Benzenesulfonamide, N-[2-[[(phenylamino)carbonyl]amino]phenyl]-

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

There was no evidence of adverse effects on parental reproductive indices following repeated oral exposure in a study according OECD 422. The NOAEL was set as 1000 mg/kg bw/day in parental and F1 animals under the conditions of the test.

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to Annex IX, 8.7.3 of REGULATION (EC) No 1907/2006 OF THE EUROPEAN PARLIAMENT (REACH), an extended one-generation reproductive toxicity study having regard to the likely route of human exposure, shall be proposed for substances within the tonnage band 100 - 1000 tpa, if the 90-day study or other available studies on reproduction indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.
As discussed in more detail below, there is no evidence of substance-related effects with regard to reproductive toxicity as demonstrated in the available OECD TG 422 study, OECD TG 414 study and the Repeated Dose 90-DayToxicity study in rats.
A study according OECD TG 422 is available. The substance was administered repeatedly by oral gavage at dose levels of 0 (control group), 110, 330, and 1000 mg/kg bw/day from 14 days before mating through mating for 42 days in males and satellite females, and 14 days before mating through gestation and parturition until Day 4 of lactation in females to determine the repeated dose toxicity and reproductive and developmental toxicity, as well as reversibility of the changes observed. Each test group consisted of 12 males (including 5 males for the control and 1000 mg/kg bw/day groups in the recovery test) and 12 females (5 females each were added for the control and 1000 mg/kg bw/day groups in the recovery test). The control group received the vehicle (0.5 w/v% methylcellulose aqueous solution) only. No death occurred during the study. No changes related to the test substance treatment were observed in the clinical signs, functional observational battery, body weight, food consumption, hematological examination, blood chemical examination, necropsy, organ weight or histopathological examination in males or females or urinalysis in males. In the parental animals, no changes related to the test substance treatment were observed in the estrous cycle, copulation index, fertility index, gestation index, gestation length, number of corpora lutea or implantation sites, implantation index, delivery index, nor in the parturition or maternal behavior. In the offspring, no changes related to the test substance treatment were observed in the sex ratio, birth index, or viability index on day 4 of postpartum, nor in the external examination or body weight. The NOAEL for repeat dose toxicity was considered to be 1000 mg/kg bw/day in both sexes under the conditions of this study. The NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day in parental animals and offspring under the conditions of this study.
The substance was examined for its possible prenatal developmental toxicity in a study according OECD TG 414/GLP in rats. Groups of 26 sperm-positive female Han: of Wistar origin rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle 1% aqueous methylcellulose. The treatment volume was 5 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The substance in 1% aqueous methylcellulose was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 °C) at the concentrations of 20 and 250 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 106 and 109 % of nominal concentrations at both analytical occasions confirming proper dosing. In total, on gestation day 20 there were 23 evaluated litters in the control, 300 and 1000 mg/kg bw/day groups, respectively, as well as 22 in the 100 mg/kg bw/day group. One non pregnant female was moribund in the course of the study in the 100 mg/kg bw/day group after clinical signs such as hunched back, piloerection and reduced activity, as well as dyspnoea and lying on the side due to an unknown reason. Necropsy revealed no macroscopic changes in the organs. The other animals had no clinical signs and there were no treatment related pathological changes found. The food consumption and body weight of the animals was similar in each group. The mean of thyroid weight was statistically significantly lower in the 1000 mg/kg bw/day group (p<0.05), however the value was above the historical control level. Moreover, the treatment did not result in histological changes of the thyroid gland in any of the dose groups and the measured hormone levels were similar in the groups. There were no treatment related differences observed in the pre- and post implantation loss. The number of implantations and viable fetuses as well as their sex distribution was similar in the groups. The fetal weight was equal in the groups. There were no significant differences in the ano-genital distance and placental weight parameters. There were no significant differences in the incidence of body weight retarded (smaller) fetuses. At fetal examinations, there were no significant differences in the incidence of malformations and variations during external, visceral and skeletal examinations at any dose level. Based on the observations the NOAEL for maternal toxicity was 1000 mg/kg bw/day and the NOAEL for developmental toxicity including teratogenicity was 1000 mg/kg bw/day.
Effects on reproductive organs were also evaluated in an oral 90-day repeated dose toxicity study. The substance was administered orally (by gavage) to Wistar rats once a day at doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day for 90 days. A test item related influence on the estrous cycle was not detected (100, 300 and 1000 mg/kg bw/day). Histological examination did not reveal test item related lesions in male and female reproductive organs or tissues of animals administered with 1000 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period. Based on these observations the NOAEL was determined as 1000 mg/kg bw/day for male and female Han: WIST rats.
In conclusion, based on the available study results, adverse effects were observed neither on fertility parameters in male and female animals nor on development of the offspring. Therefore, according to Column 1, Section 8.7.3, Annex IX of REACH Regulation an extended one-generation study is not proposed also due to animal welfare reasons.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-08-19 to 2014-12-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

22 March 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino Breeding Centre).
- Age at study initiation: (P) 9 wks
- Weight at study initiation: (P) Males: 320.9-367.7 g; Females: 214.9-240.8 g
- Housing: Males and females (except during the gestation and lactation periods); stainless steel cages. For females during the gestation and lactation periods; polymethylpentane cages.
- Diet: ad libitum (except during the urine collection and during measurement of motor activity) autoclaved sterilized pellet diet (CRF-1, oriental yeast co. Ltd.). Animals were fasted on the day before scheduled necropsy.
- Water: ad libitum, except during the measurement of motor activity
- Acclimation period: 6 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 24.3
- Humidity (%): 48.6 to 69.8
- Air changes (per hr): 10 to 20
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

other: 0.5% w/v% methyl cellulose aqueous solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing preparations were prepared once every 4-8 days (based on the results of previous stability and homogeniety assessments). The test substance was initially ground in an agate pestle and mortar, and a few drops of vehicle added. This suspension was then transferred into a measuring cylinder. The appropriate volumes of the vehicle was added to the measuring cyclinder and mixed to achieve the required concentrations which were then added into brown glass vials and stored refrigerated until use.

