Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a study performed according to the OECD 421 guideline, a closely-related substance was administered daily by oral administration (gavage) to male and female CD rats from before mating, through mating and gestation until Day 6 post-partum at dose levels of 100, 300 and 1000 mg/kg bw/day. The NOAEL for the substance was 1000 mg/kg/day (the limit dose) for reproductive performance and offspring growth and survival.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 july 2012 to 04 january 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1995
Deviations:
yes
Remarks:
Necropsies for the males were scheduled for Week 6 of treatment instead of Week 5. The OECD 421 guideline requires a necropsy of the males after a dosing period of at least 4 weeks. Therefore this deviation did not affect the the integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 10 weeks
- Weight at study initiation: 344 to 386 g (males) and 241 to 278 g (females)
- Fasting period before study:no
- Housing: Females: 5 per cage during pre-pairing then individually housed except during pairing,
Males: 5 per cage except during pairing,
- Diet: Ad libitum, standard rodent diet (SDS VRF1 Certified). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated
- Photoperiod (hrs dark / hrs light): 12 h continuous light and 12 h continuous dark/24 h.

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): homogeneous and stable suspensions were obtained with corn oil as a vehicle
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurs or 14 days has elapsed
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage 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:
Homogeneity and stability of the dosage forms: homogeneity of the test substance in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 10 mg/mL and 200 mg/mL. Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 day and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the three samples remained within 7% of the initial time zero value and the coefficient of variation was less than 4%.

Test item concentrations: the test item concentrations in the administered dosage forms analyzed in the first and last weeks of treatment remained within an acceptable range of +10% to -15% when compared to the nominal values.
Duration of treatment / exposure:
In the males:
- 2 weeks before mating,
- during the mating period (up to 2 weeks),
- until sacrifice in week 6,

In the females:
- 2 weeks before mating,
- during the mating period (up to 2 weeks),
- during pregnancy,
- during lactation until day 6 post-partum inclusive
Frequency of treatment:
daily
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 13 weeks for males, 13 weeks for females, approximately.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels used in this study (0, 100, 300 and 1000 mg/kg/day) were selected on the basis of the results of a 14-day repeated- dose preliminary study in the CD rat. In that study, the substance was generally well tolerated at dose levels up to 1000 mg/kg/day with no noteworthy effect of treatment on clinical signs, bodyweight gain, food consumption, macroscopic findings or organ weights.
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
-detailed physical examination was performed weekly. F0 females were also examined on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded during acclimatisation, on the day that treatment commenced (Week 0), weekly thereafter and before necropsy. The weight of each F0 female was also recorded on Days 0, 3, 7, 10, 14, 17 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded on a weekly basis from the first day of treatment until pairing. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.
For each F0 female, the weight of food supplied, that remaining and an estimate of any spilled was also recorded for the periods Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and Days 1-3 and 4-6 of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal.

WATER CONSUMPTION: No


Oestrous cyclicity (parental animals):
For 15 days before pairing, daily vaginal smears were taken from all females. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (all groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- microscopic evaluation of stages of the spermatogenic cycle and testicular interstitial cells (control and high-dose groups)
- detailed qualitative examination was made of the testes, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
The males were sacrificed during week 6 of treatment. The body and selected organs were weighed and a complete macroscopic post-mortem examination was performed. A microscopic examination was performed on the kidneys, liver and epididymis from the males in the control- and high dose groups, testes from all groups and on all macroscopic lesions.

The dams were sacrificed on day 7 of lactation, the body and selected organs were weighed and a complete macroscopic examination was performed.
A microscopic examination was performed on the kidneys, liver and ovaries in the control and high-dose groups and on all macroscopic lesions.

GROSS NECROPSY
On completion of the treatment period all surviving F0 males and females were killed by carbon dioxide asphyxiation.
- males: after the end of the pairing period (during week 6 of treatment ),
- females: on day 7 of lactation.,
- females which had not delivered: on day 25 after mating (after a body weight recording to check for a possible un-noticed delivery),
- mothers with litter dying entirely: as appropriate.

For females, the numbers of implantation sites in each uterine horn was counted. For females failing to produce a viable litter, the number of uterine implantation sites was re-checked after staining with ammonium sulphide (modification of the Salewski staining technique).

Pups were sacrificed by an intraperitoneal injection of sodium pentobarbital:
- surviving pups: on day7 of age.
- Premature deaths if not autolysed or cannibalised.

