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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-23 to 2018-01-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate
EC Number:
276-770-9
EC Name:
Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate
Cas Number:
72705-24-9
Molecular formula:
C26H24N4O9S3.H3N.Na
IUPAC Name:
ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number: #13298465
- Expiration date of the lot/batch: Mar 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

FORM AS APPLIED IN THE TEST
suspension

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected. This Wistar rat strain (Crl:WI(Han)) was selected since extensive historical control data were available on these Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at supply: males: about 10- 11 weeks, females: about 9 weeks
- Weight at study initiation: (P) Males: ca 356.1 - 386.9 g; Females: ca. 203.2 - 221.6 g
- Housing: During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany;
During pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III;
For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material
- Diet: ad libitum, Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water: ad libitum
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: water containing 0.5 % sodium carboxymethyl cellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, 0.5 % sodium carboxymethyl cellulose in drinking water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were produced weekly, at least.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks (maximum)
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At the time points mentioned above, one sample from the mid concentration was additionally taken for concentration control analysis. The samples collected at the beginning of the administration period and during the lactation period were analyzed.
The concentrations of the test item in the samples were calculated by means of their nitrogen content (determination of nitrogen after Kjeldahl digestion by titration with sulfuric acid). The results demonstrated the correctness of the concentrations of the test item in 0.5 % sodium carboxymethyl cellulose in drinking water. The concentrations of the samples were in compliance with the expected concentrations (recovery of 103-114 %).
Duration of treatment / exposure:
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days and once daily on Saturdays, Sundays and public holidays
- Parameters checked: dead or moribund animals

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily
- Parameters checked: signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity, parturition and lactation behavior of the dams

BODY WEIGHT: Yes
- Time schedule for examinations: During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week.

FOOD CONSUMPTION AND COMPOUND INTAKE
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
• Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was analysed during clinical observations in all animals prior to the administration period and thereafter at weekly intervals. Besides that, a functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period, during which the urine excreted within 2 minutes was examined for its amount and colour. For both observations, the animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes.
- To determine urine constituents semiquantitatively the dry chemical reactions on test strips (Combur-Test 10 M; Sysmex, Norderstedt, Germany) were evaluated with a reflection photometer (Miditron M; Sysmex, Norderstedt, Germany).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning, samples were taken from the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Parameters checked in table 3 were examined.

CLINICAL CHEMISTRY: - Time schedule for collection of blood: in the morning, samples were taken from the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
- Parameters checked in table 4 were examined.

Oestrous cyclicity (parental animals):
For all females of the pool estrous cycle normality was evaluated before the beginning of the administration. In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there was evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups/litter; If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, determination of thyroid hormone concentrations in one male and one female pup/litter

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities, organs were assessed macroscopically
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
The tissues indicated in Table 2 were prepared for microscopic examination.
Statistics:
weight parameters (see table 5):
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Reproductive indices:
Male mating index, male fertility index, female mating index, female fertility index, gestation index, live birth index, post implantation loss
Offspring viability indices:
viability index, survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All male and female animals of test group 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) showed yellowish discolored feces from study day 4 onwards until the end of the treatment period. This finding was considered treatment-related but not adverse.

Dermal irritation (if dermal study):
not examined
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):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period in males of test group 1 (100 mg/kg bw/d) alanine aminotransferase (ALT) activities and potassium levels were significantly decreased, but the changes were not dose-dependent. In males of test group 3 (1000 mg/kg bw/d) ALT was also significantly decreased, but the mean was within the historical control ranges (males, ALT 0.55-0.92 µkat/L). Therefore, the mentioned alterations were regarded as incidental and not treatment-related.

Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings (e.g. reduced epididymes size, focal constriction in the liver, reduced size of testes, eosinophi lic infiltrate in jejunem) occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings (e.g. basal cell metaplasia in glandular stomach, cyst formation in ovaries) occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

Male reproduction data:
The male mating index calculated after the mating period for F1 litter was 100 % in test groups 0, 1 (100 mg/kg bw/day and 3 (1000 mg/kg bw/day) and 90 % in test group 2 (300 mg/kg bw/day).
Fertility was proven for nearly all of the F0 parental males within the scheduled mating interval to produce F1 litter. The male fertility index was 100 % in test groups 0 and 1 and 90 % in test groups 2 and 3.
Female reproduction data:
The female mating index calculated after the mating period for F1 litter was 100 % in test groups 0, 1 and 3 and 90 % in test group 2.
The mean duration until sperm was detected (GD 0) was 2.4 days for test group 0, 2.7 days for test group 1 and 2.6 days for test groups 2 and 3.
These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
The gestation index was 100 % in all test groups including control.
The rate live birth indices were 100 % in all test groups including control.
The postimplantation loss was 8.7 % in test group 0 (control), 4.9 % in test group 1 (100 mg/kg bw/d), 9.6 % in test group 2 (300 mg/kg bw/d) and 6.0 % in test group 3 (1000 mg/kg bw/d). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. A treatment-related increase in postimplantation loss was not observed in any test group.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The viability index indicating pup mortality during PND 0-4 was 95.7 % in test group 0 (control), 97.1 in test group 1 (100 mg/kg bw/d) and 100 % in test groups 2 and 3 (300 and 1000 mg/kg bw/d).
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

The mean number of delivered F1 pups per dam was equally distributed among all test groups. No significant deviations occurred.
The viability index indicating pup mortality during PND 0-4 was 95.7 % in test group 0 (control), 97.1 in test group 1 (100 mg/kg bw/d) and 100 % in test groups 2 and 3 (300 and 1000 mg/kg bw/d).
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences including the significant deviation in test group 3 were regarded to be spontaneous in nature.
Anogenital distance and anogenital index were not affected in all F1 pups. The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
In the tested male and female pups at PND 13, no treatment-related alterations of T4 and TSH levels were observed.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test substance-related adverse effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion