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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Jan. 14, 2008 to Feb. 14, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

464-700-1

EC Name:

-

Cas Number:

607724-42-5

Molecular formula:

Hill formula: C28H24N5Na5O23S7 CAS formula: C28H29N5O23S7.5Na

IUPAC Name:

Pentasodium 4-hydroxy-3-(2-methoxy-5-(2-sulfooxyethanesulfonyl)phenylazo)-7-(sulfomethylamino)-8-(2-sulfo-4-(2-sulfooxyethanesulfonyl)phenylazo)naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

see below

Test animals

Species:

rat

Strain:

other: Crl:(Wi) BR-Wistar rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CHARLES RIVER/EUROPE/LABORATORIES INC., TOXI-COOP 1103 Budapest, Hungary Cserkesz utca 90.
- Age at study initiation: Males: 9-10 weeks, Females: 6-8 weeks
- Weight at study initiation: Virgin female rats of comparable weight were used for mating. The weight variation was 198-285 g on the first day (Day 0) of gestation.
- Housing: Type II and III polypropylene/polycarbonate cages, with stainless steel covers equipped by self-feeding basket
- Bedding: laboratory bedding material, changed twice a week
- Diet: Ssniff SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance", ad libitum
- Water: tap water, as for human consumption, ad libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 30-70%
- Photoperiod: 12 h light/dark cycle

IN-LIFE DATES: From: Jan. 14, 2008 To: Feb. 14, 2008

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was suitable for oral (by gavage) administration when suspended in distilled water. The test substance was administered at appropriate concentrations prepared with the vehicle. The formulations of each concentration were used for the treatment within 60 minutes after preparation.

VEHICLE: distilled water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling for analytical control of dosing was made on the second and last week of the treatment period by the Analytical Laboratory of LAB Research Ltd. The analysis was carried out using HPLC. All the formulations proved to be homogeneous. Measured concentrations varied between 99 and 104 % of the nominal concentrations.

Details on mating procedure:

The oestrus cycle of female animals were examined before pairing. After acclimatisation, the females were paired according to their oestrus cycle to males in the morning for 3 h (1 male: 1 to 3 females) until the number of sperm positive females / group achieved 22. Vaginal smears were prepared from each female, stained with 1% aqueous methylene blue solution and examined for presence of sperm. The day of mating was regarded as Day 0 of pregnancy (vaginal plug and/or sperm in the vaginal smear). Sperm positive females were separated and caged in groups of 2 to 4 animals.

Duration of treatment / exposure:

The test substance was administered to the sperm positive females from Day 5 up to and including the Day 19 of gestation.

Frequency of treatment:

Once a day

Duration of test:

In life phase of the test was concluded by the cervical dislocation of all sperm positive females on Day 20 of gestation

No. of animals per sex per dose:

22 sperm-positive female/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for the study were selected on the basis of a dose range finding study, in which groups of 5 sperm positive female rats were administered 62.5, 100 and 1,000 mg/kg bw/day by oral gavage from Day 5 to Day 19 post coitum. The 1,000 mg/kg bw/day dose of the test substance was slightly toxic to the dams as it led to slightly reduced body weight and body weight gain, while no effect on the intrauterine development of the embryos and foetuses at any of the doses was seen. Hence the 1,000 mg/kg bw/day was considered suitable for the top dose in the main teratology study. (Study code: 07/583-105PE)
- Rationale for animal assignment: The sperm positive females were allocated to each experimental group on each mating day in such a way that the group averages of the body weight were as similar as possible on the Day 0 of pregnancy. Females paired with the same male were allocated to different groups on the same mating day.

Examinations

Maternal examinations:

Clinical Observations:
A careful clinical observation was made after dosing at least once a day and a cage side clinical observation was made in the afternoon. Individual observation included the check of behaviour and general condition. Checking for death was made at least once daily.

Body Weight:
The body weight of the male animals was not measured.
The body weight of the female rats was measured at least once in the pre-mating period, but was not statistically evaluated. Body weight of sperm positive females was measured on gestation Days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g). Corrected body weight was calculated on the 20th day of pregnancy (body weight on Day 20 minus the weight of the gravid uterus).

Food Consumption:
The food consumption was measured between gestation Days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

Examination for Sign of Implantation:
On gestation Days 13 and/or 14 the sperm positive females were checked for the presence of vaginal bleeding which indicates the implantation of conceptuses.

Necropsy:
All sperm positive females were sacrificed by CO2 gas anaesthesia followed by cervical dislocation on Day 20 of gestation. The abdomen was opened, the uterus with cervix and the left ovary were removed and weighed. The right ovary was placed into a petri dish after removal. After removing the uterus and its content gross pathology of dams' viscera was performed. There were no organs and tissues with undiagnosed macroscopic findings examined histologically.
The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted. Animals with unambiguous implantation sites but no foetuses were found, were considered as pregnant. Foetuses were removed from the opened uterus, viability assessments were performed and after cutting the umbilical cord euthanasia of the viable foetuses was made by subcutaneous injection of 0.01 mL Euthasol in the femur region. Each live foetus was weighed individually (accuracy 0.01 g), and was subjected to external examination. The placentas were examined externally. The gender of foetuses was determined according to the anogenital distance.

