Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The reproductive toxicity screening study is waived on the basis that a pre-natal developmental toxicity study is available. Furthermore the EOGRTS study is waived on the basis that no adverse effects on reproductive organs or tissues were seen in a reliable 90 day sub-chronic study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
Reproductive effects observed:
not specified
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
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Three groups of 20 female rats received doses of 100, 300 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination. Clinical observations, body weight, gravid uterine weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. It was concluded from this study that the dosage of 1000 mg/kg bw/day was the maternal no-observed-adverse-effect-level (NOAEL) and 300 mg/kg bw/day was the no-observed-adverse- effect-level (NOAEL) for embryo-fetal survival, growth and development.

The substance was administered once daily by oral gavage of control article (vehicle) or 100, 300, or 1000 mg/kg bw/day to pregnant mice (24/dose). The physical health of the maternal animal was not impaired following dose administration up to the limit dose of 1000 mg/kg bw/day. However, maternal exposure of 1000 mg/kg bw/day resulted in smaller foetuses compared with controls. The most noticeable foetal findings included a high incidence of exencephaly, together with malformations of the skull and brain, and a high incidence of unossified or incomplete ossification of the skeleton. Based on these findings, a dose level of 1000 mg/kg bw/day was considered excessive for embryo-foetal development. A no observed adverse effect level (NOAEL) for prenatal development is, therefore, established as 300 mg/kg bw/day in the absence of maternal toxicity.

Link to relevant study records

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 June to 25 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): rd 15134
- Physical state: liquid
- Analytical purity: 95.8%
- Lot/batch No.: 1023R12118
- Expiration date of the lot/batch: 01 May 2018
- Storage condition of test material: room temperature
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: accredited commercial supplier
- Age at study initiation: approx 70 days on day 0 of gestation
- Weight at study initiation: 220-290g
- Fasting period before study:
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used for toxicity phase males throughout the study and for embryo-fetal phase females during the acclimatisation and gestation periods.
Grid bottomed cages were used during pairing.

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Five days before commencement of pairing or treatment (as appropriate)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23ºC
- Humidity (%): 40-70%
- Air changes (per hr): dark Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours

IN-LIFE DATES: From: 15 June 2015 To: 20 August 2015
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 20, 60 or 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): not stated
- Purity: not stated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 10 and 200 mg/mL were analysed to assess the stability and homogeneity of the test substance in the liquid matrix. Formulations from 10 mg/mL to 200 mg/mL were stable for up to 1 day at ambient temperature (nominally +21ºC) and 16 days refrigerated (nominally +4ºC).
Samples of each formulation prepared for administration on Day 1 and on the last day of treatment for Phase II of the embryo fetal phase II were analysed for achieved concentration of the test substance.
The method of analysis and results are presented in the report
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Length of cohabitation: until positive evidence of mating
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy:Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm referred to as day 0 of pregnancy
Duration of treatment / exposure:
Males were treated once daily, at approximately the same time each day for 14 days.
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Frequency of treatment:
Daily at approximately the same time each day
Duration of test:
Females until day 20 of gestation. Males 14 days
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:
6 females per group for phase I and 14 females per group for Phase II. 3 males for the 14-day toxicity phase
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): animals allocated randomly upon arrival
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal to monitor general health at the following time point:
Embryo-fetal phase Days 0, 5, 12, 18 and 20 after mating.
14 day toxicity phase Day 1 (pre-dose) then twice weekly.

BODY WEIGHT: Yes
- Time schedule for examinations:The weight of each animal was recorded as follows:
Embryo-fetal phase: Days 0, 3, 6-20 after mating.
14 day toxicity phase: Day -3, immediately before treatment commenced, twice weekly and at termination.

POST-MORTEM EXAMINATIONS: Yes
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

- Sacrifice on gestation day: Embryo-fetal phase Animals were killed on Day 20 after mating. 14 day toxicity phase Day 15 of study, following completion of 14 day of treatment.
- Organs examined: As detailed above
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
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No
Statistics:
For the embryo-fetal phase; the following data types were analysed at each timepoint separately:
Body weight, using absolute values and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight
The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons the F1 approximate test was applied.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre-treatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made. For all other comparisons the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.
For litter size and survival indices and fetal, placental and litter weight and gravid uterine weight data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See Tables 2 and 3 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Number of abortions:
no effects observed
Description (incidence and severity):
See Table 6 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data. Rats do not abort foetuses but resorb them
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Early and late implantation losses were not statistically significant compared to control animal group
See Table 6 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Early and late resorption values not statistically significant compared to control animal group
See Table 6 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Early or late resorptions:
no effects observed
Description (incidence and severity):
See Table 6 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead foetuses were noted on termination at day 20 of gestation in either the control group or any of the treatment groups.