HISTOPATHOLOGY
A microscopic examination was performed on:
- Liver, kidneys,epididymides and ovaries in animals of the control- and high-dose groups sacrificed at the end of the treatment period and for female that were sacrificed prematurely,
- all macroscopic lesions of all the animals of the low- and intermediate-dose groups sacrificed on completion of the treatment
period,
- Testes of males from control, low-, mid- and high-dose groups,

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

ORGAN WEIGHTS:
The body weight of each animal sacrificed as scheduled was recorded before sacrifice, and liver, kidney,epididymides (L and R), prostate, testes (L and R), seminal vesicles and ovaries (L and R) were weighed (wet) as soon as possible after dissection.The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.
Postmortem examinations (offspring):
SACRIFICE: on day 7 of age

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No
Statistics:
- For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit.
- For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

# The following sequence of statistical tests was used for bodyweight, food consumption, organ weights and litter data:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test was not significant at the 1% level, Williams' test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test was applied. If the H1 approximate test was not significant at the 1% level, Shirley's test was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.
For litter data if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
# For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied.
# Sex ratio were analysed by generalised mixed linear model with binomial errors.
Reproductive indices:
Percentage Mating = 100 * (Number animals mating/animals paired)
Fertility index = 100 * (Number animals achieving pregnancy / Number of animals mated)
Gestation index = 100 * (Number of live litters born / Number pregnant)
Offspring viability indices:
- Post-implantation survival index= 100 * (Number of pups born / Number of uterine implantation sites)
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
- Live birth index = 100 * (Number of live born pups on Day 1/ Number of delivered pups)
- Viability index = 100 * (Number of surviving pups on day 7 / Number of live born pups on Day 1)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no deaths and no particular clinical signs.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was no effect of treatment on mean bodyweight gain and foood consumption of all groups of males receiving the test substance. Overall bodyweight, bodyweight change and food consumption of females receiving the test substance at 100, 300 or 1000 mg/kg/day were lower than that of the Controls during the first week of treatment although a dose response was not apparent. There was no conclusive effect of treatment during the second week of treatment, and weight gain and food consumption throughout Days 1-7 of lactation was similar in all groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Oestrous cycle length was unaffected by treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- There was no effect of treatment on the weight of testes and epididymides.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Statistically significant increased prostate weight for males receiving the test substance at 1000 mg/kg/day was noted. As the prostate was not assessed microscopically, the toxicological significance of these slight increases was uncertain, however in the absence of any effects on fertility or reproductive performance, in isolation these changes were considered not to be adverse.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no findings at macroscopic examination that could be attributed to treatment with the test substance.

HISTOPATHOLOGY (PARENTAL ANIMALS)

- Slight to minimal bilateral tubular atrophy, with partial or complete depletion of germ cells from a few scattered tubules, was respectively seen at
300 mg/kg/day in 1 animal and at 1000 mg/kg/day in 2 animals. In the epididymides minimal amounts of degenerated spermatogenic cells in the ducts were also observed in the two males treated with 1000 mg/kg/day and were considered secondary to the changes seen in the testes.
Although the distribution pattern was suggestive of a treatment effect, these changes were small in incidence or severity and no particular cell- or spermatogenic stage specificity was observed. As reported in literature, tubular atrophy in the testes, even more extensive, can sometimes be seen as background finding in young adult rats (Creasy, 2011). Although an effect of the treatment cannot completely be discounted, the minor changes in the testes were more likely to be considered as background changes.
- A mammary adenocarcinoma was detected in 1 female at 300 mg/kg/day. This tumour was considered incidental based on the occurrence in a single animals and the lack on any proliferative changes in the mammary gland in the other treated animals. Although mammary adenocarcinomas are uncommon in young rats, they can occasionally occur spontaneously even at an early age, as reported in literature.
There was no evidence of test article related changes in the other examined tissues.

OTHER FINDINGS (PARENTAL ANIMALS)
Two females in the Control group and one female dosed at 300 mg/kg/day failed to litter, and were found to have no sign of any implantations pre- or post-staining on Day 25 after mating.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive performance
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
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
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
The assessment of litter responses detailed below is made based on the 8, 10, 9 and 10 litters in each of groups 0, 100, 300 and 1000 mg/kg bw/day, respectively.

VIABILITY (OFFSPRING)
There was no effect of parental treatment with the test substance at 100 or 300 mg/kg bw/day on mean number of implantations, live litter size on Day 1 and offspring survival up to Day 7 of age. At 1000 mg/kg/day, the mean number of implantations showed no adverse effect of parental treatment but the post-implantation survival index (86.1% ) was slightly lower than in Controls (94.4%) and the other study groups. This resulted in marginally low total and live litter sizes. However, none of the differences attained statistical significance, and the post-implantation survival index was within the recent background control range (lowest value 84.9%): therefore no effect of treatment was inferred.

CLINICAL SIGNS (OFFSPRING)
The general appearance and behaviour of the offspring were not affected by maternal treatment.

BODY WEIGHT (OFFSPRING)
Offspring bodyweight on Day 1 of age and subsequent bodyweight gain up to Day 7 of age was similar to control values for the litters of females receiving the test substance at 100, 300 or 1000 mg/kg/day.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of offspring killed at scheduled termination on Day 7 of age revealed no abnormalities.

OTHER FINDINGS (OFFSPRING):
There was no effect of parental treatment with the test substance on sex ratio.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
It was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for the substance was 1000 mg/kg/day (the limit dose) for reproductive performance and offspring growth and survival in the CD rat following oral gavage administration in a standard screening test.
Executive summary:

In a screening study for reproductive / developmental effects performed according to OECD 421 , the objective was to evaluate the potential toxic effects of the test substance following daily oral administration (gavage) to male and female rats from before mating, through mating and gestation until Day 6 post-partum.

Three groups of 10 male and 10 female rats received the test substance by gavage at doses of 100, 300 or 1000 mg/kg/day. Males were treated for 15 days before pairing up to the day before necropsy after a minimum treatment period of five consecutive weeks.  Females were treated daily for a minimum of 15 days before pairing, throughout mating and gestation until Day 6 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. 

During the study, clinical condition, bodyweight, food consumption, organ weights, macroscopic and microscopic pathology investigations were undertaken in all adults.  Oestrous cycles and gestation length were assessed and parturition observations were performed for females.  The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed and macroscopic pathology investigations were undertaken.  

There were no deaths, and the general appearance and behaviour of the animals were not affected by treatment.  There were no adverse effects of treatment on adult bodyweights, bodyweight gains or food consumption and no effects on oestrous cycles, pre-coital interval, mating performance, fertility, conception rate or fertility index; gestation length and gestation index.

At 1000 mg/kg/day, the post-implantation survival index was slightly decreased compared to controls (86.1% versus 94.4% in controls). However, this slightly low value remained in the historical range of the laboratory (84.9% to 100%). As a consequence, the mean live litter size on Day 1 was slightly lower than in controls (14.3 versus 15.1 in controls).

There were no effects of treatment on offspring survival and sex ratio and offspring bodyweight gain up to Day 7 of age. There were no macropathological findings in the adults or offspring. Analysis of the weight of the selected organs at scheduled termination revealed statistically significant increased prostate weight for males receiving 1000 mg/kg/day.  The weight of all other selected organs was unaffected by treatment with Bisamide 80005005 and there were no particular microscopic findings.

It was concluded that the NOAEL for the test substance was 1000 mg/kg/day (the limit dose) for reproductive performance and offspring growth and survival in the CD rat following oral gavage administration in a standard screening test.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is considered to be reliable with a klimisch score of 1.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Screening study for reproductive / developmental effects (OECD 421) / Read-across :

The objective of this study was to evaluate the potential toxic effects of a closely-related substance following daily oral administration (gavage) to male and female rats from before mating, through mating and gestation until Day 6 post-partum. Three groups of 10 male and 10 female rats received the test substance by gavage at doses of 100, 300 or 1000 mg/kg/day. Males were treated for 15 days before pairing up to the day before necropsy after a minimum treatment period of five consecutive weeks.  Females were treated daily for a minimum of 15 days before pairing, throughout mating and gestation until Day 6 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. 

During the study, clinical condition, bodyweight, food consumption, organ weights, macroscopic and microscopic pathology investigations were undertaken in all adults.  Oestrous cycles and gestation length were assessed and parturition observations were performed for females.  The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed and macroscopic pathology investigations were undertaken.  

There were no deaths, and the general appearance and behaviour of the animals were not affected by treatment.  There were no adverse effects of treatment on adult bodyweights, bodyweight gains or food consumption and no effects on oestrous cycles, pre-coital interval, mating performance, fertility, conception rate or fertility index; gestation length and gestation index.

At 1000 mg/kg/day, the post-implantation survival index was slightly decreased compared to controls (86.1% versus 94.4% in controls). However, this slightly low value remained in the historical range of the laboratory (84.9% to 100%). As a consequence, the mean live litter size on Day 1 was slightly lower than in controls (14.3 versus 15.1 in controls).

There were no effects of treatment on offspring survival and sex ratio and offspring bodyweight gain to Day 7 of age. There were no macropathological findings in the adults or offspring. Analysis of the weight of the selected organs at scheduled termination revealed statistically significant increased prostate weight for males receiving 1000 mg/kg/day.  The weight of all other selected organs was unaffected by treatment with the test substance and there were no particular microscopic findings.

It was concluded that the NOAEL for the closely-related substance was 1000 mg/kg/day (the limit dose) for reproductive performance and offspring growth and survival in the CD rat following oral gavage administration in a standard screening test.


Effects on developmental toxicity

Description of key information

In a study performed according to the OECD 414 guideline, the substance was administered daily by oral administration (gavage) to pregnant female rats from Day 6 to Day 20 post-coitum at dose levels of 100, 300 and 1000 mg/kg bw/day. The NOEL for  the substance was 1000 mg/kg bw/day (the limit dose) for both maternal toxicity and embryo-fetal development.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-05-23 to 2018-08-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
yes
Remarks:
: see chapter 'any other information on materials and methods' section 7.8.2 /1
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:



Purity: Considered 100% (UVCB compound)
Correction factor: No correction factor
Expiry date: 24 April 2020
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France.
- Age: at the beginning of the treatment period, the females were 10-11 weeks old
- Mean body weight: at the beginning of the treatment period, the females had a mean body weight of 280 g (range: 230 g to 343 g).
- Fasting period before study: no
- Housing: the animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2)
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of at least 4 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 02 July 2018 to 29 August 2018.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle.
The test item dose formulations were prepared daily and stirred at least 30 minutes before administration. They were delivered to the study room at room temperature.
This preparation process was validated for a range of concentrations covering the lowest and highest concentrations used in this study.
.

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous suspensions were obtained with corn oil as a vehicle
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: High Performance Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS-MS)
Test item concentrations: the test item concentrations in the administered dose formulations analyzed in study Weeks 1, 2 and 3 of the treatment period (including the treatment period of additionnal females), were within an acceptable range of variation (-10.2% to +13.0%) when compared to the nominal values (± 15% required).
Homogeneity: The dose formulations containing the test item and prepared at 10 mg/mL and 200 mg/mL in corn oil were found to be homogeneous at room temperature and protected from light.
Stability: UVCB, dose formulation prepared daily
Details on mating procedure:
The females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as Day 0 p.c.
Duration of treatment / exposure:
Days 6 to 20 post-coitum
Frequency of treatment:
Daily
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:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose selection:
The dose levels were selected on the basis of the results of a 4-week toxicity study performed in CD rats by oral route. In this study, it was concluded that 1000 mg/kg/day represented the No Observed Effect Level (NOEL) for the test substance.
Therefore, 1000 mg/kg/day was selected as the high-dose level. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 300 and 100 mg/kg/day).

- Rationale for animal assignment: computerized stratification procedure.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each animal was checked for mortality and morbidity once a day before and after the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL OBSERVATIONS:Yes
- Time schedule: from arrival, each animal was observed once a day as part of the routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:Yes
- Time schedule: the body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

WATER CONSUMPTION: No

POST-MORTEM MACOSCOPIC EXAMINATION:Yes
- Sacrifice on Day 21 post coitum
- Examined: principal thoracic and abdominal organs.


Ovaries and uterine content:
The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
The weight of the gravid uterus was recorded for each pregnant female (with at least one live fetus) at hysterectomy.
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- Number and distribution of dead and live fetuses,
- Number and distribution of uterine scars (uterine implantation without implant)
- Gross evaluation of placentas.

The following classification was used to record:
. uterine scar: uterine implantation without implant,
. early resorption: evidence of implant without recognizable embryo,
. late resorption: dead embryo or fetus with degenerative changes,
. dead fetus: dead fetus with no degenerative changes.
Fetal examinations:
- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: half fetuses per litter
- Skeletal examinations: Yes: half fetuses per litter
- Head examinations: Yes: half fetuses per litter
- Other: number of dead and alive fetuses, fetal weight, fetal sex
Statistics:
Data were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by Fisher’s exact probability test (proportions).
Indices:
The following parameters were calculated:
For each pregnant female:
- Body weight change for different intervals
- Net body weight (presented as carcass weight) = Body weight on Day 21 post-coitum - gravid uterine weight
- Net body weight change = Body weight on Day 21 post-coitum - body weight on Day 6 post-coitum - gravid uterine weight%

For each litter:
- Total number of resorptions = Sum of uterine scars + early resorptions + late resorptions
- Total number of dead fetuses = Sum of dead fetuses
- % of dead fetuses per litter = (Total number of dead fetuses / Number of implantation sites) x 100
- Total number of live fetuses = Sum of live male + live female fetuses
- % of live fetuses per litter = (Total number of live fetuses / Number of implantation sites) x 100
- % of pre-implantation loss = (Number of corpora lutea - Number of implantations / Number of corpora lutea) x 100
- % of post-implantation loss = (Number of implantation sites - Number of live fetuses / Number of implantation sites) x 100
- Average fetal body weight= Sum of individual fetal weights / Number of live fetuses

For each group:
- % of pre-implantation loss relative to the number of corpora lutea (mean of pre-implantation loss per litter)
- % of live fetuses and % of dead fetuses (relative to total number of fetuses)
- Mean % of male fetuses per litte
- Mean and standard deviations and % relative to the number of implantation sites: resorptions plus, uterine scars, uterine scars, early resorptions, late
resorptions,
- % of post-implantation loss relative to the number of implantation sites (mean of post-implantation loss per litter), % of dams affected.

