Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 March - 13 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
24 April 2014
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No. : 147592
Purity : 92.4%
Name of test material (as cited in study report): (2E)-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl) but-2-en-1-ol
Physical state: colourless to very pale yellow liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Date of manufacture: 29 October 2013
Expiry Date: 28 October 2015
Specific details on test material used for the study:
- Name of test material (as cited in study report): (2E)-ETHYL-4-(2,2,3-TRIMETHYLCYCLOPENT-3-EN-1-YL) BUT-2-EN-1-OL
- IUPAC name: 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-(2E)-buten-1-ol
- CAS No: 106185-75-5
- Common CAS No: 28219-61-6
- EC No: 248-908-8
- Physical state: colourless to very pale yellow liquid
- Analytical purity: trans-isomer 92.4 %
- Impurities: cis-isomer 5.6%, others 2.0%
- Purity test date: 30 October 2013
- Lot/batch No.: 147592
- Expiration date of the lot/batch: 28 October 2015
- Storage condition of test material: Refrigerated (2-8 °C), protected from light and under nitrogen

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: 231-282 g
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; Solid (polycarbonate) bottom cages were used during the acclimatisation and gestation periods; Grid bottomed cages were used during pairing.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: ca. 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: Approximately 50 % of the final volume of vehicle was added to the required amount of test substance and magnetically stirred to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser. A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test substance.
- Frequency of preparation: Weekly
- Storage of preparation: Refrigerated (nominally 2-8 °C)

VEHICLE
- Concentration in vehicle: 20, 60 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity: The homogeneity and stability of formulations in the concentration range 1 to 200 mg/mL were confirmed for 15 days following refrigerated storage (2-8 °C) and for two days following ambient storage (nominally 21 ˚C) as part of another study, Huntingdon Life Sciences (OAD0002).

Achieved concentration: Samples of each formulation prepared for administration on Day 1 and last day of dosing were analysed for achieved concentration of the test substance.
Results: The mean concentrations of test substance in test formulations analysed for the study were within 3 % of nominal concentrations, confirming accurate formulation.
Details on mating procedure:
- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: Ejected copulation plug / sperm in vaginal smear referred to as Day 0 of pregnancy.
- A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
14 days (Days 6-19 p.c.)
Frequency of treatment:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for investigation in this study (0, 100, 300 and 750 mg/kg bw/day) were selected in conjunction with the Sponsor, based on the results of a previously conducted combined repeated dose toxicity study with reproductive/developmental toxicity screening test in the rat (Huntingdon Life Sciences Report No. OAD0002). Considering the signs of excessive maternal toxicity at 1000 mg/kg bw/day, the high dose level for this embryo fetal development study was set at 750 mg/kg bw/day. The doses of 100 and 300 mg/kg bw/day were selected as the low and intermediate dose levels to establish a suitable dose relationship for any treatment-related changes and to give a 3 and 2.5-fold interval between doses, respectively.
- Rationale for animal assignment: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s).
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.
Detailed observations were recorded daily at the following times in relation to dose administration:
At the end of dosing of each group; one to two hours after completion of dosing of all groups; as late as possible in the working day.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and daily from Days 6 to 20 after mating.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

WATER CONSUMPTION: Not recorded

POST-MORTEM EXAMINATIONS: Yes
- Animals were killed by carbon dioxide asphyxiation on Day 20 after mating.
- All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes; Gravid uterine weight (including cervix and ovaries)
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorption sites: Yes
- Number of late resorption sites: Yes
- Number of live fetuses: Yes
- Number of dead fetuses: Yes
Fetal examinations:
Fetuses were killed by chilling on a cool plate (approximately 0 °C).

Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. The sex of each fetus was recorded.
Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.
Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.
Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning for visceral abnormalities. IMS fixed fetuses were processed and stained with Alizarin Red for skeletal development and abnormalities.
Statistics:
See "Any other information on materials and methods incl. tables"
Indices:
Reproductive assessment
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100
Post-implantation loss (%) = [(Number of implantations – Number of live fetuses)/ Number of implantations] x 100
Historical control data:
Historical background data was used to compare the incidences of developmental toxicity.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Signs in relation to dose administration were limited to the 750 mg/kg bw/day group where a low incidence of piloerection (from Day 8-19 of gestation) and underactive behaviour (from Day 13-17 of gestation) were observed 1-2 hours after dose administration but on some occasions the signs persisted to the end of the working day; two females showed hunched posture on a single occasion.
- A dose-dependent incidence of salivation or chin rubbing was also apparent among females receiving 300 or 750 mg/kg bw/day from Day 7/8 of gestation. These findings are commonly observed following oral (gavage) administration and are considered to reflect distaste of the test formulation rather than a direct effect of treatment.
- At routine physical examination, treatment-related signs were limited to a single female receiving 750 mg/kg bw/day which showed signs of piloerection and reduced body tone immediately prior to despatch to necropsy for scheduled termination on Day 20 of gestation. There were no other treatment-related signs observed at routine physical examination.
Mortality:
no mortality observed
Description (incidence):
- Oral administration of test substance to pregnant CD rats was well tolerated and there were no premature deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Females receiving 750 mg/kg bw/day showed statistically significant mean body weight loss of 3 g (compared to mean body weight gain of 3 g in the Control group) between Day 6 and Day 7 of gestation, and statistically significant lower mean body weight gain between Day 12 and Day 13 of gestation (3 g versus 8 g in the Control group). Slightly low weight gain was also apparent from Days 17-20 of gestation (36 g versus 46 g in the Control group). On all other recording occasions, mean body weight gain was essentially similar to or slightly higher than Controls, however overall mean weight gain from Day 6 to Day 20 of gestation was statistically significantly lower than Control (-13 %). There was no effect of treatment on the weight gain of females receiving 100 or 300 mg/kg bw/day.
- At scheduled termination on Day 20 of gestation, the mean gravid uterine weight of females in the 750 mg/kg bw/day group was slightly lower than Control (-7 %) and when overall mean body weight gain was adjusted for the contribution of the gravid uterus, net body weight gain during gestation was statistically significantly lower than Control (-36 %). Mean gravid uterine weight and net mean body weight gain were similar to Control for females receiving 100 or 300 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Following the commencement of treatment on Day 6 of gestation, mean food intake among females in the 750 mg/kg bw/day group was slightly but statistically significantly lower (overall decrease of -12 %) than Control throughout the treatment period. There was no effect of treatment on the mean food intake of females receiving 100 or 300 mg/kg bw/day.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
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:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic abnormalities detected at scheduled termination on Day 20 of gestation.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The mean percentage of post-implantation losses was statistically significantly lower in all treated groups when compared to Controls. All values were within the Historical Control Data (HCD) range and in view of the direction of change, these minor differences were considered fortuitous and unrelated to treatment.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
- All females were found to be pregnant with live young at scheduled termination on Day 20 of gestation.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of early resorptions was statistically significantly lower in all treated groups when compared to Controls. All values were within the Historical Control Data (HCD) range and in view of the direction of change, these minor differences were considered fortuitous and unrelated to treatment.
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
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality:
- Oral administration of test substance to pregnant CD rats was well tolerated and there were no premature deaths.

Clinical signs:
- Signs in relation to dose administration were limited to the 750 mg/kg bw/day group where a low incidence of piloerection (from Day 8-19 of gestation) and underactive behaviour (from Day 13-17 of gestation) were observed 1-2 hours after dose administration but on some occasions the signs persisted to the end of the working day; two females showed hunched posture on a single occasion.
- A dose-dependent incidence of salivation or chin rubbing was also apparent among females receiving 300 or 750 mg/kg bw/day from Day 7/8 of gestation. These findings are commonly observed following oral (gavage) administration and are considered to reflect distaste of the test formulation rather than a direct effect of treatment.
- At routine physical examination, treatment-related signs were limited to a single female receiving 750 mg/kg bw/day which showed signs of piloerection and reduced body tone immediately prior to despatch to necropsy for scheduled termination on Day 20 of gestation. There were no other treatment-related signs observed at routine physical examination.

Body weight:
- Females receiving 750 mg/kg bw/day showed statistically significant mean body weight loss of 3 g (compared to mean body weight gain of 3 g in the Control group) between Day 6 and Day 7 of gestation, and statistically significant lower mean body weight gain between Day 12 and Day 13 of gestation (3 g versus 8 g in the Control group). Slightly low weight gain was also apparent from Days 17-20 of gestation (36 g versus 46 g in the Control group). On all other recording occasions, mean body weight gain was essentially similar to or slightly higher than Controls, however overall mean weight gain from Day 6 to Day 20 of gestation was statistically significantly lower than Control (-13 %). There was no effect of treatment on the weight gain of females receiving 100 or 300 mg/kg bw/day.
- At scheduled termination on Day 20 of gestation, the mean gravid uterine weight of females in the 750 mg/kg bw/day group was slightly lower than Control (-7 %) and when overall mean body weight gain was adjusted for the contribution of the gravid uterus, net body weight gain during gestation was statistically significantly lower than Control (-36 %). Mean gravid uterine weight and net mean body weight gain were similar to Control for females receiving 100 or 300 mg/kg bw/day.

Food consumption:
- Following the commencement of treatment on Day 6 of gestation, mean food intake among females in the 750 mg/kg bw/day group was slightly but statistically significantly lower (overall decrease of -12 %) than Control throughout the treatment period. There was no effect of treatment on the mean food intake of females receiving 100 or 300 mg/kg bw/day.

Gross pathology:
- There were no treatment-related macroscopic abnormalities detected at scheduled termination on Day 20 of gestation.

Litter responses:
- All females were found to be pregnant with live young at scheduled termination on Day 20 of gestation.

Reproductive assessment:
- There was no effect of maternal treatment with test substance at any dose level investigated on litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- or post-implantation losses. Sex ratio, as assessed by the percentage of males per litter, was generally comparable in all groups and in line with expectations.
- More particularly, early resorptions and post-implantation loss were statistically significantly reduced in all treated groups compared to control group but they cannot be considered as an effect of the treatment as they are within the Historical Control Data (HCD).

