Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to internationally recognized guidelines and conformed to required GLP standards.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): undecenyl methoxycrylene
- Chemical name: 2-cyano-3-(4'-methoxyphenyl)-3-phenylpropenoic acid, 10-undecen-1-ol ester
- Physical state: amber coloured viscous liquid
- Analytical purity: 99%
- Lot/batch No.: ABP5032410
- Date received: 12 April 2010
- Expiration date of the lot/batch: 12 April 2012
- Storage condition of test material: room temperature in the dark
- Other: The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.

Test animals

Species:
rat
Strain:
other: Wistar:HsdHan:WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
See Section 7.5.1 of this IUCLID file for complete details.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
(Arachis Oil BP)
Details on exposure:
See Section 7.5.1 of this IUCLID file for complete details.
Details on mating procedure:
On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See Section 7.5.1 of this IUCLID file for complete details
Duration of treatment / exposure:
See Section 7.5.1 of this IUCLID file for complete details
Frequency of treatment:
daily
Details on study schedule:
See Section 7.5.1 of this IUCLID for complete details
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bwt/day
Basis:
other: nominal administered
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
See Section 7.5.1 of this IUCLID for complete details
Positive control:
None

Examinations

Parental animals: Observations and examinations:
The following observations are relevant to the reproductive effects portion of the OECD 422 guideline. Please refer to Section 7.5.1 of this IUCLID file for a complete description of all observations and examinations.

Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post
partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Oestrous cyclicity (parental animals):
Not determined
Sperm parameters (parental animals):
The following were removed from animals that were killed at the end of the study: Prostate, Seminal vesicles, Epididymides, and Testes.

Also, sections of testes and epididymides from all control and 1000 mg/kg/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined histopathologically.

Litter observations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post
partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
Pathology
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Refer to Section 7.5.1 of this IUCLID file for a complete desciption of histopathological analyses.
Postmortem examinations (offspring):
All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Data for males and females prior to pairing, and functional performance test data, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Bartletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was
found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test. Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

Probability values (P) were calculated as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
p≥0.05 (not significant)
Reproductive indices:
Reproductive Indices

Mating Performance and Fertility:
The following were calculated from the individual data during the mating period of the parental generation:
i) Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii) Fertility Indices:

For each group the following were calculated:
Mating Index (%) = Number of animals mated / Number of animals paired x 100
Pregnancy Index (%) = Number of pregnant females / Number of animals mated x 100

Gestation and Parturition Data

The following were calculated for individual data during the gestation and parturition period of the parental generation.

i) Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii) Parturition Index The following was calculated for each group:
Parturition Index (%) = Number of females delivering live offspring / Number of pregnant females x 100

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%)

Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

Pre–implantation loss = Number of corpora lutea - number of implantation sites number of Corpora Lutea x 100

Post–implantation loss = Number of implantation sites - Total number of offspring born / Number of implantation sites x 100

ii) Sex Ratio (% males)
Calculated for each litter on Days 1 and 4 post partum: Number of male offspring / Total number of offspring x 100.
Offspring viability indices:
Live Birth and Viability Indices

For each litter as follows:

Live Birth Index (%) = Number of offspring alive on Day 1 / Number of offspring born x 100

Viability Index (%) = Number of offspring alive on Day 4 / Number of offspring alive on Day 1 x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see Section 7.5.1 of this IUCLID file
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see Section 7.5.1 of this IUCLID file
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Section 7.5.1 of this IUCLID file
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

See Section 7.5.1 of this IUCLID file

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no treatment-related effects on reproductive performance.

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)

No treatment-related effects observed in litter weights, offspring weights or surface righting measurements.

There were no treatment-related macroscopic abnormalities detected in interim death or interim kill offspring.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: There were no significant treatment-related effects on F1 offspring.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Summary of Effects on Reproductive Performance

Observations Dose Level (mg/kg bwt/day)
0 (control) 100 300 1000
Mated Pairs (n) 10 10 10 10
Females showing evidence of copulation (n) 10 10 10 10
Pregnant females (n) 10 9 10 10
Conception days 1-5 (n) 10 9 10 10
Gestation = 22 Days (n) 1 0 0 0
Gestation = 22 1/2 Days (n) 6 4 5 4
Gestation = 23 Days (n) 2 1 2 4
Gestation = 23 1/2 Days (n) 1 4 3 2
Dams with live young born (n) 10 9 10 10
Dams with live young at Day 4 post partum (n) 10 9 10 10
Corpora lutea/dam (mean) 14.5 14.3 14.1 13
Implants/dam (mean) 14.1 13.2 13.1 11.7
Live offspring/dam at Day 1 post partum (mean) 13.1 11.6 11.1 11
Live offspring/dam at Day 4 post partum (mean) 12.7 11.6 11.1 10.8
Sex ratio: % males at Day 1 post partum (mean) 47.9 53.7 47.7 57.4
Sex ratio: % males at Day 4 post partum (mean) 48.5 53.7 47.7 57.7
Litter weight (g) at Day 1 post partum (mean) 73.54 71.79 63.97 66.57
Litter weight (g) at Day 4 post partum (mean) 106.49 107.51 93.21 94.18
Male offspring weight (g) at Day 1 post partum (mean) 5.8 6.52 5.97 6.27
Male offspring weight (g) at Day 4 post partum (mean) 8.62 9.75 8.76 8.99
Female offspring weight (g) at Day 1 post partum (mean) 5.48 6.04 5.7 5.95
Female offspring weight (g) at Day 4 post partum (mean) 8.27 9.23 8.44 8.74
LOSS OF OFFSPRINT
Pre-implantation (corpora lutea minus implantations)
0 7 6 6 6
1 2 1 1 0
2 1 1 1 0
3 0 0 1 0
4 0 0 1 1
5 0 0 0 2
7 0 1 0 0
Pre-natal (implantations minus live births)
0 5 2 2 7
1 3 4 3 1
2 1 1 2 1
3 0 1 0 0
4 0 0 4 0
5 1 1 1 0
Post natal (live births minus offspring alive on Day 4 post partum)
0 7 9 10 8
1 2 0 0 2
2 1 0 0 0
3 0 0 0 0

Applicant's summary and conclusion

Conclusions:
No treatment-related effects were detected on mating performance or fertility in parental animals and no significant effects were detected in the F1 offsping. Therefore, a NOEL for reproductive toxicity was established at 1000 mg/kg/day.