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Key value for chemical safety assessment

Effects on fertility

Description of key information

In a repeated dose oral toxicity study in rats (Wistar, OECD TG 407), the substance was adimistered via gavage to 5 rats/sex/dose at 0, 100, 300, 1000 mg/kg bw in PEG 400 for 4 weeks. No adverse effects on reproductive organs or tissue were observed at 1000 mg/kg bw.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: subacute study
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD TG 407
GLP compliance:
yes (incl. QA statement)
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
other: PEG 400
Details on mating procedure:
not applicable because it is a subacute study
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days
Frequency of treatment:
once a day
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw and day
Basis:
other: actual dose received
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: adverse effects on reproductive organs or tissues
Remarks on result:
other: Generation: not applicable because it is a subacute study (migrated information)
Reproductive effects observed:
not specified
Conclusions:
No adverse effects on reproductive organs or tissues were observed in a subacute oral study with rats up to and including 1000 mg/kg and bw.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
No extended one-generation reproductive toxicity study (EU B.56, OECD TG 443) is required because the 28-day study indicates no adverse effects on reproductive organs or tissue. Data waiver is claimed.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Under the experimental conditions of the study (OEDC TG 414), oral (gavage) administration of the test item to the pregnant Wistar rat from implantation through to the day before caesarean section at 100, 300 or 1000 mg/kg/day was well tolerated with no evidence of maternal toxicity in any group. Similarly, there was no evidence of developmental toxicity in any group. The No Observed Effect Level (NOEL) for both maternal and developmental toxicity was the limit dose of 1000 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Stability under test conditions: analytically verified
- Solubility and stability of the test substance in the solvent/vehicle: analytically verified

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI(Han)
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Age at mating: 10-13 weeks
- Housing: individually in plastic cages (meeting EU Directive 2010/63/EU requirements with autoclaved sawdust as bedding.
- Diet and water: ad libitum
- Acclimation period: 6days between animal arrival and start of treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): approximately > 35 %
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: polyethylene glycol 400
Details on exposure:
Administration volume: 5 mL/kg bw; indivudual dose volumes were adjusted using the latest body weight;
Appearance of formulations: colorless clear solution 80, 100, 300 mg/kg bw); light yellow clear solution (1000 mg/kg bw)
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared weekly and stored at room temperature. For each concentration, the test item and then the vehicle were weighed and then stirred magnetically in a container until the test item was completely dissolved.. No correction was made for the purity/composition of the test item.




Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate samples (2 x 2 mL) were taken from each formulation, including the vehicle control, used on the first day of treatment of the main study phase. Duplicate samples (2 x 2 mL) were also taken from the low dose formulation of the second preparation. The samples were kept at room temperature (15 to 25 °C) until analysis.
One set of samples (1 x 2 mL) was analysed at the Test Facility, within the defined stability period (see above), using a validated method. For each group, the volume required for HPLC was taken and analysed in duplicate from the 2 mL sample. The transfer of the analytical method is the subject of a separate study (Test Facility study number AB21317).





Details on mating procedure:
Untreated females were mated at the Supplier with a documented day of mating. They were received at the Test Facility on Day 0 of gestation.
Duration of treatment / exposure:
Days 6 - 20 of gestation, inclusive
Frequency of treatment:
Once daily
Duration of test:
from day 0 to necropsy at day 21 of gestation.
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on results of the dose range finding study.

Four groups of 6 females were exposed to 0, 100, 300 and 1000 mg/kg bw/day for Days 6 to 20 of gestation inclusive by oral gavage. Rationale for dose selection: based on results of a repeated dose toxicity study in male and female rats (OECD TG 407). Animals were treated by daily oral gavage at 100, 300 and 1000 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day. The same doses, including the limit dose of 1000 mg/kg/day, were therefore selected for this dose-range-finding phase.

- Maternal findings

No mortality occurred during the study period.
No toxicologically significant clinical signs were noted up to and including 1000 mg/kg bw/day.There was no test item-related clinical sign in any group. Hypersalivation associated or not with abnormal foraging or pedalling were observed in 5,6,6 and 6 (out of 6) animals in group 1 to 4 respectively. These findings were clearly associated with the vehicle (Polyethylene glycol 400). Incidental clinical signs such as hairloss, scabs were observed in all groups. One female given 300 mg/kg/day (no. 13) had red vaginal discharge on Gestation Day 21.
No test item related effect was observed on body weight gain up to and including 1000 mg/kg bw/day.
Food consumption was unaffected by treatment up to and including 1000 mg/kg bw/day.
No macroscopic findings were noted at necropsy.
Mean gravid uterus weight was higher in the 1000 mg/kg/day group compared with other groups due to an incidentally higher mean live litter size
Despite intergroup differences due to the small group size, there was no adverse test item-related effect on embryo-foetal survival in any group. The percentage post-implantation loss in the treated groups was lower than in the concurrent control group due to one control female (no. 3) with no viable foetuses that disproportionately influenced the control value. As a consequence of the incidental differences in the pre- and post-implantation data, the mean live litter size was superior to that in the concurrent control group in all test item groups, particularly in the 1000 mg/kg/day group.

- Fetal findings

Litter sizes were within normal limits for all groups.
The male : female ratios were equal in litters of all groups.
Fetal body weights were unaffected by treatment up to and including 1000 mg/kg bw/day.
External examination of the fetuses did not show any abnormalities.



Maternal examinations:
CLINICAL EXAMINATIONS: Yes
- Time schedule: All animals were observed daily for clinical signs.
During the treatment period, the animals were observed before and at least twice after dosing to detect any clinical signs or reaction to treatment. A full clinical examination was performed on each weighing day.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 9, 12, 15, 18 and 21 of gestation.

FOOD CONSUMPTION: Yes
- Days 0-6, 6-9, 9-12, 12-15, 15-18 and 18-21 during gestation.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:The number and distribution of live and dead fetuses; the weight of each fetus; foetal sex

Resorptions were classified on the basis of the presence (late) or absence (early) of foetal or decidual tissue in addition to placental tissue.
Fetal examinations:
External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter ]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [half per litter ]

For caesarean data, the group mean values were calculated on a litter basis. Foetal observation data are presented as the percentage of affected foetuses and percentage of affected litters.
Foetal abnormalities are categorised as follows:
– Malformations - structural defects which are rare in the control population and are thought to be life threatening or of major physiological consequence.
– Anomalies - minor abnormalities or defects which are relatively rare in the control population and/or are considered not to be of major physiological consequence.
– Variations - minor abnormalities, defects or alternative forms which are either common in the control population or are of no known physiological consequence.

Statistics:
Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) was determined depending upon:
– the normality of the data distribution tested by the Shapiro-Wilk's test
– the homogeneity of the variances across groups tested by the Bartlett's test.
Non- or log-transformed data were analysed by parametric methods.
Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
The number of resorptions and all litter-based percentages were analysed using non-parametric methods, i.e. Kruskal-Wallis test followed by non-parametric Dunnett’s test if the Kruskal-Wallis was significant.
Selected incidence data were analysed using a chi2 test for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
Microsoft Excel® (2003 or higher) was employed to present certain results.
Indices:
pregnancy indidence: number of mated females - number of pregnant females - number of females with live foetuses
For each litter the following calculations were performed:
Percent Pre-implantation loss, percent Post-implantation loss
For caesarean data, the group mean values were calculated on a litter basis. Foetal observation data are presented as the percentage of affected foetuses and percentage of affected litters.
Historical control data:
Yes - are reported in the Appendices of the study report
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Hypersalivation was noted for essentially all females in the vehicle, 100, 300 and 1000 mg/kg/day groups. This was associated with abnormal foraging in 4/22, 21/22, 22/22 and 22/22 females in the vehicle, 100, 300 and 1000 mg/kg/day, respectively. These findings were clearly associated with the vehicle (Polyethylene glycol 400) and possibly slightly exacerbated by the test item.
Incidental clinical signs such as red vaginal discharge or localized hairloss were observed for one control female, two females given 100 mg/kg/day and two females given 300 mg/kg/day
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weight gain in the treated groups was comparable with, or superior to, that in the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean food consumption in the treated groups was comparable with, or superior to, that in the control group.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
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, non-treatment-related
Description (incidence and severity):
A slightly lower mean gravid uterus in the 100 mg/kg/day group compared with the control was incidental.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical signs
dead fetuses
early or late resorptions
effects on pregnancy duration
food consumption and compound intake
food efficiency
gross pathology
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was one foetus from the 1000 mg/kg/day group (dam no. 172) with an omphalocele and malrotated hindlimb. These findings are part of the background of changes for the strain of rat and were considered incidental due to their isolated nature.
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Litter data

- Pregnancy incidence:There were 22, 20, 21 and 19 pregnant females at terminal caesarean in the control, 100, 300 and 1000 mg/kg/day groups, respectively, all of which had viable foetuses.
- Pre-implantation data: The pre-implantation data (corpora lutea count, number of implantations and the corresponding percentage pre-implantation loss) were comparable in all groups.
- Post-implantation data: There was no test item-related effect on embryo-foetal survival in any group. Mean live litter size was consequently comparable in all groups.
- Foetal data: Mean foetal weight was comparable in all groups. There was no test item-related influence on foetal sex ratio in any group.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
Abnormalities:
no effects observed
Developmental effects observed:
no

The number of foetuses (litters) submitted to the different examinations were as follows:

 Group  1  2  3  4
 External examinatiom  259 (22)  205 (20)  233 (21)  221 (19)
         
 Visceral examination        
 - Body  137 (22)  107 (20)  122 (21)  116 (19)
 - Head  122 (22)  98 (20)  111 (21)  105 (19)
         
 Skeletal examination        
 - Body  137 (22)  107 (20)  122 (21)  116 (19)
 - Head  137 (22)  107 (20)  122 (21)  116 (19)
Executive summary:

Under the experimental conditions of the study (OEDC TG 414), oral (gavage) administration of the test item to the pregnant Wistar rat from implantation through to the day before caesarean section at 100, 300 or 1000 mg/kg/day in PEG 400 was well tolerated with no evidence of maternal toxicity in any group. Similarly, there was no evidence of developmental toxicity in any group. The No Observed Effect Level (NOEL) for both maternal and developmental toxicity was the limit dose of 1000 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available study results a classification according to Regulation (EC) No 1272/2008, Annex I is not warranted.

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