Historical control data:
Cf attached document
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were observed.
Clinical signs (namely bent tail, cutaneous lesion and/or abnormal growth of teeth) were considered to be unrelated to the test item, as they were not dose-related and/or reported in isolated animals.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects on mean body weight and mean body weight change (see chapter 'any other information on results including tables' section 7.8.2 /2 for mean body weight and body weight changes tables).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no effects on mean food consumption (see chapter 'any other information on results including tables' section 7.8.2 /3 for mean food consumption tables).
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
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):
There were no test item-related macroscopic findings.
Enlarged placenta was noted in two fetuses from one female treated at 1000 mg/kg/day. These findings were considered to be related to the small size of the litter and of the fetuses which weighed 6.79 g and 6.84 g, respectively, and therefore were not considered to be test item-related.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no effects on mean gravid uterus and carcass weights and on mean net body weight change (see chapter 'any other information on results including tables' section 7.8.2 /4 for mean carcass weight, net body weight change and gravid uterus weight tables).
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no test item-related effects on pre and post implantation losses at any dose-level (see chapter 'any other information on results including tables' section 7.8.2 /5 for hysterectomy data).
Number of dams with pre-implantation loss: 17,11,14,11 at 0,100, 300 or 1000 mg/kg/day, respectively
Number of dams with post-implantation loss: 16, 12, 9, 9 at 0, 100, 300 or 1000 mg/kg/day, respectively
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
One female given 100 mg/kg/day had total resorption. As this finding occurred at the low-dose only, this was considered not to be test item-related (see chapter 'any other information on results including tables' section 7.8.2 /5 for hysterectomy data).
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no test item-related effects on early or late resorptions (see chapter 'any other information on results including tables' section 7.8.2 /5 for hysterectomy data).
Number of dams with resorptions: 16,12, 9, 9 at 0, 100, 300 or 1000 mg/kg/day, respectively.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no dead fetuses (see chapter 'any other information on results including tables' section 7.8.2 /5 for hysterectomy data).
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Number of pregnant females at hysterectomy: 24, 23, 22, 21 at 0, 100, 300 or 1000 mg/kg/day, respectively (see chapter 'any other information on results including tables' section 7.8.2 /5 for hysterectomy data).
As the test item was administered after implantation, the non-pregnant status for 1 female given 100 mg/kg/day, two females given 300 mg/kg/day and three females given 1000 mg/kg/day was considered to be fortuitous and not to be test item-related.
Key result
Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no effects on mean fetal body weight (see chapter 'any other information on results including tables' section 7.8.2 /6 for foetal body weight and sex ratio tables).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no effects on sex ratio (% of males fetuses) (see chapter 'any other information on results including tables' section 7.8.2 /6 for foetal body weight and sex ratio tables).
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no changes in litter size and weights compared to controls
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-treatment related external malformations (see chapter 'any other information on results including tables' section 7.8.2 /7 for litter and fetal incidences of external malformations table).
In the 100 mg/kg/day group, one litter had one fetus with omphalocele. This external malformation was observed with an incidence similar to the Historical Control Data and without any dose relationship. Therefore, this was considered to be unrelated to the test item.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related skeletal malformations (see chapter 'any other information on results including tables' section 7.8.2 /8 for litter and fetal incidences of skeletal malformations table).
In the 300 mg/kg/day group, one litter had one fetus with cervical rib and one other litter had two fetuses with absent lumbar vertebra. As these skeletal malformations were observed without any dose relationship and with an incidence similar to the Historical Control Data, they were considered to be unrelated to the test item.

Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related soft tissue malformations (see chapter 'any other information on results including tables' section 7.8.2 /9 for litter and fetal incidences of soft tissue malformations table).
In the control group, one litter had one fetus with marked renal dilated pelvis and marked dilated ureter. These tissue malformations were observed with an incidence similar to the Historical Control Data.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
External variations:
There were no external variations.

Skeletal variations:
There were no test item-related skeletal variations (see chapter 'any other information on results including tables' section 7.8.2 /10 for litter and fetal incidences of skeletal variations table).
When compared with controls, variations were observed at a slightly higher fetal and/or litter incidence in the 300 and/or 1000 mg/kg/day group [i.e. incomplete ossification of supraoccipital, incomplete ossification of parietal, dumbbell ossification of thoracic vertebra(e) centrum, ossification point on lumbar vertebra(e), extra sternebral ossification site and incomplete ossification of rib and unossified forepaw proximal phalanx]. These variations did not impact mean fetal body weights and were observed with incidences similar or very close to the Historical Control Data and/or were not statistically significant.

Visceral variations:
There were no test item-related soft tissue variations (see chapter 'any other information on results including tables' section 7.8.2 /11 for litter and fetal incidences of soft tissue variations table).
When compared with controls, dilated renal pelvis and ureter were observed at higher litter and fetal incidences from 300 mg/kg/day. As these variations were observed with incidences similar to the Historical Control Data and were not statistically significant, they were therefore considered to be unrelated to the test item.
As the other tissue variations were noted at a lower incidence than that recorded in controls, they were also considered to be unrelated to the test item.

Cartilage
There were no test item-related cartilage findings (see chapter 'any other information on results including tables' section 7.8.2 /12 for litter and fetal incidences of cartilage findings table).
In the 1000 mg/kg/day group and when compared with controls, there were decreases [i.e. cartilage of rib(s) present] or increases [i.e. cartilage of rib(s) absent, misshapen cartilage rib(s) and/or cartilage of forepaw proximal phalanx present] in the litter and fetal incidences of cartilage findings. These findings did not impact mean fetal body weights. They were not statistically significant and with an incidence similar or lower than the controls and/or Historical Control Data. They were therefore considered to be unrelated to the test item treatment.

Key result
Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 7.8.2/ 2 : Mean body weight and body weight changes (g)

Dose level (mg/kg/day)

0

100

300

1000

Body weight (g)

 

 

 

 

Day 6 p.c.

281

281

281

280

 

-

(0)

(0)

(0)

Day 9 p.c.

292

292

292

290

 

-

(0)

(0)

(-1)

Day 12 p.c.