See Table 6 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No rat gave birth prior to termination at day 20.

See Table 6 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): It is stated in the results section 3.3.1 (Reproductive assessment) of the report that -

"All females were pregnant. The following assessment is based on the 20, 20, 20 and 20 females with live young at termination on Day 20 of gestation in the Control group, and at 100, 300 and 1000 mg/kg/day, respectively. "

From this information it is also apparant that there were no early deliveries or still births.

This shown in Table 6 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
It is stated in the results section 3.3.1 (Reproductive assessment) of the report that -

"All females were pregnant. The following assessment is based on the 20, 20, 20 and 20 females with live young at termination on Day 20 of gestation in the Control group, and at 100, 300 and 1000 mg/kg/day, respectively. "

See Table 6 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Details on maternal toxic effects:
Maternal toxic effects:no effects
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male, female and overall fetal weights were statistically significantly lower for females receiving 1000 mg/kg/day, when compared with Controls.
At 1000 mg/kg/day, mean fetal weight was lower than in Controls (11% lower) reflecting the fact that the mean fetal weight in the majority of litters in this group was less than 3.40 grams whereas in the Control group most litters had a mean fetal weight above 3.70 grams. This difference was considered to reflect an effect of treatment on fetal growth and not to be due to the slightly higher mean litter size in the 1000 mg/kg/day group.
See Table 7 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Among females allocated to the 100, 300 or 1000 mg/kg/day groups, the mean numbers of corpora lutea were slightly higher than in Controls; as the number of corpora lutea reflect the number of eggs shed and treatment did not start until Day 6 after mating these differences were due to chance as they were established before the start of treatment. Principally as a result of these differences which were due to chance, the mean numbers of implantations and live fetuses in all treated groups were slightly higher than in Controls and the difference for live fetuses in the 1000 mg/kg/day group attained statistical significance.
See Table 6 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The sex ratio was 45.6% males for the 300 mg/kg/day group compared to 57.1% for the control group and 52.7% and 52.8% for the 100 mg/kg/day and 1000 mg/kg/day, respectively.
See Table 6 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Gravid uterine weights, embryo-fetal survival and litter and placental weights were unaffected by treatment up to 1000 mg/kg/day. Intergroup differences in gravid uterine weight reflected intergroup differences in litter size which were due to chance.
See Table 7 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
External malformations:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day there were two fetuses in two litters with the major abnormality short/threadlike tail.
See Table 8 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There was evidence of a slight disturbance of fetal development at 1000 mg/kg/day characterized by the increased incidence of large nasofrontal suture; thoracic vertebral abnormality; short supernumerary cervical rib and 14th rib; delayed/incomplete ossification/unossified cranial centres, cervical, thoracic and sacral caudal vertebrae, sternebrae, pelvic bones, metacarpals/metatarsals and a decrease in ossified cervical vertebral centra; variation in lens shape; small/absent lobe of thyroid; partially undescended lobe of thymus; small/absent renal papilla and dilated ureter when compared to concurrent control and the incidences were outside of the Historical Control Data with the exception of delayed/incomplete ossification/unossified cervical vertebrae.
Although not adverse in isolation, many of these findings would usually be associated with a significant decrease in body weight signifying a delay in fetal development. However, this was not the case on this study with some abnormalities present in fetuses with weights at the upper end of the Control range. There was a very high incidence (107 fetuses in 20 litters) of large nasofrontal suture (an abnormality not seen in the Control group or in the Control groups on the 11 studies included in the HCD) and high incidences of delayed/incomplete ossification/unossified cranial centres, sacrocaudal vertebrae, sternebrae other than 5th/6th, pelvic bones, meta tarsals/carpals compared with the HCD range. The wide range of abnormalities involved, the occurrence in fetuses with a high fetal weight and the high incidences indicate a treatment related disturbance of development which is potentially adverse.
Sixteen fetuses (7 litters) at 300 mg/kg/day, and 5 fetuses (3 litters) at 100 mg/kg/day, had a large nasofrontal suture, and in view of the very high incidence of this unusual abnormality at 1000 mg/kg/day, these finding were considered to be related to treatment at doses which did not elicit any maternal toxicity. In isolation, this finding was not considered to represent an adverse effect on fetal development.
See Tables 8-10 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Placental and litter weights were similar to Controls, and were not affected by the administration of RD 15134.
Male, female and overall fetal weights were statistically significantly lower for females receiving 1000 mg/kg/day, when compared with Controls. This
difference was considered to reflect an effect of treatment on fetal growth and not to be due to the slightly higher mean litter size in the 1000 mg/kg/day group.
There was evidence of a slight disturbance of fetal development at 1000 mg/kg/day characterized by the increased incidence of large nasofrontal suture; thoracic vertebral abnormality; short supernumerary cervical rib and 14th rib; delayed/incomplete ossification/unossified cranial centres, cervical, thoracic and sacral caudal vertebrae, sternebrae, pelvic bones, metacarpals/metatarsals and a decrease in ossified cervical vertebral centra; variation in lens shape; small/absent lobe of thyroid; partially undescended lobe of thymus; small/absent renal papilla and dilated ureter when compared to concurrent control and the incidences were outside of the Historical Control Data with the exception of delayed/incomplete ossification/unossified cervical vertebrae.
There was a very high incidence (107 fetuses in 20 litters) of large nasofrontal suture (an abnormality not seen in the Control group or in the Control groups on the 11 studies included in the HCD) and high incidences of delayed/incomplete ossification/unossified cranial centres, sacrocaudal vertebrae, sternebrae other than 5th/6th, pelvic bones, meta tarsals/carpals compared with the HCD range. The wide range of abnormalities involved, the occurrence in fetuses with a high fetal weight and the high incidences indicate a treatment related disturbance of development which is potentially adverse.
Sixteen fetuses (7 litters) at 300 mg/kg/day, and 5 fetuses (3 litters) at 100 mg/kg/day, had a large nasofrontal suture, and in view of the very high incidence of this unusual abnormality at 1000 mg/kg/day, these finding were considered to be related to treatment at doses which did not elicit any maternal toxicity. In isolation, this finding was not considered to represent an adverse effect on fetal development.
See Tables 8-10 in the document "2016-06-07 RD 15134 Embryo-fetal development study in the rat Tables 2-10", attached in the background data section below, for data
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Abnormalities:
not specified
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
It was concluded from this study that the dosage of 1000 mg/kg/day was the maternal no-observed-adverse-effect-level (NOAEL) and 300 mg/kg/day was the no-observed-adverse-effect-level (NOAEL) for embryo-fetal survival, growth and development.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 June 2020 - 26 June 2020
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:
adopted 25 June 2018
Deviations:
yes
Remarks:
One thyroid for a control animal was reported as missing after macroscopic examination. Fetuses from the litter of one control animal did not have fetal sex recorded prior to evisceration. These deviations did not impact study integrity.
GLP compliance:
yes
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: RL 750/18
- Expiration date of the lot/batch: 24 October 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Sealed container, 15 to 25°C, protected from light
Species:
mouse
Strain:
other: Crl:CD-1(ICR)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Margate, United Kingdom.
- Age at study initiation: 8 to 10 weeks old
- Weight at study initiation: at least 25 g
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied, or Used for Scientific Purposes.
- Diet (e.g. ad libitum): ad libitum access to VRF1 diet
- Water (e.g. ad libitum): ad libitum via water bottles
- Acclimation period: Acclimation was limited by mated status, and an inspection was performed by the Named Animal Care and Welfare Officer (NACWO) before the start of dosing to ensure suitability for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): maintained in the specified ranges of 19 to 25°C
- Humidity (%): maintained in the specified ranges of 40 to 70%
- Air changes (per hr): minimum of 15 air changes/hour
- Photoperiod (hrs dark / hrs light): cycle of 12 hours of light and 12 hours of dark.

