5-[[4-chloro-6-[(3-sulphophenyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonic acid, sodium salt

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September to 25 November 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

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

241-164-5

EC Name:

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

Cas Number:

17095-24-8

Molecular formula:

C26H25N5O19S6.4Na

IUPAC Name:

Tetrasodium 4-amino-5-hydroxy-3,6-bis[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

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

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

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

Details on results:

Clinical examinations
The dams of the compound group and the control dams showed no impairment to behaviour and General condition and survived until the scheduled study end. All dams in the compound group excreted black-discoloured faeces on at least eight days during and after the treatment period, mostly, however, in the period from day 8 - 19 of pregnancy. Blue-discoloured urine was voided by three dams from days 8 - 10 or days 8 - 11 of pregnancy. One dam from the compound group displayed pallor from day 8 - 11 of pregnancy. Compound solution escaped from the mouths of two dams after the 3rd and/or 4th administration, presumably due to the dosing method. The amounts, however, were small and without relevance for assessing the test compound. Local abdominal hair loss was noted in another dam from the compound group on days 20 and 21 of pregnancy.
The administration of C. I. Reactive Black 5 had no influence on the feed consumption of the dams. The dams consumed comparable amounts of feed to those of the controls both during and after the treatment period.
The body weight gain of the dams treated with C. I. Reactive Black 5 was likewise unaffected. The dams treated with the test compound gained in weight to the same extent as the controls.
Autopsy of the dams
The concluding autopsy revealed a marked dilatation of one renal pelvis in one dam each from the compound and control groups. No further alterations to the internal organs could be seen in the macroscopic examination.

Maternal developmental toxicity

Number of abortions:

no effects observed

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 pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Examinations after caesarean section
All 20 dams in the compound group and 19 dams in the control group carried live conceptuses to term and were delivered by caesarean section. One control dam had no live foetuses, but merely 8 empty implantation sites in the uterus, indicating early embryonic death shortly after implantation. In this animal, the corpora lutea were quite small and could not therefore be determined exactly. The number of corpora lutea, which provides information on the number of ovulations prior to start of treatment, and the number of implants and live foetuses in the remaining dams of the compound group did not differ markedly from those of the control animals and lay in the range of our previous control values.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

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:

Examination of foetuses
The live foetuses delivered in the compound group were normally developed. Their body weights and lengths did not differ from those of the control foetuses and lay in the range of our previous control values. The number of live foetuses weighing less than 3.0 g and thus qualifying according to our definition as slightly retarded or retarded was even lower than that of the control group. The sex ratio was relatively balanced in both groups: the males predominated slightly in the control group and the females predominated slightly in the compound group.
Dead embryofoetal primordia were found in both groups. They consisted exclusively of conceptuses undergoing resorption. These had died at a relatively early stage and exhibited diameters of up to 6.3 mm (compound group) or 6.6 mm (control group). Dead foetuses were not found in either of the two groups. The number of dead embryofoetal primordia in the dams of the compound group was low and did not differ from that of the control dams.
The placentas of the foetuses in the compound group were unremarkable macroscopically and in respect of their weights, which corresponded to those of the control group.
The findings observed at the morphological examination of the foetuses show that no malformations appeared in any foetus in the compound group. One control foetus exhibited a right-sided microphthalmia.
The autopsy and body cross-section examinations revealed blood in the pericardium or abdominal cavity, completely fused or bipartite pulmonary lobes or a haematoma in a liver lobe as well as a distention of one or both renal pelves or one ureter in individual or some foetuses. Furthermore, one foetus in the compound group displayed blood in the vicinity of the right kidney. Findings confined to individual foetuses in the control group were: blood in the thoracic cavity or in the right kidney and a stomach which was relatively large and tautly filled with fluid.
The skeletons of the foetuses in the compound group were at roughly the same stage of development as those of the control foetuses. Their stage of ossification corresponded to the 21st day of pregnancy. The number of foetuses in the substance-treated group displaying a poor or missing ossification of sternebrae, 5th metacarpal bone and the ossification of fewer than two caudal vertebral centres did not differ markedly from that of the control foetuses. The number of foetuses in which individual cranial bones were not-yet or only poorly ossified was, significantly increased compared to the simultaneous control group but still lay within the range of previous control values (maximum frequency 61.5 %).
Apart from these findings, individual or several foetuses in both groups exhibited longitudinally displaced, dislocated, fragmented sternebrae or waved and/or thickened ribs while numerous foetuses displayed an additional short rib anlage, uni- or bilaterally, at the 1st lumbar vertebra. In individual foetuses of the compound group, a dysplastic 10th or 11th and 12th thoracic vertebral centre or a 14th thoracic vertebra with a short, analogous rib pair were observed. Three foetuses in the control group showed an additional short rib, left- or both-sided, at the 7th cervical vertebra.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

In this limit test, the test substance, 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 study showed that the repeated oral administration of the test substance, 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” in rats following oral administration lies at 1000 mg/kg body weight/day or above with regard to maternal and embryofoetal toxicity and teratogenicity.

No teratogenic effect was observed.