Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 October 2017 - 15 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 October 2017 - 15 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature container flushed with nitrogen
- Stability under test conditions:
stable
- Solubility and stability of the test substance in the solvent/vehicle:
stability in propylene glycol for at least 5 hours at room temperature under normal laboratory light conditions was confirmed over the concentration range 1 to 200 mg/mL (solutions). Stability for at least 6 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL (solutions). Stability for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions) at the test facility in separate studies.

OTHER SPECIFICS:
No correction for purity was used.
pH 8-9 at concentration of 1% w/w
Specific gravity / density: 1.115 kg/L
Substance changes colour slowly especially in melted aggregate condition under air.
Species:
rat
Strain:
other: Crl:Wi(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) males 10 weeks, females 11 weeks at the start of dosing
- Weight at study initiation: (P) Males: 270-304 g; Females: 188-228 g
- Fasting period before study:
none
- Housing:

Pre-mating: group-housed up to 5 animals/sex/group in polycarbonate cages (Macrolon type MIV)
Mating: on 1:1 basis in Macrolon MIII cages
Post-mating: males in their home cages with a maximum of 5 males/cage, females individually in MIII Macrolon cages.
Lactation: females in Macrolon MIII cages, pups housed with dams.
Cages contained appropriate bedding.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period:
7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
19-20
- Humidity (%):
45-71
- Air changes (per hr):
at least 10
- Photoperiod (hrs dark / hrs light):
12/12

IN-LIFE DATES: From: 6 October 2017 To: 15 December 2017
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 5 hours after completion of the preparation of the formulation. Formulations were heated to a maximum temperature of 80 ±5°C for maximally 17 minutes to obtain visual homogeneity. Formulations were
released for dosing when they had reached a temperature of 40°C or lower. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: based on trial formulations conducted at the testing facility
- Amount of vehicle (if gavage): 5 mL/kg bw
- Relative density: 1.036
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of duplicate samples were analysed by a validated procedure (LC-DAD), with a lower limit of quantification of 1.00 mg/g and the calibration curve in the range from 1.00 to 25.0 mg/L. Preparation of formulations was considered acceptable if the mean accuracy was in the range 90 – 110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurred, for maximum 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post-coitum
Duration of treatment / exposure:
Males: 29 days (2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy)
Females that delivered: 50-56 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and 14 or 16 days after delivery, up to and including the day before scheduled necropsy
Females which failed to deliver: 52 days (female without evidence of mating) or 41 days (two non-pregnant females and a female with implantations only).
Frequency of treatment:
Once daily
Duration of test:
Ca. 16 weeks
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 (concurrent vehicle controls)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on the results of a 14-day dose range finding study with the test item with female Wistar Han rats and a 28-day repeated dose oral toxicity study with the test item in Wistar Han (female) and Wistar (male) rats, and in an attempt to produce graded responses to the test item. A NOAEL could not be established for the 28-day study due to adverse findings in the kidneys of males at dose 50, 150 and 500 mg/kg bw/day. At dose 500 mg/kg bw/day, adverse test-item related effects consisted of clinical signs, reduced body weights, increased kidney weights and an increased incidence and severity of myofiber degeneration in the skeletal muscle of males and females.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: once daily, beginning during the first administration of the test item and lasting throughout the dosing period up to the day prior to necropsy. These clinical observations were at least conducted during the intervals 0-15 minutes and 1 hour ± 15 minutes after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: yes
Time schedule: weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13

WATER CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on PND 15-16
- Organs examined:
The following organ weights were weighed at necropsy for all scheduled euthanasia female animals: thyroid including parathyroid, kidneys, thymus.
The following tissues were examined by a histopathologist:
Controls and high-dose female animals: thyroid gland, ovaries, thymus
All animals: kidneys and gross lesions/masses
Females that failed to deliver pups: cervix, coagulation gland, prostate gland, seminal vesicles, uterus and vagina.

OTHER:
Clinical chemistry:
P generation:
Time point for blood collection: on the day of scheduled necropsy
Animals fasted: females: no.
Blood samples were stored for possible T4 measurements, but no analyses were performed, because there were no treatment-related changes in T4 in F0-males, or in the weight and morphology of the thyroid in both sexes.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No, females were allowed to litter naturally
- Number of corpora lutea: no
- Number of implantations: Yes
- Number of early resorptions: no
- Number of late resorptions: no
Fetal examinations:
No fetal examinations were performed, as females were allowed to litter naturally.

The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, T4 in pups on PND 14-16

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible.

OTHER:
Clinical chemistry:
Blood was collected on PND4 (at culling) from two surplus pups (one male, one female) per litter and on PND 14-16 (scheduled necropsy) from two pups per litter (one male, one female). Blood from pups on PND4 was stored, but not analysed.
Measurement of total T4 was conducted for PND 14-16 pups. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Parametric datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Non-parametric datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). For incidence, an overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
The following offspring indices were calculated:
Post-implantation survival index (%) = 100 x (total number of offspring born/total number of uterine implantation sites)
Live birth index (%) = 100 x (bnumber of live offspring on Day 1 after littering/total number of offspring born)
Percentage live males at first litter check (%) = 100 x (number of live male pups at first litter check / number of live pups at first litter check)
Percentage live females at first litter check (%) = 100 x (number of live female pups at first litter check / number of live pups at first litter check)
Viability index (%) = 100 x (number of live offspring on Day 4 before culling/number of live offspring on Day 1 after littering)
Lactation index (%) = 100 x (number of live offspring on Day 13 after littering/number of live offspring on Day 4 after culling)
Historical control data:
Available from the same testing laboratory for the period of 2015 - June 2017.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period. Salivation was noted occasionally among treated animals, most frequently in females at 300 mg/kg bw/day. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its minor severity (slight) and the time of occurrence (i.e. immediately after dosing), and may be related to the irritancy of the test item. Flat gait was noted in most males at 300 mg/kg bw/day towards the end of the treatment period. As this clinical sign was observed on only a single occasion, it was considered not to be toxicologically relevant. Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gain and mean body weights of females treated up to 300 mg/kg bw/day were considered not to be affected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes in food consumption before or after correction for body weight were observed in females treated up to 300 mg/kg bw/day. The slightly lower mean food consumption noted in 300 mg/kg bw/day females between Days 7-13 of the lactation period could be explained by the lower food consumption of two dams with small litters and was considered not to reflect an effect of the test item on food consumption.
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:
no effects observed
Description (incidence and severity):
There were no test item-related organ weight changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In kidneys, tubular degeneration was present in a single female at 300 mg/kg bw/day at slight degree. Tubular basophilia was present in 3 females at 300 mg/kg bw/day up to moderate degree, vs. 2 in controls (up to slight degree). There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was not affected by treatment. The survival indices were 88, 94, 95 and 94% for the control, 30, 100 and 300 mg/kg bw/day groups, respectively.
For three females at 30 mg/kg bw/day, 100 mg/kg bw/day and 300 mg/kg bw/day the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from
time to time and is caused by normal resorption of these areas during lactation.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) was considered not to be affected by treatment.
Mean litter size in the 100 mg/kg bw/day group was higher than that in the control group (14.9 versus 11.0). Statistical significance was not achieved and mean litter size at 100 mg/kg bw/day remained
within normal limits. In absence of a dose-related trend, this finding was not attributed to treatment.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Duration of gestation was not affected by treatment.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Fertility index was not affected by treatment. Except for one female at 30 mg/kg bw/day and one female at 100 mg/kg bw/day, all mated females were pregnant. The fertility indices were 100, 90, 89 and 100% for the control, 30, 100 and 300 mg/kg bw/day groups, respectively.
These cases of non-pregnancy, which occurred in absence of related histopathology changes in reproductive organs, were considered to be unrelated to treatment due to their incidental occurrence and lack of a dose-related trend.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: kidneys
Description (incidence and severity):
Slight tubular degeneration in one female and tubular basophilia (up to moderate) in 3 females in the high-dose group of 300 mg/kg bw/day
Fetal body weight changes:
not examined
Description (incidence and severity):
No examinations on fetal body weights were performed, as females were allowed to litter naturally.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 99, 100, 100 and 98% for the control, 30, 100 and 300 mg/kg bw/day groups, respectively.
The numbers of dead pups at first litter check were within normal limits (one in the control group and two in the 300 mg/kg bw/day groups. The incidental pup mortality at 300 mg/kg bw/day was regarded as unrelated to treatment.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio was not affected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) was considered not to be affected by treatment.
Mean litter size in the 100 mg/kg bw/day group was higher than that in the control group (14.9 versus 11.0). Statistical significance was not achieved and mean litter size at 100 mg/kg bw/day remained within normal limits. In absence of a dose-related trend, this finding was not attributed to treatment. Body weights of pups were not affected by treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 99% for the control and 30 mg/kg bw/day groups and 100% for the higher dose groups.
One pup of the control group was found dead at PND 2 and one pup of the 30 mg/kg bw/day group went missing at PND 2 (presumably cannibalized). This postnatal loss was regarded as unrelated to treatment as it was incidental and showed no dose-related trend.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of the macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No detailed examinations were performed, but no macroscopic changes were noted at gross necropsy.
Visceral malformations:
no effects observed
Description (incidence and severity):
No detailed examinations were performed, but no macroscopic changes were noted at gross necropsy.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Mean serum T4 in female PND 14-16 pups at 300 mg/kg bw/day was statistically significantly higher than that in control pups (relative difference 19%). A possible treatment-related effect was observed in T4 values for female PND 14-16 pups. Since the change in T4 level was minimal and values remained in the historical control range, a possible effect on T4 was considered not adverse.
No other clinical chemistry parameters were examined.
Sex ratio was not affected by treatment. Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment. Treatment up to 300 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects on development at the highest tested dose of 300 mg/kg bw/day.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Formulation analysis:

The concentrations analyzed in the formulations of low, mid-dose and high dose groups were in agreement with the target concentrations (i.e. mean accuracies 99.7% ( n = 6), 100.3% ( n = 2) and 97.6% (n = 6), respectively). The formulations of low-dose and high-dose groups prepared were homogeneous (i.e. coefficient of variation 0.8% and 1.4%, respectively).

Conclusions:
In a GLP-compliablt OECD guideline 421 study with rats, no developmental toxicity was observed at the highest tested dose of 300 mg/kg bw/day, which was considered to be the NOAEL for developmental effects. In maternal animals, tubular degeneration in kidneys was evidenced in one female at 300 mg/kg bw/day, and tubular basophilia was present in 3 females at 300 mg/kg bw/day. The NOAEL for maternal toxicity was therefore set at 100 mg/kg bw/day.
Executive summary:

In a GLP-compliant OECD guideline 421 study, male and female Crl:WI(Han) rats were treated with solutions of the test substance in propylene glycol at dose levels of 0, 30, 100 and 300 mg/kg bw/day for two weeks prior to mating, during mating and subsequent gestation and lactation (up to PND 15-16). No mortality occurred and no clinical signs related to treatment were noted. There were no adverse effects on body weight, body weight gain or food consumption in maternal animals. At necropsy, tubular degeneration in one female (up to slight) and tubular basophilia (up to moderate) in three females were seen at the highest dose level of 300 mg/kg bw/day. No other adverse effects were noted. Based on this the NOAEL for maternal toxicity was set at 100 mg/kg bw/day. There were no adverse effects on post-implantation survival, pup viability, litter size, pup body weights, sex ratio, areola/nipple retention or anogenital distance up to and including the highest tested dose of 300 mg/kg bw/day. No changes were noted at gross macroscopy of the pups. The highest tested dose of 300 mg/kg bw/day was therefore considered to be the NOAEL for developmental toxicity.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Colourless solidified melt
Details on test material:
Expiry date: 25 October 2018
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability in propylene glycol for at least 5 hours at room temperature under normal laboratory light conditions was confirmed over the concentration range 1 to 200 mg/mL (solutions). Stability for at least 6 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL (solutions). Stability for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions) at the test facility in separate studies.

OTHER SPECIFICS:
No correction for purity was used.
pH 8-9 at concentration of 1% w/w
Specific gravity / density: 1.115 kg/L
Substance changes colour slowly especially in melted aggregate condition under air.

