Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14. Sep to 25 Nov 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): C.I. Reactive Blue 19
- Physical state: solid
- Appearence: dark powder

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: 65 to 75 days
- Weight at study initiation: 193.2 +/- 14 g
- Fasting period before study: NA
- Housing: single
- Diet: Altromin 1310 ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 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:
yes
Details on analytical verification of doses or concentrations:
homogeneity and stability of test formulations were analytically verified
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
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
23 to 24 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: test item was tolerated in the acute and subacute studies without adverse effects
The study compound was suspended fresh daily in a concentration of 200 g/L in distilled water and administered daily to all the animals within three hours of preparation.
All animals received the same volume of liquid, 5 ml/kg body weight. After each weighing of the animals, the compound doses were adjusted according to body weight. The stability and homogeneity of the soiutions were guaranteed over a period of 4 hours

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

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Gross pathological findings:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
20 dams in the compound group and 19 dams in the control group carried live conceptuses to term and were delivered by caesarean section
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
black discoloration of faeces and red discoloration of urine due to excretion of dye

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food efficiency
gross pathology
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
early or late resorptions
dead fetuses
changes in number of pregnant
necropsy findings

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
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

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
No adverse effects were observed after daily administration of 1000 mg/kg/day Reactive Blue 19 in dams or their fetuses.
Maternal NOEL: 1000 mg/kg/day
Fetal NOEL: 1000 mg/kg/day
Executive summary:

In this limit test, C.I. Reactive Blue 19, 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 were then examined morphologically for developmental disorders.

The studies showed that the repeated oral administration of C.I. Reactive Blue 19, 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 Blue 19 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.