Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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
Details on species / strain selection:
Wistar Hannover RccHan®:WIST rats
Sex:
male/female
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
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.
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.
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
Details on study schedule:
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)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and 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.
Oestrous cyclicity (parental animals):
Stage of Estrus: All females during the mating period via a vaginal smear. Smearing of individual females was discontinued when sperm are found.
Sperm parameters (parental animals):
Spermatology Stage Evaluation: A qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control and high-dose groups was performed. If test-item-related morphologic changes are detected in a high-dose animal, the mid- and lowdose groups will be examined to establish a no-effect level, if possible, at extra cost.
Litter observations:
During the lactation phase, clinical observations were recorded daily for all surviving offspring,
together with litter size and offspring weights; and surface righting reflex was assessed.
Postmortem examinations (parental animals):
The necropsy included the examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and the observation of the organs both in situ and after evisceration.
Organ weights were recorded on the scheduled necropsy dates for all animals and their organ to terminal-body-weight ratios and organ to brain-weight ratios were determined
Postmortem examinations (offspring):
The necropsy included the examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and the observation of the organs both in situ and after evisceration.
Organ weights were recorded on the scheduled necropsy dates for all animals and their organ to terminal-body-weight ratios and organ to brain-weight ratios were determined
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.
Reproductive indices:
1. Mating Performance and Fertility/ The following parameters were calculated from the individual data during the mating period of
the parental generation:
i) Pre-coital time
ii) Fertility Indices
iii) Implantation Losses (%)

2. Gestation and Parturition Data: The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
i) Gestation Length
ii) Gestation Index
Offspring viability indices:
Calculation of Post–natal loss, Viability Index (%) and Live birth index (%)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Males: No clinical signs obnserved at 100, 300 and 1000 mg/kg

Females: No clinical signs were observed during the prepairing period. Female 70 at 300 mg/kg had alopecia during pregnancy and lactating and female 79 at 1000 mg/kg had chromodacryorrhea and hypotrichosis during pregnancy and lactating. Moreover, female 44 from the control group, which started delivery on day 23 of pregnancy,
showed decreased motor activity, abnormal gait, hunched back, paleness, ruffled fur, chromodacryorrhea, diarrhea, hypothermia and palpebral ptosis and was sacrificed for ethical reasons after observing that all pups were dead at delivery. Female 55 at 100 mg/kg, which lost its litter, had hunched back, abnormal gait and ruffled fur on
days 2-3 of lactation.
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
Description (incidence and severity):
Food consumption was similar in all groups.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
Water consumption monitored but no result is presented. No effect expected.
Ophthalmological findings:
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:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Description (incidence and severity):
No effect expected as Fertility: 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.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Treatment with the test item caused no indication of toxicity in the testes. The qualitative examination of the stages of spermatogenesis in the testis did not reveal any test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating performance: Mean precoital time was longer at 100 and 300 mg/kg (4.5 and 4.6 days, respectively), however median precoital time was similar with respect to the control group. No differences were recorded at 1000 mg/kg.

No treatment-related differences from the control group were recorded in the mean of implantation sites per litter or corpora lutea or between right and left horn.

Significantly higher preimplantation losses were recorded at 100 mg/kg. No differences were recorded at 300 or 1000 mg/kg. This finding is not considered test-item related as it is not dose dependent.

No treatment-related differences from the control group were recorded in the percentage of postimplantation losses.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Male : systemic toxicity Female : systemic toxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Male : fertility and mating performance Female : breeding

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality 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 lower 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
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences were recorded between groups or within sexes. In the control group, one runt pup (no. 15 from litter no. 50) was observed and was devoured on day 2 postpartum.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The percentage of pups showing milk in the stomach was similar within groups 1, 2 and 4.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No differences from the control group were recorded in mean body weight
Gross pathological findings:
no effects observed
Description (incidence and severity):
No alterations were recorded in the morphological examination of the pups.
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Regarding postural reflexes in the pups, no treatment-related differences compared to the control animals was recorded in the percentage of fetuses with positive response in the surface-righting reflex (righting reflex).

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Remarks on result:
other: Higher postnatal losses on day 4 postpartum were recorded at 300 and 1000 mg/kg, but they were not dose-dependant.

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

PARENTAL: F0

General Tolerability

No mortality was recorded in any sex. One male was sacrificed after showing signs of illness, such as pallor, hunched back, blood secretion from nose and ruffled fur. This animal had findings related to an incorrect administration such as red-brownish areas in two lobes of lungs.

 

No test-item-related clinical signs were recorded.

 

Functional Performance Test

Slightly lower locomotor activity was recorded in males at 300 and 1000 mg/kg. No differences were recorded in females.

 

No relevant differences from the control group were recorded in the grip strength test in either sex.

 

Sensory Reactivity Assessments

No differences from the control group were recorded.

 

Behavior Assessments

No differences from the control group were recorded.

 

Food Consumption

No relevant differences from the control group were recorded.

 

Body Weights

No test-item-related differences from the control group were recorded in males. Lower body-weight gain was recorded in females at 1000 mg/kg during the prepairing period and afterwards body-weight gain was similar to the control group.

 

 

LABORATORY INVESTIGATIONS

Hematology

No relevant differences were observed.

 

Blood Chemistry

No relevant differences were observed.

 

A trend to higher values of creatine kinase was recorded in males and females.

 

REPRODUCTIVE PERFORMANCE

Mating performance

No test item-related differences were recorded in mean or median precoital time.

 

Fertility

No test item-related differences were recorded in the percentage of mating, gestation index, fertility index or conception rate.

 

Reproduction data

No test-item-related differences in corpora lutea, implantation sites, pre- and postimplantion losses or sex ratio were observed with respect to the control group.

 

Breeding data

The length of pregnancy (days) was similar in all groups.

 

The presence of milk on day 1 postpartum was significantly lower at 300 mg/kg.

 

 

FILIAL: PUPS

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.

 

Body Weight

No test item-related differences were recorded.

 

Morphological Examination

No test-item-related alterations were recorded.

 

Motor Development

No test item-related alterations were recorded.

PATHOLOGY

Macroscopic Findings

No findings were recorded in the offspring at necropsy.

 

Small thymus was recorded in all groups, including the control group. However, the number of animals affected was higher at 300 and 1000 mg/kg.

 

All remaining lesions recorded were considered to be within the normal range of background alterations observed in rats of this strain and age and under the experimental conditions used in this study.

 

Organ Weights

No noticeable differences were observed. However, lower thymus weights were recorded in females at 1000 mg/kg

 

Histopathology

No test item-related alterations were recorded.

 

Sperm Stage Evaluation

No test item-related alterations were recorded.

Applicant's summary and conclusion

Conclusions:
For F0 generation

Males:

The dose of 1000 mg/kg can be considered the NOAEL (No Observed Adverse Effect Level) for systemic toxicity.

The dose of 1000 mg/kg can be considered the NOEL (No Observed Effect Level) for fertility and mating performance.

Females:

The dose of 1000 mg/kg can be considered the NOAEL for systemic toxicity.

The dose of 1000 mg/kg can be considered the NOAEL for fertility and mating performance and 100 mg/kg for breeding.

For F1 generation

The dose of 1000 mg/kg can be considered the NOEL.
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.

No mortality was recorded in any sex. No test-item-related differences from the control group were recorded in males. No test item-related alterations were recorded.

The NOEL was 1000 mg/kg/day for male (mating and reproductive performance) and female (breeding).