Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2014
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD guideline 422 (combined repeated dose toxicity with reproduction/developmental toxicity)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Hannover RccHan®:WIST rats
Details on test animals and environmental conditions:
The females were nulliparous and nonpregnant
Cage card and individual ear tattoo
Acclimatization: 7 days (males) and 6 days (females) between arrival and treatment start

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The concentration of dose formulation was determined in samples taken from the formulation to be administered to Groups 1 to 4 at the start and end of treatment by GC-FID analysis (on 6 February 2013 and 11 March 2013) and the homogeneity (top/middle/bottom) was also determined in the samples taken at the start of treatment.
Details on mating procedure:
During the mating period the females were housed with males within each dose group (one male:one female) until evidence of copulation was observed for a period of 18 hours.
Duration of treatment / exposure:
Males: two weeks prior to mating and at least up to and including the day before sacrifice (minimum of 28 days).
Females: two weeks prior to mating and at least up to and including the day before sacrifice (day 4 postpartum).
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 - Vehicle (corn oil)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
DAY 0 - F0: First treatment then Pre-pairing
DAY 15 - F0: Mating then Gestation
DAY 37 - Delivery
DAY 41 - F0 females: day 4 postpartum (grip strength, motor activity and sensory reactivity to stimuli)
and F1 necropsy - F0 males (grip strength, motor activity and sensory reactivity to stimuli)
DAY 42 - F0 males (necropsy and blood sampling) - F0: females Day 5 postpartum (necropsy and
blood sampling)

Examinations

Maternal examinations:
During acclimatization (on 1 February 2013), the animals were examined by a veterinary surgeon.
Only animals without any visible signs of illness will be used.
Ovaries and uterine content:
Stage of Estrus: All females during the mating period via a vaginal smear. Smearing of individual females was discontinued when sperm are found.
Fetal examinations:
Offspring Litter Size and Viability: No test-item-related differences were recorded in live and total pups at first litter check. There were no differences in sex ratio with respect to the control group.

Higher postnatal losses on day 4 postpartum were recorded at 300 and 1000 mg/kg, but they were not dose-dependant
Statistics:
The following statistical methods were used to analyze body weight, clinical laboratory data, organ weights, postnatal development and reproduction data, as well as macroscopic findings:
● The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
● The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data cannot be assumed to follow a normal distribution.
● Fisher's exact-test was applied to the macroscopic findings.
● Armitage/Cochran Trend Test for non-neoplastic lesions, if appropriate.
● Anova and Dunnett tests were applied for functional performance tests.
● Bonferroni-test was applied to some reproduction parameters.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
No mortality was recorded in males and females treated with the test item or in the control groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males: There were no differences in body weight or body-weight gain compared to the control group.
Females: Significantly lower body-weight gain was recorded at 1000 mg/kg during prepairing. No differences were recorded in the remaining groups. No differences in body weight or body-weight gain were recorded compared to the control group during pregnancy or lactating in any test-item administered group.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption monitored but no result is presented. No effect expected.
Ophthalmological findings:
not specified
Description (incidence and severity):
monitored but not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: No test-item related differences with respect to the control group were recorded. Animal no. 10, which showed a decrease in red blood cells, hemoglobin and an increase in the reticulocyte and platelet count due to the bleeding from nose recorded as a clinical sign, was not included in the mean.

Females: No noteworthy changes were recorded.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No changes of toxicological relevance were recorded in the clinical pathology analysis or organ weights.

Males: Although slightly lower glucose levels were observed in males receiving 300 and 1000 mg/kg, it was not considered toxicologically relevant because all individual data are within the range considered normal for this parameter. A decrease in creatine kinase values was observed at 1000 mg/kg. The difference was not statistically significant. No noticeable differences were recorded in the remaining parameters.

Females: No noteworthy changes were recorded. An increase in creatine kinase values were observed at 1000 mg/kg. This difference was not statistically significant. The high mean values recorded at 300 mg/kg are related to the high individual values recorded in one animal (no. 63).
Urinalysis findings:
no effects observed
Description (incidence and severity):
See clinical biochemistry findings for creatine kinase observation
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Lower locomotor activity was recorded in males treated at 300 and 1000 mg/kg, but it was not statistically significant.
Immunological findings:
not examined
Description (incidence and severity):
See Hematology for immune cells observations
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All macroscopic and microscopic findings recorded were considered to be within the range of normal background lesions that may be seen in rats of this strain and age and under the experimental conditions used in this study. The yellowish/whitish/pale discoloration of the gastric mucosa was not correlated with histological findings and was considered to be linked to the color of administered vehicle.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test-item-related differences in pre- and postimplantion losses
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
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
Description (incidence and severity):
No treatment-related differences were recorded in the percentage of mating, gestation index, fertility index or conception rate in females at the three doses with respect to the control group.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Remarks:
male and female
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
histopathology: non-neoplastic
pre and post implantation loss
effects on pregnancy duration
changes in pregnancy duration
changes in number of pregnant
other: developmental toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): 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
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related differences from the control group were recorded in the mean of total dead pups
in the first litter check and living pups. The number and mean of dead pups at 1000 mg/kg was lowe
r than in the remaining groups (1 pup vs 20 pups). Therefore, the live birth index was significantly
higher at 1000 mg/kg compared to the control group.
During the first 4 days postpartum, the percentage and mean of postnatal losses was slightly higher at
300 and 1000 mg/kg. The resulting viability index was lower than in the control group (78 and 88.2%,
respectively, versus 95.1% in the Control group). These differences were statistically significant at
300 mg/kg.
No differences were recorded at 100 mg/kg. No differences in sex ratio were recorded.

Higher postnatal losses on day 4 postpartum were recorded at 300 and 1000 mg/kg, but they were not dose-dependant
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
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
changes in postnatal survival
external malformations
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The dose of 1000 mg/kg can be considered the NOEL.
No differences from the control group were recorded in mean body weight.
No morphological findings were recorded.
Regarding motor development, no differences from the control group were recorded.
No macroscopic findings were recorded at necropsy.
Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

The test item Phytosqualan was administered orally (by gavage) to three groups, each consisting of ten male and ten female RccHan®:WIST rats, daily for at least 4 weeks (including two weeks prior to mating, through mating, pregnancy and early lactation for females) at the following dose levels: 0, 100, 300 and 1000 mg/kg/day.

The dose of 1000 mg/kg can be considered the NOEL. No differences from the control group were recorded in mean body weight. No morphological findings were recorded. Regarding motor development, no differences from the control group were recorded. No macroscopic findings were recorded at necropsy.