VEHICLE
- Concentration in vehicle: 0, 11, 33 and 100 mg/mL
- Amount of vehicle: Dose volume of 10 mL/kg bw/day
- Lot/batch no.: PDG1792

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Until evidence of mating was apparent (upto 6 days).
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of gestation.
- 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 initial test substance preparation, 15 mL of each of the analytical samples were tested for homogeneity and analytical determination. The measured concentrations were within acceptable ranges (actual values 98.4 to 100.3% of nominal).

Duration of treatment / exposure:

Males: From 14 days before mating (Days 1 to 15) until the day before the necropsy through the mating period (42 days in total).
Females: From 14 days before mating (Days 1 to 15) until Day 4 of lactation (day of delivery was Day 0 of lactation) through the mating and pregnancy periods and delivery. One female not which did not deliver was kept until day before the necropsy.

Frequency of treatment:

Parental males and females daily by oral gavage.

Details on study schedule:

- Age at mating of the mated animals in the study: approximately 11 weeks

Dose / conc.:

110 mg/kg bw/day (nominal)

Dose / conc.:

330 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- A total of 106 rats were used on study (48 males and 58 females).
- Each test group consisted of 12 males (including 5 males for the control and 1000 mg/kg bw/day groups in the recovery test) and 12 females (5 females
each were added for the control and 1000 mg/kg bw/day groups in the recovery test).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A preceeding 14 Day repeated dose oral study with the test substance to rats, indicated that at a high dose of 1000 mg/kg bw/day would be expected to provide obvious toxicological changes.

Positive control:

no

Parental animals: Observations and examinations:

See Chapter 7.5.1, Supporting study

Oestrous cyclicity (parental animals):

Vaginal smears were collected with swabs from all test females in the morning (same time every day) from the initial dosing day to the day of mating or end of the mating period to confirm the estrous cycle. The obtained smears were collected on a plate for each animal, and stained with Giemsa.The estrous cycle was classified into diestrus (D), proestrus (P), estrus (E), and metestrus (M). The mean estrous cycle (number of
days from the estrous period to the next estrous period) and the number of the estrous period during the test period were calculated.

Sperm parameters (parental animals):

Not examined

Litter observations:

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.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities.

Postmortem examinations (parental animals):

Necropsy and histopathology procedures are described in Chapter 7.5.1, Supporting study

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on Day 4 of lactation.
- These animals were subjected to postmortem macroscopic examination (external anomalies).

GROSS NECROPSY
- Gross necropsy consisted of a check for external anomalies, including that in the oral cavity.

Statistics:

Histopathological findings were analysed using Fisher's exact probability test. Copulation index, fertility index, gestation index, delivery index and sex ratio were analysed using the Chi-squared test. Implantation index, stillborn rate, external anomaly index, external anomaly typing index, live birth index, and viability index were analysed using Wilcoxon's rank sum test.

Number of rearing, grip strength, motor activity, body weights, food consumption, urinalysis (reagent strip), hematology, blood chemistry, absolute and relative organ weight, estrous cycle, number of estrus, days until copulation, gestation length, number of corpora lutea, number of implantations, number of delivered offspring, number of live offspring, body weight of offspring (both sexes) were initially analysed using Bartlett's test. When the data was homogenous the Dunnet's multiple comparison test was applied, and when not homogenous, Steel's multiple comparison test was applied for the control group and each test group. For the urinalysis reagent strip, Steel's test was performed.

Reproductive indices:

Compulation index (%), fertility index (%), gestation length, implantation index (%), gestation index (%) and delivery index (%) were the determined reproductive indices.

Offspring viability indices:

Live birth index (%), stillborn rate (%), Viability index on Day 4 (%), Sex ration (%), external anomaly index (%), and external anomaly typing index (%), were the determined offspring viability indices.

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal clinical signs were observed in either sex of any group throughout the
dosing or recovery period, including the gestation and lactation periods of pregnant
females.

Mortality:

no mortality observed

Description (incidence):

No death occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant difference was observed in any sex in the treated-groups as compared to the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant difference was observed in any sex in the treated-groups as compared to the control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed in any treated group.
A low value of eosinophil ratio was observed in males of the 110 mg/kg bw/day group and above; however, as no significant change was noted in eosinophil count or leukocyte count and no related changes were observed in any examination, it was not considered to be toxicologically significant. A high value of reticulocyte ratio was observed in males of the 330 mg/kg bw/day group; however, the change was not considered to be treatment-related because there was no dose-dependency. No statistically significant difference was observed in females of any treated-group.
At the end of recovery period: A low value of white blood cell count was observed in satellite females of the 1000 mg/kg bw/day group, however, it was judged to be toxicologically insignificant, as this was no observed at the end of the dosing period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed in any treated group.
A high value of inorganic phosphorus was observed in males of the 1000 mg/kg bw/day group. However, it was judged to be toxicologically insignificant, as no related changes were observed in the other blood chemistry items and were observed in any examinations. A low value of sodium was observed in males of the 110 and 1000 mg/kg bw/day groups. However, it was judged to be toxicologically insignificant, as the change was noted in only a value that was slightly higher than the historical data in the test facility (min-max: 143.6 to 144.7 mmol/L; 2010 to 2013), and no related changes were observed in any examinations. A low value of albumin and a high value of creatinine were observed in females of the 330 mg/kg bw/day group; however, these were not considered to be treatment-related because there was no dose-dependency.
At the end of recovery period: A high value of ASAT was observed in males of the 1000 mg/kg bw/day group, and a low value of Ca and a high value of Na were observed in females of the 1000 mg/kg bw/day group. However, the changes were judged to be toxicologically insignificant, as these were not observed at the end of the dosing period.