309

312

312

309

 

-

(+1)

(+1)

(0)

Day 15 p.c.

328

333

332

329

 

-

(+2)

(+1)

(0)

Day 18 p.c.

375

383

381

374

 

-

(+2)

(+2)

(0)

Day 21 p.c.

425

434

434

422

 

-

(+2)

(+2)

(-1)

Body weight change (g)

 

 

 

 

Days 6 - 9 p.c.

+11

+11

+11

+10

Days 9 - 12 p.c.

+17

+20

+20

+19

Days 12 - 15 p.c.

+19

+20

+20

+20

Days 15 - 18 p.c.

+46

+50

+49

+45

Days 18 - 21 p.c.

+51

+51

+52

+48

Days 6 - 21 p.c.

+144

+153

+153

+143

 

-

(+6)

(+6)

(-1)

p.c.: post-coitum.

-: not applicable.

( ): in brackets, percentage difference vs.controls.


 

Table 7.8.2/3: Mean food consumption (g/animal/day)

Dose level (mg/kg/day)

0

100

300

1000

. Days 6 - 9 p.c.

21

21

23

21

. Days 9 - 12 p.c.

24

23

26

24

. Days 12 - 15 p.c.

26

26

27

26

. Days 15 - 18 p.c.

34

31

34

31

. Days 18 - 21 p.c.

29

29

31

29

p.c.: post-coitum.

 

Table7.8.2/4: Mean carcass weight, net body weight change and gravid uterus weight (g)

Dose level (mg/kg/day)

0

100

300

1000

Gravid uterus weight

98

106
(+8)

100
(+2)

97
(-1)

Carcass weight

328

328
(0)

333
(+2)

326
(-1)

Net body weight change
from Day 6
p.c.

+47

+47

+52

+46

( ): in brackets, percentage difference vs. controls.

p.c.: post-coitum.

(a): weights are rounded values.

Table7.8.2/5a: Pregnancy status

Dose level (mg/kg/day)

0

100

300

1000

Number of females

24

24

24

24

Non-pregnant females

0

1

2

3

Females with total resorption

0

1

0

0

Females with live fetuses at term

24

22

22

21

 

Table7.8.2/5b: Hysterectomy data

Dose level (mg/kg/day)

0

100

300

1000

Number of pregnant
females at hysterectomy

24

23

22

21

Number of females with live
fetuses at termination

24

22

22

21

Number of females with total
resorption

0

1

0

0

Mean number of corpora lutea

14.0

15.2

13.7

13.1

Mean number of implantation sites

12.8

13.3

12.8

12.2

Mean pre-implantation loss (%)

9.2

11.2

6.5

7.3

Number of females with
pre-implantation loss

17

11

14

11

Mean number of live fetuses

11.9

12.6

12.1

11.8

Dead fetuses (%)

0.0

0.0

0.0

0.0

Mean number of implantation scars

0.0

0.0

0.0

0.0

Mean number of early resorptions

0.7

0.7

0.7

0.4

Mean number of late resorptions

0.2

0.1

0.0

0.1

Mean number of resorptions + scars

0.9

0.7

0.7

0.5

Number of females with resorptions

16

12

9

9

Mean post-implantation loss (%)

7.4

9.4

5.4

3.7

Number of females with
post-implantation loss

16

12

9

9

 

Table7.8.2/6: fetal body weight and sex ratio

Dose level (mg/kg/day)

0

100

300

1000

Mean fetal body weight (g)

5.88
-

5.82
(-1)

5.94
(+1)

5.92
(+1)

Mean fetal body weight
of males (g)

6.05

-

5.97

(-1)

6.09

(+1)

6.09

(+1)

Mean fetal body weight
of females (g)

5.74

-

5.64

(-2)

5.75

(0)

5.78

(+1)

Mean percentage
of male fetuses (%)

46.6

52.3

52.1

47.5

( ): in brackets, percentage difference vs.controls.

-: not applicable.

 

Table7.8.2/7: litter (L) and fetal (F) incidence (%) of external malformations

Fetal external malformations

Dose level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

24

22

22

21

235

Live fetuses, n

286

290

266

247

2896

. omphalocele, L(F)

0 (0)

4.5 (0.3)

0 (0)

0 (0)

4.2 (0.3)(a)

Litters affected, n (%)(c)

0 (0)

1 (4.5)

0 (0)

0 (0)

8 (3.4)(b)

Fetuses affected, n (%)(c)

0 (0)

1 (0.3)

 0 (0)

0 (0)

10 (0.3)(b)

n: number.

HCD: Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017.

(a): upper litter (fetal) incidence per study; (b): mean number (percentage) of litters or fetuses affected.
(c): all malformations combined.

