Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: data from peer- reviewed journals

Data source

Reference

Reference Type:

publication

Title:

Teratologic Studies with FD & C Red No. 2 in Rats and Rabbits

Author:

M. L. KEPLINGER, P. L. WRIGHT, J. B. PLANK AND J. C. CALANDRA

Year:

1974

Bibliographic source:

TOXlCOLOGY AND APPLIED PHARMACOLOGY 28,209-215(1974)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 21 CFR [Code of Federal Regualtions], para. 9.61, Cl no. 16185-1971

Principles of method if other than guideline:

To assess the teratogenic potential of FD&C Red No 2 (Amaranth dye) meeting the specifications in 21 CFR [Code of Federal Regualtions], para. 9.61, Cl no. 16185-1971 in albino rabbits.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

EC Number:

213-022-2

EC Name:

Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

Cas Number:

915-67-3

Molecular formula:

C20H14N2O10S3.3Na

IUPAC Name:

trisodium 3-hydroxy-4-[(4-sulfonato-1-naphthyl)diazenyl]naphthalene-2,7-disulfonate

Constituent 2

Reference substance name:

2,7-Naphthalenedisulfonic acid, 3-hydroxy-4-[(4-sulfo-1-naphthalenyl) azo]-, trisodium salt

IUPAC Name:

2,7-Naphthalenedisulfonic acid, 3-hydroxy-4-[(4-sulfo-1-naphthalenyl) azo]-, trisodium salt

Constituent 3

Reference substance name:

Amaranth dye

IUPAC Name:

Amaranth dye

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Details on test material
Name of test material (as cited in study report): Amaranth dye
Molecular formula (if other than submission substance): C20H11N2Na3O10S3
Molecular weight (if other than submission substance): 604.47
Smiles notation (if other than submission substance): c1ccc2c(c1)c(ccc2S(=O)(=O)[O-])N=Nc3c4ccc(cc4cc(c3O)S(=O)(=O)[O-])S(=O)(=O)[O-].[Na+].[Na+].[Na+]
InChl (if other than submission substance): 1S/C20H14N2O10S3.3Na/c23-20-18(35(30,31)32)10-11-9-12(33(24,25)26)5-6-13(11)19(20)22-21-16-7-8-17(34(27,28)29)15-4-2-1-3-14(15)16;;;/h1-10,23H,(H,24,25,26)(H,27,28,29)(H,30,31,32);;;/q;3*+1/p-3
Substance type: Organic
Physical state: Solid

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Charles River CD
Age at study initiation: no data
Weight at study initiation: No data
Fasting period before study: the animals were fasted during the night preceding each dosing day
Housing: metal cages
Diet (e.g. ad libitum): ad libitum
Water (e.g. ad libitum): water, ad libitum
Acclimation period: No data


ENVIRONMENTAL CONDITIONS
Temperature (°C): No data
Humidity (%): No data
Air changes (per hr):No data
Photoperiod (hrs dark / hrs light):
No data
IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

oral: capsule

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: gelatin capsules

Details on exposure:

Test animals were treated with 1.5, 5 or 15 mg/kg of FD & C Red No. 2.
All females received treatment daily via gelatin capsule

Details on mating procedure:

M/F ratio per cage: On gestation day 0 each female was given 2mg/kg lutenizing hormone and was inseminated with 0.7 ml of diluted, pooled semen from proven bucks.
Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy : No data
After successful mating each pregnant female was caged (how): Bred does were housed individually for the remainder of the study.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

Day 6 through 18 of gestation.

Frequency of treatment:

daily

Details on study schedule:

no data

Doses / concentrations

Remarks:

Doses / Concentrations:
3 test groups received 1.5,5.0 or 15 mg/kg/day of FD & C Red No. 2.
Basis:
nominal conc.

No. of animals per sex per dose:

3 test groups of 17 female rabbits

Control animals:

yes, concurrent vehicle

Details on study design:

3 control groups received empty capsules

Positive control:

2 positive control groups were treated with 37.5 mg/kg/day of thalidomide

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes / No / No data: No data
Time schedule: No data
Cage side observations checked in table [No.?] were included.: No data

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data: no
Time schedule: No data

BODY WEIGHT: Yes / No / No data: Yes
Time schedule for examinations: No data

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 / No / No data: No data
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data

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

OTHER:
On day 29, all does were sacrificed and the young removed by caesarean section.
Apparently nongravid does were carefully examined for any indication of having been pregnant. Immediately after removal from the chorion, all fetuses were examined for gross abnormalities.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

Immediately after removal from the chorion, all fetuses were examined for gross abnormalities. Viable pups were weighed and placed in an incubator (37°C). Observations for viability as indicated by respiratory and paw movements were made hourly for 7 hr and again after 24 hr. At the end of the 24-hr period, all fetuses were sacrificed.

Postmortem examinations (parental animals):

All females were sacrificed at day 29 of gestation

Postmortem examinations (offspring):

At the end of the 24-hr period, all fetuses were sacrificed. All young were examined by careful dissection. Particular attention was paid to any differences in size, shape and orientation of the major organs and blood vessels which might relate to treatment. An examination of skeletal tissue was performed employing a modified method for the demonstration of skeletal tissues in embryos as described by Hurley.