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

In the qualitative analysis, no statistically significant difference was observed in males in
the treated-groups as compared to the control group.

Behaviour (functional findings):

no effects observed

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):

The various histopathological changes were observed in the animals of the control and 1000 mg/kg bw/day groups subjected to necropsy at the end of the treatment. However, these changes were judged to be not related to the test substance treatment, as such changes are nonspecifically observed in rats, and there was no apparent dose-dependency in their incidences. No histopathological change was observed in the ovary of the non-pregnant animal.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes in estrous cycle were observed in females of any treated group. One female of the 1000 mg/kg bw/day group showed continuous estrus for 4 days (from Day 5 to Day 8) and extension of proestrus for 5 days (from Day 10 to Day 14), suggesting an irregular estrous cycle. In one female of the 1000 mg/kg bw/day group, irregular estrous cycle (6-day cycle) was observed. However, no statistically significant change was observed in the mean estrous cycles or estrus count between the control group and any treated group.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no treatment-related differences in:
- Estrus cycling
- precoital time
- the number of pregnant females
- duration of gestation
- mean numbers of corpora lutae
- number of total, pre-implantation and post-implantation sites

Selected results by dose level (0, 110, 330 or 1000 mg/kg bw/day).
- females mated: 12/12, 12/12, 12/12, 12/12
- females pregnant: 12/12, 12/12, 12/12, 11/12
- females with live pups: 12/12, 12/12, 12/12, 11/12
- Copulation indices (%): 100, 100, 100, 100
- fertility Indices (%): 100, 100, 100, 91.7

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 at highest dose tested

Key result

Critical effects observed:

no

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant difference was observed between the control group and any treated group in body weight on days 0 and 4 of postpartum.

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

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No external abnormalities were observed in the offspring on Day 4 of lactation in any group.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

VIABILITY (OFFSPRING)
No statistically significant difference was noted in number of live offspring at birth, number of stillborn at birth, sex ratio, stillborn rate, or viability indexon Day 4 between the control group and any treated group. In addition, a high value of the birth index was observed in the 1000 mg/kg group; however, this change was judged to be not related to the test substance treatment because the post-implantation losses were a few.

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 adverse effects at highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

There was no evidence of adverse effects on parental reproductive indices and litter data following repeated oral exposure of the test substance, with the NOAEL set as 1000 mg/kg bw/day in parental animals and offspring under the conditions of the test.

Executive summary:

A study according OECD TG 422 was performed. The substance was administered repeatedly by oral gavage at dose levels of 0 (control group), 110, 330, and 1000 mg/kg bw/day from 14 days before mating through mating for 42 days in males and satellite females, and 14 days before mating through gestation and parturition until Day 4 of lactation in females to determine the repeated dose toxicity and reproductive and developmental toxicity, as well as reversibility of the changes observed. Each test group consisted of 12 males (including 5 males for the control and 1000 mg/kg bw/day groups in the recovery test) and 12 females (5 females each were added for the control and 1000 mg/kg groups in the recovery test). The control group received the vehicle (0.5 w/v% methylcellulose aqueous solution) only.

No death occurred during the study. No changes related to the test substance treatment were observed in the clinical signs, functional observational battery, body weight, food consumption, hematological examination, blood chemical examination, necropsy, organ weight or histopathological examination in males or females or urinalysis in males.

In the parental animals, no changes related to the test substance treatment were observed in the estrous cycle, copulation index, fertility index, gestation index, gestation length, number of corpora lutea or implantation sites, implantation index, delivery index, nor in the parturition or maternal behavior. In the offspring, no changes related to the test substance treatment were observed in the sex ratio, birth index, or viability index on day 4 of postpartum, nor in the external examination or body weight.

The NOAEL for repeated dose toxicity was considered to be 1000 mg/kg bw/day in both sexes under the conditions of this study. The NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day in parental animals and offspring under the conditions of this study.

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:

OECD TG 422, GLP

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD TG 422

A study according OECD TG 422 was performed. The substance was administered repeatedly by oral gavage at dose levels of 0 (control group), 110, 330, and 1000 mg/kg bw/day from 14 days before mating through mating for 42 days in males and satellite females, and 14 days before mating through gestation and parturition until Day 4 of lactation in females to determine the repeated dose toxicity and reproductive and developmental toxicity, as well as reversibility of the changes observed. Each test group consisted of 12 males (including 5 males for the control and 1000 mg/kg bw/day groups in the recovery test) and 12 females (5 females each were added for the control and 1000 mg/kg bw/day groups in the recovery test). The control group received the vehicle (0.5 w/v% methylcellulose aqueous solution) only.

