Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Oct 2013 - 20 Mar 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): C6SFCLA
- Physical state: clear colourless liquid
- Lot/batch No.: G2X01
- Stability under test conditions: stable during the experimental period (measured by infrared absorption spectrometry)
- Storage condition of test material: at room temperature, dark place in a tight container

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hino Breeding Center, Charles River Laboratories Japan Inc.
- Age at study initiation: 10 weeks
- Weight at study initiation: 366.9 - 416.0 g (males), 219.6 - 253.8 g (females)
- Assigned to test groups randomly: yes, under following basis: body weight distribution
- Fasting period before study: no
- Housing: in stainless steel cages and polymethylpentene cages (females during gestation and lactation period)
Before grouping: one animal per cage
After grouping: one male and one female per cage during the mating period, one dam and its itter per cage during the lactation period, one animal per cage for the other periods.
- Diet: pelleted diet CRF-1, Oriental Yeast Co., Ltd., ad libitum
- Water: tap water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5-24.3
- Humidity (%): 44.2-64.6
- Air changes (per hr): 10-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: olive oil containing 10% cremophor
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
4 mg/mL: Test substance was weighed, filled up to 50% of the final volume of the vehicle and was mixed using a magnetic stirrer. The vehicle was added to the measuring glass to adjust the final volume, and mixed by gentle turning.
0.16 and 0.8 mg/mL: Before use, the required amount of 4 mg/mL dosing formulation was placed in a measuring cylinder. The final volume was adjusted by adding a proper quantity of the vehicle to required concentrations of 0.16 and 0.8 mg/mL. The dosing formulation after preparation was mixed a few times by end-over-end rotation and taken into a brown glass vial.

VEHICLE
- Concentration in vehicle: 0.16, 0.8, and 4 mg/mL
- Amount of vehicle (if gavage): 5 mL
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): single housing
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration was determined based on an internal validated method (study no. P110999) conducted at the test facility by a GC method. Measured initial concentration was 3.939 (RSD%: 0.6). After 8 days measured concentration was 3.951 (RSD%: 0.6).
Duration of treatment / exposure:
(P) Males: 42 days (beginning during 15 days of pre-mating period)
(P) Females: maximum period of 54 days (15 days pre-mating until day 3 of lactation). The females not successfully mated was kept until day before the necropsy.
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0.8, 4, 20 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a preliminary study with 20, 30, and 40 mg/kg bw/day 1 female died on day 1 of lactation in the high dose group. No further effects were observed in the parent and F1 generation. Furthermore in a 28-day study a NOAEL of 10 mg/kg bw/day was established for repeated dose toxicity. Thus, 20 mg/kg bw/day was chosen as the high dose level in the main study.

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (before and after dosing) during the reatment period and once a day at the necropsy day

BODY WEIGHT: Yes
- Time schedule for examinations:
males: day 1, 8, 15, 22, 29, 36, and 43
females: day 1, 8, 15; day 0, 7, 14, and 20 of gestation; day 0 4 of lactation

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Vaginal smears were collected with swabs from all females in the morning (same time, everyday) from the initial dosing day to the day of successful copulation or end of the mating period to confirm the estrous cycle. The estrous cycle was classified into diestrus (D), proestrus (P), estrus (E) and metestrus (M). The mean estrous cycle and the number of estrous period during the test period were calculated.
Sperm parameters (parental animals):
Parameters examined in male parental generation: testis weight, epididymis weight
Litter observations:
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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at day 43
- Female animals: All surviving animals at day 4 of lactation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
Males: Testes and epididymides were weighed. Heart, testes, epididymides, prostate (ventral) and seminal vesicle (including coagulating gland)) were fixed.
Females: Ovaries were weighed. Heart, ovaries and gross lesions were fixed.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at day 4 post-partum.
- These animals were subjected to external examinations for gross abnormalities.
Statistics:
Sex ratio, Copulation index, fertility index, gestation index, delivery index: Chi-Square test
Group mean and SD: Bartlett´s test, Dunnett´s multiple comparison test, Steel´s multiple comparison test
Histopathology: Fisher exact test
Implantation index, stillborn index, external anomaly index, external anomaly typing index, live birth index, viability index: Wilcoxon´s rank sum test
Reproductive indices:
copulation index: (no with successful copulation/no cohabited)x100
fertility index: no pregnant/no successful copulation)x100
gestation length: days until completion of delivery from day 0 of gestation
implantation index: no implantations/no corpora lutea)x100
gestation index: no of pregnant animals delivered live offsprings/no of pregnant animals)x100
delivery index: no of delivered offsprings/no of implantations)
Offspring viability indices:
live birth index: (no of live offspring at birth/no of implantations)x100
stillborn index: no of stillborn/total no of delivered offspring)x100
viability index: no of live offspring on day 4/no of live offspring at birth)x100
sex ratio: no of male live offspring/no of female live offspring
External anomaly index: (no of offspring with external anomaly/no of observed offspring)x100
External anomaly typing index: (no of offspring with external anomaly by each type/no of observed offspring)x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
20 mg/kg bw/day: 2 dead females
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
20 mg/kg bw/day: degeneration/necrosis in the myocardial cell