Statistics:

Fetal viability data were subjected to 2 types of statistical analysis. First, in a chi-square analysis, the number of resorption sites per implantation site of each test animal was compared to the expected incidence. The expected incidence was calculated from data obtained from the 3 untreated control groups. The second analysis utilized the ranking method of Weil. For this analysis the individual viability indices of each test group were ranked with individual viability indices of each control group.

Reproductive indices:

no data

Offspring viability indices:

no data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):No signs of toxicity were seen throughout the study.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): The body weight of maternal animals was not adversely affected by any treatment and no deaths or abnormal reactions were noted during the study. Mean body weight gain of the maternal animals from day 0 to 29 of gestation were 0.7, 0.20, 0.25, 0.27, 0.19 kg for the 3 vehicle control, 2 positive control, 1.5, 5 and 15 mg/kg test groups,respectively
TEST SUBSTANCE INTAKE (PARENTAL ANIMALS: No data
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): No data
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): No data
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS: No data
ORGAN WEIGHTS (PARENTAL ANIMALS): No data
GROSS PATHOLOGY (PARENTAL ANIMALS): Fetal viability data were subjected to 2 types of statistical analyses. Neither method disclosed any significant differences p < 0.05) between test and untreated control groups. The resultsof these analyses combined with the lack of any dose-correlated response indicated that an apparently lower fetal viability was not related to the administration of FD & C Red No. 2.
HISTOPATHOLOGY (PARENTAL ANIMALS): No data
OTHER FINDINGS (PARENTAL ANIMALS): No data

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No embroytoxic or fetotoxic effects were observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

not specified

Details on results (F1)

VIABILITY (OFFSPRING): The yield of viable pups at all dosage levels appeared to be somewhat lower than that for concurrent controls.
CLINICAL SIGNS (OFFSPRING): No data
BODY WEIGHT (OFFSPRING): Body weights and 24 hr survival of the progeny were not adversely affected by any treatment.
SEXUAL MATURATION (OFFSPRING): No data
ORGAN WEIGHTS (OFFSPRING): No data
GROSS PATHOLOGY (OFFSPRING): Treatment with FD & C Red No. 2 did not lead to any significant increase in the incidence of gross anomalies.
Observations of fetal skeletal development revealed no effect related to treatment with FD & C Red No. 2.
HISTOPATHOLOGY (OFFSPRING): No data
OTHER FINDINGS (OFFSPRING): No data

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No embroytoxic or fetotoxic effects were observed

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Studies were conducted to evaluate the teratogenic potential of FD & C Red No. 2 were conducted in albino rabbits at dosage levels of 1.5, 5.0 and 15.0 mg/kg.
These studies revealed no evidence on the adverse effects of the dye.
Hence the NOAEL (No Observed Adverse Effect Level) in rabbits was concluded to be 15.0mg/kg/day.

Executive summary:

Studies were conducted to evaluate the teratogenic potential of FD & C Red No. 2 were conducted in albino rabbits at dosage levels of 1.5, 5.0 and 15.0 mg/kg.

A total of 136 female New Zealand albino rabbits were utilized to form 8 groups of 17 animals each: 3 control groups, 2 positive control groups and 3 test groups.On gestation day 0 each female was given 2 mg/kg of luteinizing hormone and was inseminated with 0.7 ml of diluted, pooled semen from proven bucks. Bred does were housed individually for the remainder of the study.

All does received treatment daily via gelatin capsule on days 6 through 18 of the gestation period. Control animals were given empty capsules, positive controls received 37.5 mg/kg of thalidomide and test animals were treated with 1.5, 5 or 15 mg/kg of FD & C Red No. 2. Does weighed on days 0, 6, 9, 12, 15, 18 and at sacrifice (day 29).

 

The most recent weight was used in dose calculation. The animals were fasted during the night preceding each dosing day. At all other times the animals were allowed food and water ad libitum.On day 29, all does were sacrificed and the young removed by caesarean section. Apparently nongravid does were carefully examined for any indication of having been pregnant. Immediately after removal from the chorion, all fetuses were examined for gross abnormalities. Viable pups were weighed and placed in an incubator (37°C). Observations for viability as indicated by respiratory and paw movements were made hourly for 7 hr and again after 24 hr. At the end of the 24-hr period, all fetuses were sacrificed.All young were examined by careful dissection. Particular attention was paid to any differences in size, shape and orientation of the major organs and blood vessels which might relate to treatment. An examination of skeletal tissue was performed employing a modified method for the demonstration of skeletal tissues in embryos as described by Hurley.

 

The body weight of maternal animals was not adversely affected by any treatment and no deaths or abnormal reactions were noted during the study. Mean body weight gain of the maternal animals from day 0 to 29 of gestation were 0.27, 0.20, 0.25, 0.27, 0.19 kg for the 3 vehicle control, 2 positive control, 1.5, 5 and 15 mg/kg test groups, respectively.

 

Treatment with FD & C Red No. 2 did not lead to any significant increase in the incidence of gross anomalies. Body weights and 24-hr survival of progeny were not adversely affected by any treatment. Observations of fetal skeletal development revealed no effect related to treatment with FD & C Red No. 2.

 

These studies revealed no evidence on the adverse effects of the dye.

 

Hence the NOAEL (No Observed Adverse Effect Level) in rabbits was concluded to be 15.0mg/kg/day.