No death occurred during the study. No changes related to the test substance treatment were observed in the clinical signs, functional observational battery, body weight, food consumption, hematological examination, blood chemical examination, necropsy, organ weight or histopathological examination in males or females or urinalysis in males.

In the parental animals, no changes related to the test substance treatment were observed in the estrous cycle, copulation index, fertility index, gestation index, gestation length, number of corpora lutea or implantation sites, implantation index, delivery index, nor in the parturition or maternal behavior. In the offspring, no changes related to the test substance treatment were observed in the sex ratio, birth index, or viability index on day 4 of postpartum, nor in the external examination or body weight.

The NOAEL for repeated dose toxicity was considered to be 1000 mg/kg bw/day in both sexes under the conditions of this study. The NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day in parental animals and offspring under the conditions of this study.

Effects on developmental toxicity

Description of key information

Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with the substance at the dose levels of 1000, 300 and 100 mg/kg bw/day caused no mortality, clinical signs and necropsy or histopathology changes of the maternal animals. The treatment with the test item had no impact the food consumption and body weight/corrected body weight/gain. Thyroid-related parameters (FT3, FT4 TSH hormone levels and histopathology) were not affected by treatment. Statistically significantly lower thyroid weight in the 1000 mg/kg bw/day dose group was not attributed to the treatment based on historical control data and lack of changes in the hormone levels or absence of histopathology lesions.  The test item caused no differences in the intrauterine parameters. No test item related differences in the incidence of malformations and variations as well as growth retardation or placental changes over all dosing groups were observed.

Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity):       1000 mg/kg bw/day

NOAEL (developmental toxicity

including teratogenicity):           1000 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
REACH Annex IX Column 2 Section 8.7.2 states as follows: “The study shall be initially performed on one species. A decision on the need to perform a study at this tonnage level or the next on a second species should be based on the outcome of the first test and all other relevant available data.”
Based on the outcome of the first test with rats and all other relevant available data, a study on a second species, i. e. non-rodent species, is considered not necessary, as discussed in more detail below. There is no evidence of substance-related effects with regard to reproductive toxicity as demonstrated in the available Screening on Reproduction/Developmental Toxicity Study (OECD 422), the Prenatal Developmental Toxicity study (OECD 414), as well as the Repeated Dose 90-Day Toxicity study (OECD 408).
The substance was examined for its possible prenatal developmental toxicity in a study according OECD TG 414/GLP in rats. Groups of 26 sperm-positive female Han: of Wistar origin rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle 1% aqueous methylcellulose. The treatment volume was 5 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The substance in 1% aqueous methylcellulose was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 °C) at the concentrations of 20 and 250 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 106 and 109 % of nominal concentrations at both analytical occasions confirming proper dosing. In total, on gestation day 20 there were 23 evaluated litters in the control, 300 and 1000 mg/kg bw/day groups, respectively, as well as 22 in the 100 mg/kg bw/day group. One non pregnant female was moribund in the course of the study in the 100 mg/kg bw/day group after clinical signs such as hunched back, piloerection and reduced activity, as well as dyspnoea and lying on the side due to an unknown reason. Necropsy revealed no macroscopic changes in the organs. The other animals had no clinical signs and there were no treatment related pathological changes found. The food consumption and body weight of the animals was similar in each group. The mean of thyroid weight was statistically significantly lower in the 1000 mg/kg bw/day group (p<0.05), however the value was above the historical control level. Moreover, the treatment did not result in histological changes of the thyroid gland in any of the dose groups and the measured hormone levels were similar in the groups. There were no treatment related differences observed in the pre- and post implantation loss. The number of implantations and viable fetuses as well as their sex distribution was similar in the groups. The fetal weight was equal in the groups. There were no significant differences in the ano-genital distance and placental weight parameters. There were no significant differences in the incidence of body weight retarded (smaller) fetuses. At fetal examinations, there were no significant differences in the incidence of malformations and variations during external, visceral and skeletal examinations at any dose level. Based on the observations the NOAEL for maternal toxicity was 1000 mg/kg bw/day and the NOAEL for developmental toxicity including teratogenicity was 1000 mg/kg bw/day.
A study according OECD TG 422 is available. The substance was administered repeatedly by oral gavage at dose levels of 0 (control group), 110, 330, and 1000 mg/kg bw/day from 14 days before mating through mating for 42 days in males and satellite females, and 14 days before mating through gestation and parturition until Day 4 of lactation in females to determine the repeated dose toxicity and reproductive and developmental toxicity, as well as reversibility of the changes observed. Each test group consisted of 12 males (including 5 males for the control and 1000 mg/kg bw/day groups in the recovery test) and 12 females (5 females each were added for the control and 1000 mg/kg bw/day groups in the recovery test). The control group received the vehicle (0.5 w/v% methylcellulose aqueous solution) only. No death occurred during the study. No changes related to the test substance treatment were observed in the clinical signs, functional observational battery, body weight, food consumption, hematological examination, blood chemical examination, necropsy, organ weight or histopathological examination in males or females or urinalysis in males. In the parental animals, no changes related to the test substance treatment were observed in the estrous cycle, copulation index, fertility index, gestation index, gestation length, number of corpora lutea or implantation sites, implantation index, delivery index, nor in the parturition or maternal behavior. In the offspring, no changes related to the test substance treatment were observed in the sex ratio, birth index, or viability index on day 4 of postpartum, nor in the external examination or body weight. The NOAEL for repeated dose toxicity was considered to be 1000 mg/kg bw/day in both sexes under the conditions of this study. The NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day in parental animals and offspring under the conditions of this study.
Effects on reproductive organs were also evaluated in an oral 90-day repeated dose toxicity study. The substance was administered orally (by gavage) to Wistar rats once a day at doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day for 90 days. A test item related influence on the estrous cycle was not detected (100, 300 and 1000 mg/kg bw/day). Histological examination did not reveal test item related lesions in male and female reproductive organs or tissues of animals administered with 1000 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period. Based on these observations the NOAEL was determined as 1000 mg/kg bw/day for male and female Han: WIST rats.
According to ECHA Guidance on Information Requirements and Chemical Safety Assessment R7a (2017) a test on the second species might be necessary if the available data triggers for prenatal developmental toxicity. “For example (…) if developmental effects that are not sufficient to meet classification criteria to Category 1B reproductive toxicant (but maybe sufficient to Category 2 reproductive toxicant) were observed in the prenatal developmental toxicity study with the first species. (…) However, if there are no triggers and no indication of prenatal developmental toxicity in the first prenatal developmental toxicity study, no study on a second species is necessary at REACH Annex IX level”.
Based on the data available, there are no such concerns as outlined above.
In conclusion, based on the available study results, adverse effects were observed neither on fertility parameters in male and female animals nor on development of the offspring. Therefore, according to Column 2, Section 8.7.2, Annex IX of REACH Regulation a study on developmental toxicity and teratogenicity in a second species is not considered scientifically justified, also in regards to animal welfare reasons.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Species:

rabbit

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2021-03-02 to 2021-07-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

DRF study, only limited parameters evaluated

GLP compliance:

no

Remarks:

principles of GLP were followed and all data were recorded and retained

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han: WIST

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90., 1103 Budapest, Hungary
- Age at study initiation: ca. 9 weeks (females), ca.23 weeks (males for mating)
- Weight at study initiation: 196-231 g
- Fasting period before study: no
- Housing: Type II polypropylene/polycarbonate
Before and during mating: 1-2 females per cage
During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats
and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water
- Acclimation period: 7 days for females

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 23.5
- Humidity (%): 35 - 53
- Air changes (per hr): above 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: Methylcellulose (1 %)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (Methylcellulose (1 %)) at concentrations of 20 mg/mL, 60 mg/mL and 200 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility beforehand for not longer than 2 days and stored in a refrigerator (5±3 °C) until use. A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment was based on the most recent individual body weight of the animals.
VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water. Therefore, Methylcellulose (1 %) was used for preparing formulations appropriate for oral administration. Methylcellulose (1 %) is a suitable vehicle for the test item

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: one male: one to two females
- Length of cohabitation: 2 - 4 hours
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

The sperm positive females were treated from gestational day 5 to 19.

Frequency of treatment:

daily

Duration of test:

15 days

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting with 100, 300 and 1000 mg/kg bw/day is based on findings obtained in a previous repeated dose toxicity study according to OECD 422 withthe substance in the rat (see Section 7.5.1).Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day, from gestation day 0 to 20, on treatment days after treatment at approximately the same time, considering the peak period of anticipated effects after dosing.
Individual observation included the check of behavior and general condition. An inspection for signs of morbidity and mortality was made twice daily from gestation day 0 to 19 (at the same time as the clinical observation and at the end of work period) and once on gestation day 20.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the male animals was not measured. The body weight of the female rats was measured at least once in the premating 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) and evaluated. Corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- 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).

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
The abdomen was opened, the uterus with cervix and the left ovary removed
and weighed. The right ovary was placed into a Petri dish after removal. After
removing the uterus gross pathology of dams' viscera was performed. The number of corpora lutea in each ovary and implantation sites in each uterine horn, live fetuses, early and late embryonic death and fetal death were counted. Animals, in which unambiguous implantation sites, but not fetuses have been found, were considered as pregnant. Uteri that appeared empty were stained with 2% KOH solution. Fetuses were removed from the opened uterus. The fetuses were sunk in a Petridish filled up with water. Spontaneous movement of fetuses was observed as a viability assessment. Euthanasia of the fetuses was performed by hypothermia. The fetuses were washed with tap water and randomly laid on a filter paper with written ordinal numbering. Bleeding from the umbilical cord after it was cut wasobserved also as a sign of viability. Each live fetus was weighed individually (accuracy 0.01 g), and subjected to external examination. External fetal sex was determined by gross pathological examination (ano-genital distance). Placentas were examined externally and not weighed. All abnormalities found during the fetal examinations were recorded. Visceral and skeletal examinations were not part of this study. The fetuses were discarded after external examination.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

Not performed.

Fetal examinations:

- External examinations: No
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

Because of the low number of litters in the DRF study, statistical analysis was not performed.

Indices:

- Pre-implantation loss
- Post-implantation loss
- Sex distribution

Historical control data:

No

Clinical signs:

no effects observed

Description (incidence and severity):

None of the animals died and there were no clinical signs observed.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in the mean body weight parameters among the groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences in the mean food consumption among the groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Reddish mottled lungs were observed without a dose response in the control,
100 and 300 mg/kg bw/day groups and none in the 1000 mg/kg bw/day dose
group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no dose response indicated in the mean percent of pre- and postimplantation loss.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The fetal weight was similar in the groups.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution was equal.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no malformations found in the dose groups. The values for body weight retardation (evaluated as a variation) criteria were 2.53 and 2.36 g for the male and female fetuses respectively. The incidence of body weight retarded fetuses/litters was 0/0, 4/3, 6/1 and 6/1 in the control, 100, 300 and 1000 mg/kg bw/day dose groups. Based on lack of clear dose response, a test item effect was not proved.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Key result

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

not specified

Conclusions:

The dose range finding study was performed to determine the dose levels for a OECD TG 414 study in rats.