 

Table7.8.2/11: litter (L) and fetal (F) incidence (%) of soft tissue variations

Dose level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

24

22

22

21

235

Live fetuses, n

136

141

127

120

1385

. kidney: dilated pelvis, L(F)

16.7 (3.7)

13.6 (2.1)

22.7 (6.3)

28.6 (5.8)

30.8 (9.1)(a)

. ureter: dilated, L(F)

29.2 (8.8)

18.2 (5.0)

40.9 (12.6)

33.3 (6.7)

48.7 (15.1)(a)

Litters affected, n (%)(c)

12 (50.0)

6 (27.3)

10 (45.5)

8 (38.1)

70 (29.8)(b)

Fetuses affected, n (%)(c)

18 (13.2)

10 (7.1)

21 (16.5)

11 (9.2)

132 (9.5)(b)

n: number.

HCD: Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

(a): upper litter (fetal) incidence per study; (b): mean number (percentage) of litters or fetuses affected.
(c): all variations combined.

 

Table7.8.2/9: litter (L) and fetal (F) incidence (%) of soft tissue malformations

Dose level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

24

22

22

21

235

Live fetuses, n

136

141

127

120

1385

. kidney: marked dilated
 pelvis, L(F)

4.2 (0.7)

0 (0)

0 (0)

0 (0)

4.2 (0.7)(a)

. ureter: marked dilated,
 L(F)

4.2 (0.7)

0 (0)

0 (0)

0 (0)

10.3 (2.2)(a)

Litters affected, n (%)(c)

1 (4.2)

0 (0)

0 (0)

0 (0)

7 (3.0)(b)

Fetuses affected, n (%)(c)

1 (0.7)

0 (0)

 0 (0)

0 (0)

10 (0.7)(b)

n: number.

HCD: Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

(a): upper litter (fetal) incidence per study; (b): mean number (percentage) of litters or fetuses affected.

(c): all malformations combined.

 

Table7.8.2/12: litter (L) and fetal (F) incidence (%) of cartilage findings

Dose level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

24

22

22

21

235

Live fetuses, n

149

149

139

127

1511

. cartilage of rib(s) present, L(F)

16.7 (4.7)

13.6 (2.0)

18.2 (2.9)

9.5 (1.6)

16.7 (3.8)(a)

. cartilage of rib(s) absent,
 L(F)

0 (0)

0 (0)

0 (0)

4.8 (0.8)

4.3 (0.6)(a)

. cartilage of rib(s) misshapen, L(F)

4.2 (1.3)

4.5 (0.7)

4.5 (1.4)

4.8 (0.8)

0 (0)

. forepaw: cartilage of
 proximal phalanx
 present, L(F)

33.3 (13.4)

54.5 (14.1)

40.9 (15.8)

42.9 (13.4)

87.5 (50.0)(a)

Litters affected, n (%)(c)

21 (87.5)

20 (90.9)

19 (86.4)

19 (90.5)

128 (54.5)(b)

Fetuses affected, n (%)(c)

98 (65.8)

99 (66.4)

90 (64.7)

73 (57.5)

584 (38.6)(b)

n: number.

HCD: Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

(a): upper litter (fetal) incidence by study; (b): mean number (percentage) of litters or fetuses affected.
(c): all cartilage findings combined.

 

Table7.8.2/10: litter (L) and fetal (F) incidence (%) of skeletal variations

Dose level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

24

22

22

21

235

Live fetuses, n

149

149

139

127

1511

. supraoccipital: incomplete
 ossification, L(F)

0 (0)

4.5 (0.7)

0 (0)

9.5 (1.6)

8.3 (3.2)(a)

. interparietal: incomplete
 ossification, L(F)

0 (0)

9.1 (1.3)

9.1 (1.4)

9.5 (2.4)

21.1 (4.5)(a)

. thoracic vertebra(e): dumbbell
 ossification of centrum, L(F)

0 (0)

4.5 (0.7)

0 (0)

4.8 (0.8)

25.0 (4.7)(a)[DP1] 

. lumbar vertebra(e): ossification
 point, L(F)

4.2 (0.7)

9.1 (1.3)

0 (0)

9.5 (1.6)

4.2 (0.6)(a)

. extra sternebral ossification
 site, L(F)

4.2 (0.7)

13.6 (4.0)

4.5 (0.7)

23.8 (3.9)

25 (6.0)(a)

. rib: incomplete ossification, L(F)

4.2 (0.7)

4.5 (0.7)

9.1 (2.2)

9.5 (1.6)

15.8 (4.8)(a)

. forepaw: unossified proximal
 phalanx, L(F)

33.3 (13.4)

54.5 (14.1)

40.9 (15.8)

42.9 (13.4)

87.5 (50.0)(a)

Litters affected, n (%)(c)

21 (87.5)

20 (90.9)

20 (90.9)

19 (90.5)

194 (82.6)(b)

Fetuses affected, n (%)(c)

99 (66.4)

103 (69.1)

91 (65.5)

76 (59.8)

742 (49.1)(b)

n: number.

HCD: Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

(a): upper litter (fetal) incidence by study; (b): mean number (percentage) of litters or fetuses affected.

(c): all variations combined.