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) males 10 weeks, females 11 weeks at the start of dosing
- Weight at study initiation: (P) Males: 270-304 g; Females: 188-228 g
- Fasting period before study: none
- Housing:
Pre-mating: group-housed up to 5 animals/sex/group in polycarbonate cages (Macrolon type MIV)
Mating: on 1:1 basis in Macrolon MIII cages
Post-mating: males in their home cages with a maximum of 5 males/cage, females individually in MIII Macrolon cages.
Lactation: females in Macrolon MIII cages, pups housed with dams.
Cages contained appropriate bedding.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20
- Humidity (%): 45-71
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 6 October 2017 To: 15 December 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 5 hours after completion of the preparation of the formulation. Formulations were heated to a maximum temperature of 80 ±5°C for maximally 17 minutes to obtain visual homogeneity. Formulations were
released for dosing when they had reached a temperature of 40°C or lower. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: based on trial formulations conducted at the testing facility
- Amount of vehicle (if gavage): 5 mL/kg bw
- Relative density: 1.036
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurred, for maximum 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post-coitum
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of duplicate samples were analysed by a validated procedure (LC-DAD), with a lower limit of quantification of 1.00 mg/g and the calibration curve in the range from 1.00 to 25.0 mg/L. Preparation of formulations was considered acceptable if the mean accuracy was in the range 90 – 110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Duration of treatment / exposure:
Males: 29 days (2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy)
Females that delivered: 50-56 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and 14 or 16 days after delivery, up to and including the day before scheduled necropsy
Females which failed to deliver: 52 days (female without evidence of mating) or 41 days (two non-pregnant females and a female with implantations only).
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: males 12 weeks, females 13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 (concurrent vehicle controls)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected based on the results of a 14-day dose range finding study with the test item with female Wistar Han rats and a 28-day repeated dose oral toxicity study with the test item in Wistar Han (female) and Wistar (male) rats, and in an attempt to produce graded responses to the test item. A NOAEL could not be established for the 28-day study due to adverse findings in the kidneys of males at dose 50, 150 and 500 mg/kg bw/day. At dose 500 mg/kg bw/day, adverse test-item related effects consisted of clinical signs, reduced body weights, increased kidney weights and an increased incidence and severity of myofiber degeneration in the skeletal muscle of males and females.
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: once daily, beginning during the first administration of the test item and lasting throughout the dosing period up to the day prior to necropsy. These clinical observations were at least conducted during the intervals 0-15 minutes and 1 hour ± 15 minutes after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: yes
Time schedule: weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13

WATER CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

OTHER:
Clinical chemistry:
P generation:
Time point for blood collection: on the day of scheduled necropsy
Animals fasted: females: no, males: yes, overnight with a maximum of approximately 24 hours before blood sampling, but water was available

T4 examination:
Measurement of total T4 was conducted for F0-males. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because there were no treatment-related changes in T4 in F0-males, or in the weight and morphology of the thyroid in both sexes.
Oestrous cyclicity (parental animals):
Not performed, since this evaluation was already performed in the OECD guideline 407 study (section 7.5.1 of this IUCLID).
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight, stages of spermatogenesis. For the testes of all control and high-dose males and the males of low- and mid-dose group that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, T4 in pups on PND 14-16

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not performed

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not performed

OTHER:
Clinical chemistry:
F1 generation:
Time point for blood collection: PND4 (at culling) from two surplus pups (one male, one female) per litter and PND 14-16 (scheduled necropsy) from two pups per litter (one male, one female). Blood from pups on PND4 was stored, but not analysed.
Measurement of total T4 was conducted for PND 14-16 pups. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of administration).
- Females which delivered: PND 14 or 16.
- Females which failed to deliver: With evidence of mating: post-coitum Day 25-26, without evidence of mating: 24 days after the last day of the mating period.

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organ weights were weighed at necropsy for all scheduled euthanasia animals: epididymis, coagulation gland, thyroid including parathyroid, prostate gland, seminal vesicles, kidneys, testes, thymus.
The following tissues were examined by a histopathologist:
Controls and high-dose animals: epididymis, thyroid gland, ovaries, testes, thymus and seminal vesicles
All animals: kidneys and gross lesions/masses
Males that failed to sire and females that failed to deliver pups: cervix, coagulation gland, prostate gland, seminal vesicles, uterus and vagina.
For the testes of all males of controls and high-dose animals and the low- and mid-dose males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed on PND 14-16; culling was performed on Day 4.

