Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an oral combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats according to OECD Guideline 422 a NOAEL of 1000 mg/kg bw/d for reproduction/development was determined.

Link to relevant study records

Reference

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

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

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

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

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

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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.

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

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a combined repeated dose oral gavage toxicity study with the reproduction/developmental toxicity screening (OECD 422) the test item wasadministered by gavage to groups of 10 male and 10 female Wistar rats (F0 generation) at dose levels of 0 mg/kg bw/d (test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). Drinking water containing 0.5 % sodium carboxymethyl cellulose served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 during the entire study period. Fertility indices for male and female animals were not impaired by test-substance administration even at a dose level of 1000 mg/kg bw/d. In addition, live birth indices of pups in all test groups were not influenced. The viability index as indicator for pup mortality was not altered. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, there were neither treatment-related organ weight changes nor histopathological lesions. Macroscopically, a yellow discoloration of the contents was found at 100, 300 and 1000 mg/kg bw/d in the glandular stomach of males and females, and in the jejunum, colon and cecum of males only. This finding was consistent with the color of the test substance and had no histopathological correlate. Therefore, it was considered treatment-related but not adverse. All other findings 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. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance and fertility was also set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 1000 mg/kg bw/d.

Effects on developmental toxicity

Description of key information

The NOAEL for reproductive performance and fertility and for developmental toxicity was determined to be 1000 mg/kg bw/d based on the results of the reproduction/developmental toxicity screening study (OECD 422).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for reproductive or developmental toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.