Tetrasodium 4-amino-6-[[2,5-dimethoxy-4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]-5-hydroxy-3-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

14. Sep to 25 Nov 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reactive Black 5

IUPAC Name:

Reactive Black 5

Details on test material:

Test item: Reactive Black 5

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: 65 to 75 days
- Weight at study initiation: 193.1 +/- 14.2
- Fasting period before study: NA
- Housing: single
- Diet: Altromin 1310 ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22°C
- Humidity (%): 48 to 61%
- Air changes (per hr): 16 to 20
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 14. Sep To: 25. Nov 1993

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

- Frequency of preparation: daily
- Administration: within 3 hours after preparation


VEHICLE
- Concentration in vehicle: 200 mg/kg nominal
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

-

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: over night (3:30 pm to 7:30 am next day)
- Verification of same strain and source of both sexes: yes - own breeding facility
- Proof of pregnancy: sperm in vaginal smear, referred to as day 1 of pregnancy

Duration of treatment / exposure:

7. - 16. day of pregnancy

Frequency of treatment:

daily

Duration of test:

cesarean section on Day 21 of pregnancy

Doses / concentrations

Remarks:

Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal in water

No. of animals per sex per dose:

20 to 24 mated females

Control animals:

yes

Details on study design:

- Dose selection rationale: test item was tolerated in the acute and subacute studies without adverse effects

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/100 g body weight/day: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: all organs examined macroscopically
uterus - live and dead fetuses, resorption sites, placentas
ovaries - corpora lutea


OTHER:
- diameter of conceptuses undergoing resorption
- placenta weights
- presence of iron in uterus walls with ammonium sulphide to detect invisible implantation sites

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Placenta weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Body weight: Yes: all per litter
- Crown-rump length: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

comparison to actual control group and historical controls
MANOVA: body weight development, fetal weight, placental weight
PURI&SEN rank order test: food intake
Mantel-Haenszel's chi-squared test: live fetuses, intrauterine fetal death, number of implants, number of corpora lutea
multivariate analysis of variance: litter means of fetal weights, crown-rump length, placental weights
Fisher test: autopsy findings, body cross-sections, skelettal examination

Dams which had no live fetuses were excluded from the calculation of mean values and statistical evaluation

Indices:

No data

Historical control data:

Yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
black discoloration of feces and blue discoloration of urine due to excretion of dye

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
only minor anomalies or variation within the historical range of spontaneous findings were observed

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No adverse effects were observed after daily administration of 1000 mg/kg/day Reactive Black 5 in dams or their fetuses.
Maternal NOAEL: 1000 mg/kg/day
Fetal NOEL: 1000 mg/kg/day

Executive summary:

In this limit test, Reactive Black 5, which rapidly reacts to Reactive Black 5 bis-vinyl in aqueous solution, dissolved in distilled water, was administered orally by stomach tube in a single daily dose of 1000 mg/kg body weight to a group of 20 pregnant female Wistar rats from the 7th - 16th day of pregnancy. A simultaneous control group of the same size received the vehicle without test compound. On the 21st day of pregnancy, the dams were killed and delivered by caesarean section. The foetuses delivered by caesarean section were then examined morphologically for developmental disorders.

The studie showed that the repeated oral administration of C.I. Reactive Black 5, at a dose of 1000 mg/kg body weight in the sensitive phase of organogenesis for the conceptuses, did not lead to any impairment of the general physical condition of the dams or impaired intrauterine development of conceptuses.

The morphological examination of the foetuses with regard to stage of development, outwardly detectable anomalies as well as anomalies of the internal organs and the skeleton showed no indication of an embryotoxic or teratogenic effect of the compound. The findings observed are to be regarded as spontaneous in origin.

On the basis of the results of this limit test, the “no observed adverse effect level” for C.I. Reactive Black 5 in rats following oral administration lies at 1000 mg/kg body weight with regard to maternal and embryofoetal toxicity and teratogenicity.

No teratogenic effect was observed.