GROSS NECROPSY:
Pups that died before scheduled termination were examined externally and sexed (both externally and internally).

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Parametric datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Non-parametric datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). For incidence, an overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
The following reproductive indices were calculated:
Mating (%) = 100 x (number of females mated/number of females paired)
Precoital time = Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%) = 100 x (number of pregnant females/number of mated females)
Gestation index (%) = 100 x (number of females with living pups on Day 1/number of pregnant females)
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
The following offspring indices were calculated:
Post-implantation survival index (%) = 100 x (total number of offspring born/total number of uterine implantation sites)
Live birth index (%) = 100 x (bnumber of live offspring on Day 1 after littering/total number of offspring born)
Percentage live males at first litter check (%) = 100 x (number of live male pups at first litter check / number of live pups at first litter check)
Percentage live females at first litter check (%) = 100 x (number of live female pups at first litter check / number of live pups at first litter check)
Viability index (%) = 100 x (number of live offspring on Day 4 before culling/number of live offspring on Day 1 after littering)
Lactation index (%) = 100 x (number of live offspring on Day 13 after littering/number of live offspring on Day 4 after culling)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period. Salivation was noted occasionally among treated animals, most frequently in females at 300 mg/kg bw/day. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its minor severity (slight) and the time of occurrence (i.e. immediately after dosing), and may be related to the irritancy of the test item. Flat gait was noted in most males at 300 mg/kg bw/day towards the end of the treatment period. As this clinical sign was observed on only a single occasion, it was considered not to be toxicologically relevant. Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight gain of males at 300 mg/kg bw/day was reduced in the first treatment week, resulting in a 5% lower mean body weight at Day 8 of the pre-mating period (differences from the control group were statistically significant). Thereafter, 300 mg/kg bw/day males showed a normal body weight gain.
Body weight gain and mean body weights of males treated up to 100 mg/kg bw/day and females treated up to 300 mg/kg bw/day were considered not to be affected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption (before and after correction for body weight) was reduced in males at 300 mg/kg bw/day in the first week of the treatment period.
No treatment-related changes in food consumption before or after correction for body weight were observed in males treated up to 100 mg/kg bw/day and females treated up to 300 mg/kg bw/day. The slightly lower mean food consumption noted in 300 mg/kg bw/day females between Days 7-13 of the lactation period could be explained by the lower food consumption of two dams with small litters and was considered not to reflect an effect of the test item on food consumption.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Serum levels of T4 in F0-males were considered not to be affected by treatment. The statistically significantly lower mean T4 value noted at 100 mg/kg bw/day was considered unrelated to treatment due to the lack of a dose-related trend.
No other clinical chemistry parameters were 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
Description (incidence and severity):
Statistically significantly lower weights of the seminal vesicles (absolute and relative to body weight), possibly test item-related, were noted in males treated at 300 mg/kg bw/day (-17% absolute and -15% relative to body weight). There were no other test item-related organ weight changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In kidneys, tubular degeneration was present in males starting at 100 mg/kg bw/day up to moderate degree, and in a single female at 300 mg/kg bw/day at slight degree. Tubular basophilia was present at increased incidence and severity in males starting at 100 mg/kg bw/day up to marked degree, and in females at 300 mg/kg bw/day up to moderate degree.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
Not performed, since this evaluation was already performed in the OECD guideline 407 study (section 7.5.1 of this IUCLID). In that study, no effects on oestrous cycle were observed.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis, except in one male at 100 mg/kg bw/day who also did not sire. This male showed bilateral tubular atrophy and
mineralization in the testes and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the reproductive failure.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Four couples had no offspring: 1/10 couples at 30 mg/kg bw/day (one female not pregnant) and 3/10 couples at 100 mg/kg bw/day (one female with no evidence of mating, one female with implantation site only and one female not pregnant). For the female with no evidence of mating, the male showed bilateral tubular atrophy and mineralization in the testes and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the reproductive failure. For the other couples no abnormalities were seen in the reproductive organs which could account for their lack of offspring. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Details on results (P0)