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
2 females of the 20 mg/kg bw/day group died on day 1 and 2 of lactation. Mass was noted in abdominal subcutis in one dam (0.8 mg/kg bw/day) from day 17 of gestation. This change was not judged to be treatment-related as there was no dose-dependency.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No significant difference was observed. High values of food consumption were observed in males of the high dose group on days 8 and 15, but was considered to be not toxicologically significant.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No significant difference was observed.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No significant difference was observed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No significant difference was observed. In one female of the high dose group no mating was confirmed (no histopathological finding in the ovary and testis/epididymides of the male counterpart were observed). Diestrus continued after cohabitation in 1 female of the 4 mg/kg bw/day group, which was seen as incidental.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No significant difference was observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Edema were found in the lungs of the death females. No further abnormaliteis were observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Moderate and diffuse degeneration/necrosis in the myocardial cell, and moderate and diffuse inflammatory cellular infiltration in the myocardium were noted in the dead dams. In addition, mild/moderate and diffuse fibrosis in the myocardium was noted in the dams. In the alveolus of lung, moderate oedema was noted in the dead dams. In 9 surviving males and 7 surviving females of the high dose group similar effects were observed but in mild severity. Fibrosis in the myocardium was noted in 1 male and 6 females of the 20 mg/kg bw/day group.
Several changes were noted in all groups including the control group and were thus seen as incidental.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related changes were observed at the highest dose tested.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Death and degeneration/necrosis in the myocardial cell at the high dose of 20 mg/kg bw/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Low values of birth index were noted in 3 dams of the 20 mg/kg bw/day group (75%, 71.43%, and 72.73%). These changes were within the normal range of the historical control data and were judged as non treatment-related.

CLINICAL SIGNS (OFFSPRING)
No effects were observed.

BODY WEIGHT (OFFSPRING)
No effects were observed.

GROSS PATHOLOGY (OFFSPRING)
No effects were observed.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related changes were observed at the highest dose tested.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Parameter

Controls

0.8 mg/kg bw/day

4 mg/kg bw/day

20 mg/kg bw/day

Histopathology

 

 

 

Fibrosis, myocardium, diffuse, mild

0

0

0

0

0

0

1

6*

 

Cell infiltration; myocardium, inflammatory, diffuse, mild

0

0

0

0

0

0

9*

7*

 

Cell infiltration; myocardium, inflammatory, focal, mild

3

0

1

0

4

0

1

0

 

Degeneration/necrosis; myocardial cell, diffuse, mild

0

0

0

0

0

0

9*

7*

Reproductive Performance

 

Fertility index

%

100

100

100

100

Number of implantation sites

Mean

14.67±1.44

14.25±1.36

14.67±0.78

15.18±2.23

Number of corpora lutea

Mean

15.5±1.62

15.17±2.17

15.67±1.5

15.64±2.46

Implantation index

%

94.9

94.6

94.0

97.2

Delivery index

%

94.3

97.3

94.2

88.3

Birth index

%

93.7

97.3

93.7

85.8

Stillborn index

%

0.60

0

0.69

3.0

Gestation index

%

100

100

100

100

Litter size

Mean

13.8±1.7

13.8±1.1

13.8±1.4

13.1±2.8

Sex ratio

m/f

1.58

1.34

1.32

1.12

Viability index

%

98.6

97.2

98.8

100

Copulation index

%

100

100

100

91.7

*: p<0.01

Applicant's summary and conclusion