 

Table7.8.2/8 : litter (L) and fetal (F) incidence (%) of skeletal malformations

Dose level (mg/kg/day)

0

100

300

1000

HCD

Dams with live fetuses, n

24

22

22

21

235

Live fetuses, n

149

149

139

127

1511

. cervical vertebra(e): cervical rib, L(F)

0 (0)

0 (0)

4.5 (0.7)

0 (0)

0 (0)

. lumbar vertebra(e): absent, L(F)

0 (0)

0 (0)

4.5 (1.4)

0 (0)

5.3 (2.4)(a)

Litters affected, n (%)(c)

0 (0)

0 (0)

2 (9.1)

0 (0)

13 (5.5)(b)

Fetuses affected, n (%)(c)

0 (0)

0 (0)

3 (2.2)

0 (0)

15 (1.0)(b)

n: number.

HCD: Historical Control Data (control data collected from 11 studies covering a period ranging from August 2016 to December 2017).

(a): upper litter (fetal) incidence per study; (b): mean number (percentage) of litters or fetuses affected.

(c): all malformations combined.

Conclusions:
The No Observed Effect Level (NOEL) for maternal parameters and for embryo-fetal development was considered to be 1000 mg/kg/day in absence of test item treatment effects in the study.
Executive summary:

In a prenatal development toxicity study performed according to OECD 414 and in compliance with Good Laboratory Practice, the objective was to evaluate the potential toxic effects of the test substance on the pregnant female rat and on embryonic and fetal development, following oral administration (gavage) from Day 6 to Day 20 post-coitum, inclusive. 

Three groups of 24 time-mated female rats received the test substance at doses of 100, 300 or 1000 mg/kg bw /day from Day 6 to 20 post-coitum. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume throughout the same period. Test substance concentration was checked four times in formulations given to the animals

Animals were killed on Day 21 of gestation for reproductive assessment and fetal examination.

Clinical observations, bodyweight and food consumption were monitored. Adult females were examined macroscopically at necropsy on Day 21 of gestationand the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. All fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities.

The test item concentrations in the analyzed dose formulations were within an acceptable range of variation (between -10.2% and +13.0%) when compared to the nominal values (± 15% required).

At hysterectomy on Day 21 post-coitum., there were 24/24, 22/24, 22/24 and 21/24 pregnant dams with live fetuses in the groups treated at 0,100, 300 or 1000 mg/kg/day,respectively. The non-pregnant status of 1 female given 100 mg/kg/day, two females given 300 mg/kg/day and three females given 1000 mg/kg/day was not test-item related as the test item was administered after implantation.

There were no test item treatment-related effects in the dams in terms of mortality, clinical signs, necropsy findings, body weight, food consumption, carcass weight, gravid uterus weight, net body weight change, or hysterectomy data. There were no test item treatment-related effects in the litters in terms of sex ratio, fetal body weight, external, visceral and skeletal variations or malformations or cartilage findings.

The No Observed Effect Level (NOEL) for maternal parameters and for embryo-fetal development was considered to be 1000 mg/kg/day in absence of test item treatment effects in the study.

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:
The key study is considered to be reliable with a klimisch score of 1.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Prenatal development toxicity study (OECD 414):

The objective of this study was to evaluate the potential toxic effects of the test substance on the pregnant female rat and on embryonic and fetal development, following oral administration (gavage) from Day 6 to Day 20 post-coitum, inclusive. 

Three groups of 24 time-mated female rats received the test substance at doses of 100, 300 or 1000 mg/kg bw /day from Day 6 to 20 post-coitum. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume throughout the same period. Test substance concentration was checked four times in formulations given to the animals

Animals were killed on Day 21 of gestation for reproductive assessment and fetal examination.

Clinical observations, bodyweight and food consumption were monitored. Adult females were examined macroscopically at necropsy on Day 21 of gestationand the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. All fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities.

The test item concentrations in the analyzed dose formulations were within an acceptable range of variation (between -10.2% and +13.0%) when compared to the nominal values (± 15% required).

At hysterectomy on Day 21 post-coitum., there were 24/24, 22/24, 22/24 and 21/24 pregnant dams with live fetuses in the groups treated at 0,100, 300 or 1000 mg/kg/day,respectively. The non-pregnant status of 1 female given 100 mg/kg/day, two females given 300 mg/kg/day and three females given 1000 mg/kg/day was not test-item related as the test item was administered after implantation.

There were no test item treatment-related effects in the dams in terms of mortality, clinical signs, necropsy findings, body weight, food consumption, carcass weight, gravid uterus weight, net body weight change, or hysterectomy data. There were no test item treatment-related effects in the litters in terms of sex ratio, fetal body weight, external, visceral and skeletal variations or malformations or cartilage findings.

The No Observed Effect Level (NOEL) for maternal parameters and for embryo-fetal development was considered to be 1000 mg/kg/day in absence of test item treatment effects in the study.

Justification for classification or non-classification

No effects on reproductive performance and offspring growth and survival were observed at the limit dose of 1000 mg/kg bw/day in a screening study for reproductive /developemental effects performed in rats on a closely-related substance.

No effects on embryo/fetal development were observed at the limit dose of 1000 mg/kg bw/day in a developmental toxicity study performed in rats.

On the basis of this results and according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures, no classification is warranted with respect to reproductive toxicity.