Executive summary:

In the present DRF study for a subsequent OECD TG 414 study, oral treatment of pregnant Han: WIST rats with the dose level of 1000, 300 and 100 mg/kg bw/day  caused neither death nor clinical signs and macroscopical alterations in the organs.
The test item had no impact on the food consumption and the body weight
parameters. Pre- and post-implantation loss, and number of viable fetuses as well as their sex distribution was not affected by the test item.The test item did not induce any malformations. External appearance of placentas was not affected by the test item. The occurrence of body weight retarded (smaller fetuses) in the 100, 300 and 1000 mg/kg bw/day groups with a moderate incidence was not proved to be due to the test item, based on lack of clear dose response.
Based on these preliminary observations, the doses for the main Prenatal
Developmental Toxicity Study were selected as follows:
Group 1 Vehicle control
Group 2 100 mg/kg bw/day
Group 3 300 mg/kg bw/day
Group 4 1000 mg/kg bw/day

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-05-11 to 2021-06-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han: WIST

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90., 1103 Budapest, Hungary
- Age at study initiation: ca. 10 weeks (females), 33-34 weeks (males for mating)
- Weight at study initiation: The group averages of the body weight of the females were as similar as possible on the first day of gestation (182-232 g)
- Fasting period before study: no
- Housing:
Before mating: 1-2 females per cage, 2 males per cage
During mating: 1 male and 1-2 females / cage
During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: ad libitum, ssniff® SM Rat/Mouse-Zucht+Haltung" Autoclavable
complete feed for rats and mice – breeding " produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water
- Acclimation period: 6 days for females

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 - 24.6
- Humidity (%): 44 - 61
- Air changes (per hr): above 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: Methylcellulose (1 %)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (Methylcellulose (1 %)) at concentrations of 20 mg/mL, 60 mg/mL and 200 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility beforehand for not longer than 2 days and stored in a refrigerator (5±3 °C) until use. A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment was based on the most recent individual body weight of the animals.
VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water. Therefore, Methylcellulose (1 %) was used for preparing formulations appropriate for oral administration. Methylcellulose (1 %) is a suitable vehicle for the test item

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean concentration of formulation samples was in the range of 106-109 % compared to the corresponding nominal concentration (within the acceptance criteria). The formulations were considered as homogenous since the difference of the measured concentrations of parallel samples was not more than 15% (1.2-3.3%).

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: one male: one to two females
- Length of cohabitation: 2 - 4 hours
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

The sperm positive females were treated from gestational day 5 to 19.

Frequency of treatment:

daily

Duration of test:

15 days

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

26

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the dose range finding study, where oral treatment of pregnant Han: WIST rats with the dose levels of 1000, 300 and 100 mg/kg bw/day of NKK-1304 revealed neither maternal nor fetal toxicity.
- Fasting period before blood sampling for (rat) dam thyroid hormones: no
- Time of day for (rat) dam blood sampling: in the morning on the day of necropsy within a short timeframe (not exceeding two hours).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for behavior and general condition; twice daily for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: 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). The corrected body weight was calculated for the 20th day of pregnancy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- 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).

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Thyroid glands together with the parathyroid glands of all sperm positive females were weighed and recorded with a precision of 0.001 g after fixation in 4 % (v/v) buffered formaldehyde solution.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected: least 1.0 mL blood
-Other: Serum samples were divided into two aliquots (one for FT3 and FT4 as well as one for TSH level determination) and stored between -15 to -30 °C

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: The anogenital distance (AGD) of each fetus was determined (accuracy 1 mm).

Statistics:

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there is a positive result, the inter-group comparisons are performed using the Mann-Whitney U-test. Chi2 test was performed if feasible.

Indices:

- Pre-implantation loss
- Post-implantation loss
- Sex distribution
- External abnormalities/litter
- Visceral abnormalities/litter
- Skeletal abnormalities/litter

Historical control data:

Yes, provided by test laboratory.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One female in the 100 mg/kg bw/day group was moribund and euthanized in the course of the study on g.d. 11. This female had clinical signs from g.d. 9 such as hunched back, piloerection and reduced activity as well it was lying on the side and had dyspoea on g.d. 11. Considering that none of the other animals died, was moribund or had clinical signs in the experimental groups, this was judged as unrelated to the test item.

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight/gain (including corrected body weight/gain) values were similar in the groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no reductions in the food consumption observed. In the treatment period, there were no statistically significant differences indicated. Before the treatment period, from g.d. 3 to 5, a slightly but statistically significantly higher (p<0.05) mean food consumption was seen in the 300 and 1000 mg/kg bw/day which was considered as biologically not relevant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no statistically significant differences observed in the measured TSH, FT3 and FT4 hormone levels in the control, 100, 300 and 1000 mg/kg bw/day groups.

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean thyroid weight (including relative values) was statistically significantly lower (0.0265 g, p<0.05) in the 1000 mg/kg bw/day dose group. However, according to the historical control database, the value was above the historical control range (0.0219-0.0224 g). Considering this, and that there were no differences in the thyroid and TSH hormone levels indicated, as well as there were no lesions found in the thyroids at histopathology, it was not considered as treatment-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dilated renal pelvis occurred with low incidences and without a dose response in one control female. Therefore, these were considered as unrelated to the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

According to the expert’s evaluation, there were no test item- related lesions observed upon histological examinations of the thyroid tissue in all treatment groups compared to the control. There were no other organs examined since no lesions were detected in the treatment groups during necropsy.