IN-LIFE DATES: From: To: 11 June 2020 to 26 June 2020
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations of 10 and 200 mg/mL were found to be stable and homogenous for 1 day at ambient temperature (15 to 25°C) and refrigerated (2 to 8°C) for 16 days.
Details on mating procedure:
Mating (overnight at the supplier’s laboratory) was confirmed by the presence of a vaginal plug in situ, or other evidence of mating, if necessary.
Duration of treatment / exposure:
Animals were dosed from GD 6 through 17. Females were maintained to GD 18, when they were sacrificed, and their uterine contents were examined.
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control (vehicle - corn oil)
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Maternal examinations:
Health monitoring: All animals were observed at the beginning and end (nominal) of the working day for signs of ill health or overt toxicity.
Clinical examination: Each animal was given a detailed physical examination daily from the start of dosing until necropsy. An individual record of the clinical condition of each animal was maintained.
Post-dose observations: Animals were observed daily immediately after dosing, upon return to the home cage.
Body weights: Body weights were recorded on GD 5, 6, 7, 8, 9, 12, 15, 17, and 18.
Food consumption: The amount of food consumed was determined at the following intervals: GD 6 to 7, 7 to 8, 8 to 9, 9 to 12, 12 to 15, 15 to 17, and 17 to 18. Food consumption was calculated as g/animal/day.
Total thyroxine (T4) and total thyroid triiodothyronine (T3) were measured.
Thyroid weight and histopathology.

