the treatment effect patterns observed in the first experiment. was fed to each treatment group for 2 weeks prior to breeding, 1-14 days during breeding (males and females housed together in pairs), then to the females during gestation and lactation, the males being discarded after confirmation of breeding (determined by the presence of sperm in the daily vaginal lavage of the females and designated as day zero of gestation). The experimental diets were freely available throughout postnatal development to the offspring of all delivering dams (up to the end of the experiments at 90-110 days of age of the offspring). Litters of fewer than eight live offspring were not retained beyond day 1 (birth was designated post natal day zero). Litters of more than 12 were reduced to 12 using a random selection procedure with sex restriction, i.e., in a way that tended to equalize the number of males and females in each litter. Body weights were measured at weekly intervals except during breeding, and food consumption was measured every third day on an arbitrarily selected subset of each treatment group (usually the first 5-15 pairs of animals enrolled in the study in each group). On the day following birth all litters were examined and data collected on litter size, sex distribution, weight, and number of dead or malformed offspring. Behavioural landmarks were evaluated according to the Cincinnati psychoteratogenicity test system for rats. The experiment was replicated after 2 years using the same exposure regimen, but without positive control in the second experiment and using different versions of the Cincinnati psychoteratogenicity test system in the two experiments.No adverse effects were found in the present experiments on the test chemical on parental weight, food consumption, or reproductive performance. The offspring were evaluated for growth and behavioral development. There was an increase in preweaning mortality in the high and mid-dose groups in the first experiment, but this effect was not replicated in the second experiment. No adverse effects of the test chemical on offspring growth or adult regional brain weight were observed. Thus, it was concluded that no evidence was obtained in these two experiments to suggest that the test chemical was a developmental psychotoxin in rats at doses of up to 1.0% of the diet. Hence, the NOAEL can be considered to be 1% (1000 mg/kg/day) in diet.