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

There was no increase indicated in the mean of pre- and post-implantation loss. According to the Chi square evaluation, the number of pre-implantation loss was statistically significantly lower in the 100 mg/kg bw/day dose group and statistically significantly higher in the 300 mg/kg bw/day dose group without a dose response, hence not considered as treatment related.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no differences in the body weight of the fetuses.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

The number of male fetuses was statistically significantly higher, and the number of female fetuses statistically significantly lower in the 300 mg/kg bw/day group versus control. This was considered as not relevant, since the sex distribution was near to 50-50%.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of litters with malformed fetuses was 2/23, 1/22, 1/23 and 1/23 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively. There were no statistically significant differences in the incidence of malformations over all dosing groups.
The number of evaluated fetuses was 240, 241, 238 and 233 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Variations: There were three pale fetuses found in the control and one with neck edema in the 100 mg/kg bw/day group.

Malformations: One fetus had umbilical hernia in the control group.

Growth retardation (small fetuses): There was no significant of dose related difference in the incidence of small fetuses.

Placentas, amniotic fluid: In one litter (in the same as with the three pale fetuses), the amniotic fluid was brownish and two of the placentas had clotted bloody margin in the control group.

Considering the low incidences at external examination, and lack of dose response, the findings were not attributed to an effect of the test item.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of examined fetuses was 121, 121, 119 and 115 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

There was no statistically significant increase observed in the skeletal abnormalities (variations and malformations).

Malformations:
There was one malformed fetus found in each group including control.

One fetus had vertebra agenesis (5 lumbar vertebrae) and a misshapen LI vertebra in the 300 mg/kg bw/day group. This was found in the same litter with the fetus with hydronephrosis.

One fetus had bent scapula in the control group. One fetus had also bent scapula and in addition bent radius, ulna, femur, tibia and fibula in the 100 mg/kg bw/day dose group.

One fetus had hyperflexion of the left forelimb in the 1000 mg/kg bw/day group.

Considering the low incidences or lack of dose response, the skeletal findings were not proved as treatment related.

Variations:
Delayed ossification of skull bones, sternebra, vertebrae, pelvic girdle, metacarpal/metatarsal, as well as slightly open palatine, misaligned or bipartite ossification of sternebra, wavy ribs or interrupted 14th rib, bipartite or dumb-bell shaped ossification of vertebral centra as well as slightly bent scapula were observed without statistically significant differences among the dose groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of examined fetuses was 119, 121, 119 and 118 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. There were no statistically significant increases in the incidence of visceral variations and malformations.

Variations:
Slightly dilated IIIrd ventricle was recorded in one fetus in the 300 mg/kg bw/day group.

Bilateral hydroureter was found in two fetuses in the 100 mg/kg bw/day group. Hydroureter accompanied with dilated renal pelvis was observed in one fetus in the control and one in the 100 mg/kg bw/day group.

Malformations
Marked hydroureter and in addition hydronephrosis was observed in one fetus in the 300 mg/kg bw/day group.

Considering the low incidences or lack of dose response, the findings at visceral examination were not judged as treatment related.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity including teratogenicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Based on the observations the NOAELs from a OECD TG 414 study in rats were determined as follows:
NOAEL (maternal toxicity): 1000 mg/kg bw/day
NOAEL (developmental toxicity including teratogenicity):1000 mg/kg bw/day

Executive summary:

The substance was examined for its possible prenatal developmental toxicity in a study according OECD TG 414/GLP. Groups of 26 sperm-positive female Han: of Wistar origin rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle 1% aqueous methylcellulose. The treatment volume was 5 mL/kg bw.

A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The substance in 1% aqueous methylcellulose was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 °C) at the concentrations of 20 and 250 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 106 and 109 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. External fetal sex was determined by gross pathological examination and compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue alterations). The anogenital distance was measured. In addition, indication of incomplete testicular descent / cryptorchidism was noted in male fetuses.

About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

Results

In total, on gestation day 20 there were 23 evaluated litters in the control, 300 and 1000 mg/kg bw/day groups, respectively, as well as 22 in the 100 mg/kg bw/day group.

One non pregnant female was moribund in the course of the study in the 100 mg/kg bw/day group after clinical signs such as hunched back, piloerection and reduced activity, as well as dyspnoea and lying on the side due to an unknown reason. Necropsy revealed no macroscopic changes in the organs.

The other animals had no clinical signs and there were no treatment related pathological changes found.

The food consumption and body weight of the animals was similar in each group.

The mean of thyroid weight was statistically significantly lower in the 1000 mg/kg bw/day group (p<0.05), however the value was above the historical control level. Moreover, the treatment did not result in histological changes of the thyroid gland in any of the dose groups and the measured hormone levels were similar in the groups.

There were no treatment related differences observed in the pre- and post implantation loss. The number of implantations and viable fetuses as well as their sex distribution was similar in the groups.

The fetal weight was equal in the groups. There were no significant differences in the ano-genital distance and placental weight parameters. There were no significant differences in the incidence of body weight retarded (smaller) fetuses.

At fetal examinations, there were no significant differences in the incidence of malformations and variations during external, visceral and skeletal examinations at any dose level.