Mating index was considered not to be affected by treatment. Except for one female at 100 mg/kg bw/day, all females showed evidence of mating. The mating indices were 90% for the 100 mg/kg bw/day group and 100% for the other groups. The unsuccessful mating of this female was due to infertility of the male she was paired with. This unsuccessful mating was considered to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend. Precoital time was not affected by treatment. Except for female at 100 mg/kg bw/day with no evidence of mating, all females showed evidence of mating within four days. Number of implantation sites was considered not to be affected by treatment. One female of the 100 mg/kg bw/day group had only one implantation site and no offspring. This incidental finding in an intermediate dose group animal was regarded as unrelated to treatment. Fertility index was not affected by treatment. Except for one female at 30 mg/kg bw/day and one female at 100 mg/kg bw/day, all mated females were pregnant. The fertility indices were 100, 90, 89 and 100% for the control, 30, 100 and 300 mg/kg bw/day groups, respectively. These cases of non-pregnancy, which occurred in absence of related histopathology changes in reproductive organs, were considered to be unrelated to treatment due to their incidental occurrence and lack of a dose-related trend.
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at the highest dose of 300 mg/kg bw/day.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed remained within the range considered normal for pups of this age, and were therefore regarded as unrelated to treatment.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 99, 100, 100 and 98% for the control, 30, 100 and 300 mg/kg bw/day groups, respectively. The numbers of dead pups at first litter check were within normal limits (one in the control group and two in the 300 mg/kg bw/day group). The incidental pup mortality at 300 mg/kg bw/day was regarded as unrelated to treatment.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 99% for the control and 30 mg/kg bw/day groups and 100% for the higher dose groups. One pup of the control group was found dead at PND 2 and one pup of the 30 mg/kg bw/day group went missing at PND 2 (presumably cannibalized). This postnatal loss was regarded as unrelated to treatment as it was incidental and showed no dose-related trend.
Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not affected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Mean serum T4 in female PND 14-16 pups at 300 mg/kg bw/day was statistically significantly higher than that in control pups (relative difference 19%). A possible treatment-related effect was observed in T4 values for female PND 14-16 pups. Since the change in T4 level was minimal and values remained in the historical control range, a possible effect on T4 was considered not adverse.
No other clinical chemistry parameters were examined.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of the macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio was not affected by treatment. Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment. Treatment up to 300 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects on development at the highest tested dose of 300 mg/kg bw/day

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Formulation analysis:

The concentrations analyzed in the formulations of low, mid-dose and high dose groups were in agreement with the target concentrations (i.e. mean accuracies 99.7% ( n = 6), 100.3% ( n = 2) and 97.6% (n = 6), respectively). The formulations of low-dose and high-dose groups prepared were homogeneous (i.e. coefficient of variation 0.8% and 1.4%, respectively).

Applicant's summary and conclusion

Conclusions:
In a GLP-compliablt OECD guideline 421 study with rats, no adverse effects on reproduction and development were observed at the highest tested dose of 300 mg/kg bw/day, which was considered to be the NOAEL for reproductive and developmental effects. The NOAEL for general toxicity was set at 30 mg/kg bw/day based on adverse changes in kidneys (tubular degeneration and basophilia) in male rats starting from 100 mg/kg bw/day.
Executive summary:

In a GLP-compliant OECD guideline 421 study, male and female Crl:WI(Han) rats were treated with solutions of the test substance in propylene glycol at dose levels of 0, 30, 100 and 300 mg/kg bw/day for two weeks prior to mating, during mating and subsequent gestation and lactation (up to PND 15-16). No mortality occurred and no clinical signs related to treatment were noted. Reduced body weight gain, correlated with reduced food consumption was seen in teh high-dose males during the first treatment week, but returned back to normal thereafter. Renal toxicity in males was seen starting from 100 mg/kg bw/day and was characterized by the presence of tubular degeneration in males (up to moderate). In females a single case of tubular degeneration (slight) was seen at 300 mg/kg bw/day. Tubular basophilia was also seen in all males at 100 and 300 mg/kg bw/day and in 3 females (vs 2 in controls) at 300 mg/kg bw/day. Based on this the NOAEL for general toxicity was set at 30 mg/kg bw/day. No adverse effects on reproduction and development were observed at the highest tested dose of 300 mg/kg bw/day, which was considered to be the NOAEL for reproductive and developmental effects.