Ovaries and uterine content:

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

Fetal examinations:

- Body weight: Yes: all per litter
- External examinations: Yes: all per litter
- Visceral examinations (including head): Yes: half per litter
- Skeletal examinations: Yes: half per litter
-

Statistics:

The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.

A foetus was considered as retarded in body weight, when its weight was below the average minus twofold standard deviation of all control foetuses.

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 consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, findings of slightly pale areas on the liver was recorded for the dams with a statistical significance in the 1000 mg/kg bw/day dose group.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

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:

At necropsy, findings of slightly pale areas on the liver was recorded for the dams with a statistical significance in the 1000 mg/kg bw/day dose group.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

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

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

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

Any other information on results incl. tables

Details on maternal observations:

There was no maternal mortality or maternal clinical signs in any group. The test substance had no effect on the body weight, body weight gain, gravid uterine weight, corrected body weight, corrected body weight gain or food consumption of dams.

At necropsy, findings of slightly pale areas on the liver was recorded for the dams with a statistical significance in the 1000 mg/kg dose group.

 

Details on foetal observations:

The intrauterine mortality and sex distribution of foetuses were unaffected by the treatment. There was no difference between groups in the number of corpora lutea or implantations. The body weight of foetuses was similar in all experimental groups. Reddish discolouration of the amniotic epithelium was recorded for the majority of the foetuses in the 1000 mg/kg dose group that suggested that the coloured test substance (or a coloured metabolite) crossed the placenta. Oedematous placentas were found only in the 1000 mg/kg bw/day dose group, this was attributed to the treatment with the test substance. Three multiple malformed foetuses were found at external examination in the 1000 mg/kg bw/day dose group. Two of these foetuses were found in one single litter and had visceral multiple malformations. All of the three foetuses had severe skeletal malformations. The malformations were of very low incidence and taking into account the findings from the whole study and historic data, were considered to reflect spontaneous malformations and are not likely to be related to test substance.

Applicant's summary and conclusion

Conclusions:

The substance was found to have no developmental toxicity / teratogenic effects. The NOAEL for maternal toxicity and developmental toxicity / teratogenic effects was determined to be 1000 mg/kg bw/day.

Executive summary:

A study was conducted to assess the developmental toxicity of the test substance according to OECD Guideline 414, in compliance with GLP.

Groups of 22 sperm-positive female rats were treated orally in one control group and three dose levels of 62.5, 250 and 1000 mg/kg bw/day (5 mL/kg) from Day 5 to Day 19 post-coitum. Rats were examined daily for morbidity and clinical signs. Body weight was recorded on Days 0, 3, 5, 8, 11, 14, 17 and 20 of gestation. Food consumption was determined on Days 0-3, 3-5, 5-8, 8-11, 11-14, 14-17 and 17-20 of gestation. Caesarean section and gross-pathology were performed on Day 20 of pregnancy. Implantations, early and late resorptions, live and dead foetuses in each uterine horn and the number of corpora lutea in each ovary were recorded. Each foetus was weighed and examined for sex and gross external abnormalities. The placentas were examined externally. About half of each litter was subjected to visceral examination and the other half to skeletal examination (with double staining). All abnormalities found during the foetal examinations were recorded.

No maternal mortality or maternal clinical signs were recorded in any group. The test substance had no effect on the body weight, body weight gain, gravid uterine weight, corrected body weight, corrected body weight gain or food consumption of dams. At necropsy, slightly pale areas on the liver were recorded for the dams with a statistical significance in the 1000 mg/kg bw/day dose group. The intrauterine mortality and sex distribution of foetuses were unaffected by the treatment. There was no difference between groups in the number of corpora lutea or implantations. The body weight of foetuses was similar in all experimental groups. Reddish discolouration of the amniotic epithelium was recorded for the majority of the foetuses in the 1,000 mg/kg dose group, suggesting that the test substance (or a coloured metabolite) crossed the placenta. Oedematous placentas were found only in the 1000 mg/kg bw/day dose group. This was attributed to treatment with the test substance. Three multiple malformed foetuses were found at external examination in the 1000 mg/kg bw/day dose group. Two of these foetuses were found in one single litter and had visceral multiple malformations. All of the three foetuses had severe skeletal malformations. The malformations were of very low incidence. Taking into account the findings from the whole study and historic data, they were considered to reflect spontaneous malformations rather than related to test substance exposure.

Under the study conditions, the substance was found to have no developmental toxicity / teratogenic effects. The NOAEL for maternal toxicity and developmental toxicity / teratogenic effects wasdetermined to be 1000 mg/kg bw/day.