Conclusion

Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with the substance at the dose levels of 1000, 300 and 100 mg/kg bw/day caused no mortality, clinical signs and necropsy or histopathology changes of the maternal animals. The treatment with the test item had no impact the food consumption and body weight/corrected body weight/gain. Thyroid-related parameters (FT3, FT4 TSH hormone levels and histopathology) were not affected by treatment. Statistically significantly lower thyroid weight in the 1000 mg/kg bw/day dose group was not attributed to the treatment based on historical control data and lack of changes in the hormone levels or absence of histopathology lesions.  The test item caused no differences in the intrauterine parameters. No test item related differences in the incidence of malformations and variations as well as growth retardation or placental changes over all dosing groups were observed.

Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity):       1000 mg/kg bw/day

NOAEL (developmental toxicity

including teratogenicity):           1000 mg/kg bw/day

Effect on developmental toxicity: 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:

OECD TG 414, GLP

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

DRF for OECD TG 414 (rat)

The substance was examined for its possible prenatal developmental toxicity. Groups of seven to eight spermium-positive female Han: WIST rats were treated by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of seven spermium positive females was included and the animals were given the vehicle 1% Methylcellulose. The treatment volume was 5 mL/kg bw. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when spermium was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day (g.d.) 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were examined externally.

Results: On gestation day 20, a total of 7, 7, 6 and 7 litters in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively, were evaluated.

There occurred no mortality during the study period.

None of the animals showed any clinical signs.

There were no significant differences among the groups.

There was no test item related effect in the number of implantations or percent pre- and post-implantation loss. There was no test item effect indicated in the number of viable fetuses and in their sex distribution. 

The weight of fetuses was similar in the groups.

There were no malformations found at external examinations. Variations in form of growth retardations occurred only in the test item treated groups, however without a dose response, hence likely incidentally. There were no placental changes observed.

In conclusion, oral treatment of pregnant Han: WIST rats with the dose level of 1000, 300 and 100 mg/kg bw/day caused neither death nor clinical signs and macroscopical alterations in the organs. The test item had no impact on the food consumption and the body weight parameters. Pre- and post-implantation loss, and number of viable fetuses as well as their sex distribution was not affected by the test item. The test item did not induce any malformations. External appearance of placentas was not affected by the test item. The occurrence of body weight retarded (smaller fetuses) in the 100, 300 and 1000 mg/kg bw/day groups with a moderate incidence was not proved to be due to the test item, based on lack of clear dose response.

Based on these preliminary observations, the doses for the main Prenatal Developmental Toxicity Study were selected as follows:

Group 1 Vehicle control

Group 2 100 mg/kg bw/day

Group 3 300 mg/kg bw/day

Group 4 1000 mg/kg bw/day

OECD TG 414 (rat)

The substance was examined for its possible prenatal developmental toxicity in a study according OECD TG 414/GLP. Groups of 26 sperm-positive female Han: of Wistar origin rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day, respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle 1% aqueous methylcellulose. The treatment volume was 5 mL/kg bw.

A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The substance in 1% aqueous methylcellulose was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3°C) at the concentrations of 20 and 250 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 106 and 109 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. External fetal sex was determined by gross pathological examination and compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue alterations). The anogenital distance was measured. In addition, indication of incomplete testicular descent / cryptorchidism was noted in male fetuses.

About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

Results

In total, on gestation day 20 there were 23 evaluated litters in the control, 300 and 1000 mg/kg bw/day groups, respectively, as well as 22 in the 100 mg/kg bw/day group.

One non pregnant female was moribund in the course of the study in the 100 mg/kg bw/day group after clinical signs such as hunched back, piloerection and reduced activity, as well as dyspnoea and lying on the side due to an unknown reason. Necropsy revealed no macroscopic changes in the organs.

The other animals had no clinical signs and there were no treatment related pathological changes found.

The food consumption and body weight of the animals was similar in each group.

The mean of thyroid weight was statistically significantly lower in the 1000 mg/kg bw/day group (p<0.05), however the value was above the historical control level. Moreover, the treatment did not result in histological changes of the thyroid gland in any of the dose groups and the measured hormone levels were similar in the groups.

There were no treatment related differences observed in the pre- and post implantation loss. The number of implantations and viable fetuses as well as their sex distribution was similar in the groups.

The fetal weight was equal in the groups. There were no significant differences in the ano-genital distance and placental weight parameters. There were no significant differences in the incidence of body weight retarded (smaller) fetuses.

At fetal examinations, there were no significant differences in the incidence of malformations and variations during external, visceral and skeletal examinations at any dose level.

Conclusion

Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with the substance at the dose levels of 1000, 300 and 100 mg/kg bw/day caused no mortality, clinical signs and necropsy or histopathology changes of the maternal animals. The treatment with the test item had no impact on the food consumption and body weight/corrected body weight/gain. Thyroid-related parameters (FT3, FT4 TSH hormone levels and histopathology) were not affected by treatment. Statistically significantly lower thyroid weight in the 1000 mg/kg bw/day dose group was not attributed to the treatment based on historical control data and lack of changes in the hormone levels or absence of histopathology lesions. The test item caused no differences in the intrauterine parameters. No test item related differences in the incidence of malformations and variations as well as growth retardation or placental changes over all dosing groups were observed.

Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity):       1000 mg/kg bw/day

NOAEL (developmental toxicity

including teratogenicity):           1000 mg/kg bw/day

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008
The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for reproductive and developmental toxicity under Regulation (EC) No 1272/2008, as amended for the seventeenth time in Regulation (EU) 2021/849.