Ovaries and uterine content:
•Pregnancy status
•Gravid uterus weight
•Terminal body weight (recorded for adjusted gravid uterus weight calculations only and have not been reported)
•Number of corpora lutea
•Number and intrauterine position of implantations subdivided into: Live fetuses, Early intrauterine deaths, Late intrauterine deaths, Dead fetuses
Fetal examinations:
Individual fetal and placental weights and anogenital distances were recorded, and fetuses were examined externally and sexed.
Approximately one half of the fetuses in each litter, selected by systematic sampling, were dissected, and the viscera were examined and fixed in Bouin’s fluid. Heads were then examined by the Wilson sectioning method.
The skeletons of the remaining fetuses were eviscerated and stained with Alizarin Red S, examined for skeletal abnormalities in 50% glycerol and retained in glycerol/propylene glycol.
Historical control data:
Included.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
One control animal was sacrificed in a moribund condition on GD 1, prior to the start of dose administration, due to the severity of clinical observations (squinting eyes; thin body condition; hunched posture; fast respiration; and abnormal color, yellow skin of the uro-genital area). Macroscopically, thin, clear fluid (approximately 1 mL) was observed in the abdominal cavity. The cause of demise was not determined, however, as dose administration did not commence until GD 6, and as this was a control group animal, there was no association with the substance.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No substance-related effect for total thyroxine or total thyroid triiodothyronine levels was noted.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No substance-related thyroid weight or thyroid weight ratio differences were recorded.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No microscopic findings that related to the substance were recorded.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Substance-related effects on fetal weights were evident following administration of 1000 mg/kg bw/day, compared with controls. Mean male (-13%), female -15%), and combined fetal weights (-14%), adjusted for litter size, were significantly lower from litters maternally exposed to 1000 mg/kg bw/day, compared with controls (P < 0.001). Due to the magnitude of the reduction in fetal weight, this finding was considered adverse.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Adverse malformations of the head, skull, and brain were recorded for fetuses following maternal exposure to 1000 mg/kg bw/day. A high incidence of exencephaly for fetuses from litters maternally exposed to 1000 mg/kg bw/day, which attained statistical significance (P < 0.05). The brain malformation of disorganised cranial cavity structures was also observed in litters maternally exposed to 1000 mg/kg bw/day. A high incidence of skull malformations, which consisted of small orbital sockets and open eyes; misshapen frontal, interparietal, parietal, squamosal regions; and absent supraoccipital and presphenoid regions, were also noted for three fetuses from three litters maternally exposed to 1000 mg/kg/day. Three fetuses from litter maternally exposed to 1000 mg/kg bw/day were also recorded with a large and / or wide interfrontal sutural bone.

Malformations :
0 (Control): 3 foetuses had malformations (22%)
100 mg/kg bw/day: 2 foetuses had malformations (14%)
300 mg/kg bw/day: 3 foetuses had malformations (22%)
1000 mg/kg bw/day: 13 foetuses had malformations (99%)


Please see below for details on malformations and historical control data.
Visceral malformations:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Substance-related skeletal variations, which mostly consisted of unossified or incomplete ossification of the skeleton, were noted in the skull, sternebra, cervical central arch, thoracic centrum, and the limbs following maternal exposure to 1000 mg/kg/day. Most of the incidences were higher than the historical control data ranges and not previously observed in the historical control data ranges to cover the period of this study.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: sternum
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
Adverse substance-related effects were observed in foetuses following maternal exposure to 1000 mg/kg bw/day in the absence of maternal toxicity. Based on these findings, a dose level of 1000 mg/kg bw/day was considered excessive for embryo-fetal development. A no observed adverse effect level (NOAEL) for prenatal development is, therefore, established as 300 mg/kg bw/day.
Executive summary:

The substance was administered once daily by oral gavage of control article (vehicle) or 100, 300, or 1000 mg/kg bw/day to pregnant mice (24/dose). The physical health of the maternal animal was not impaired following dose administration up to the limit dose of 1000 mg/kg bw/day. However, maternal exposure of 1000 mg/kg bw/day resulted in smaller foetuses compared with controls. The most noticeable foetal findings included a high incidence of exencephaly, together with malformations of the skull and brain, and a high incidence of unossified or incomplete ossification of the skeleton. Based on these findings, a dose level of 1000 mg/kg bw/day was considered excessive for embryo-foetal development. A no observed adverse effect level (NOAEL) for prenatal development is, therefore, established as 300 mg/kg bw/day in the absence of maternal toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Sufficient to meet the requirements.
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

The substance is classified according to GHS as Category 2 for reproductive toxicity (H361 - suspected of damaging fertility or the unborn child) on the basis of two reliable prenatal developmental toxicity studies on the rat and mouse.

Additional information