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Remarks on result:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: Food intake, body weight, and uterus weight
Description (incidence and severity):
The mean food intake of females receiving 750 mg/kg/day was slightly but statistically
significantly lower than Control. The mean weight gain during gestation was
13% lower than Control, mean gravid uterine weight was 7% lower than Control and net
weight gain when adjusted for the contribution of the gravid uterus was 36% lower than
Control.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In all treated groups, a dose dependent marginal decrease in mean male, female and overall fetal weights was apparent with statistical significance and values slightly below the lower limit of the HCD attained in the 300 and 750 mg/kg bw/day groups. As there is no other effects observed, this light decrease could be considered as a non adverse effect.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): In all treated groups, a dose dependent marginal decrease in mean male, female and overall fetal weights was apparent with values slightly below the lower limit of the HCD range in all treated groups and statistical significance attained in the 300 and 750 mg/kg/day groups. As there is no other effects observed, this light decrease could be considered as a non adverse effect.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio, as assessed by the percentage of males per litter, was generally comparable in all groups and in line with expectations.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
Description (incidence and severity):
not relevant for OECD 414
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Across all treated groups there was a slight increase in the incidence of incomplete ossification of sternebrae, sacrocaudal vertebral arches and pelvic bones compared to concurrent control; the mean percentage of fetuses per litter with affected
sternebrae in the 100 and 750 mg/kg/day groups (73.7% and 85.2%, respectively) exceeded the upper limit of the Historical Control Data (HCD) of 69.2%. In addition, the fetal incidence of incomplete ossification of the pelvic bones in the 750 mg/kg/day exceeded the upper limit of the HCD range (12 fetuses in 10 litters). This incomplete ossification was considered likely to relate to the marginal decrease in mean fetal weight throughout the treated groups, representing a slight delay in maturation.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Placental, litter and fetal weights:
- In all treated groups, a dose dependent marginal decrease in mean male, female and overall fetal weights was apparent with statistical significance and values slightly below the lower limit of the HCD attained in the 300 and 750 mg/kg bw/day groups.
- Mean placental weights were unaffected by maternal treatment in all treated groups.

Fetal pathology:
- There was no effect of treatment on the incidence of major abnormalities or minor visceral abnormalities. Across all treated groups there was a slight increase in the incidence of incomplete ossification of sternebrae, sacrocaudal vertebral arches and pelvic bones compared to concurrent control; the mean percentage of fetuses per litter with affected sternebrae in the 100 and 750 mg/kg bw/day groups (73.7 % and 85.2 %, respectively) exceeded the upper limit of the Historical Control Data (HCD) of 69.2 %. In addition, the fetal incidence of incomplete ossification of the pelvic bones in the 750 mg/kg bw/day exceeded the upper limit of the HCD range (12 fetuses in 10 litters). This incomplete ossification was considered likely to relate to the marginal decrease in mean fetal weight, especially at 750 mg/kg bw/day, representing a slight delay in maturation.
A slight increase in the incidence of 13/14 or 14/14 lumbar ribs was also observed across all treated groups compared to concurrent control; the fetal incidence in the 300 and 750 mg/kg bw/day groups slightly exceeded the upper limit of the HCD range (18 fetuses in 10 litters).

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect of the treatment

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 7.8.2/1: Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (g) during gestation

 

Dose (mg/kg bw/day)

 

Body weight on Day 6

Terminal body weight on Day 20

Body weight change Days 6-20

Gravid uterine weight

Adjusted body weight Day 20

Adjusted body weight Day 20

Statistical test

 

Av

Wi

Wi

Wi

Wi

Wi

0 (control)

Mean

288

412

124

91

321

33

SD

13.6

23.8

15.1

15.7

14.7

8.6

N

20

20

20

20

20

20

100

Mean

291

416

125

94

322

31

SD

13.8

23.4

14.0

11.2

18.7

9.6

N

20

20

20

20

20

20

300

Mean

289

414

124

94

320

30

SD

15.3

24.4

13.0

12.0

17.3

6.7

N

20

20

20

20

20

20

750

Mean

285

391**

106**

85

306*

21**

SD

16.6

23.8

15.4

8.8

20.5

12.4

N

20

20

20

20

20

20

 

Table 7.8.2/2: Food consumption - group mean values (g/animal/day) during gestation

 

Dose (mg/kg bw/day)

 

Day 0-2

Day 3-5

Day 6-9

Day 10-13

Day 14-17

Day 18-19

Statistical test

 

Av

Av

Wi

Wi

Wi

Wi

0 (control)

Mean

25

26

22

25

28

27

SD

2.6

2.9

2.2

2.5

2.4

2.5

N

20

20

20

20

20

20

100

Mean

25

26

23

25

28

27

SD

2.7

2.4

2.4

2.6

2.6

3.6

N

20

20

20

20

20

20

300

Mean

25

27

22

25

27

27

SD

1.9

2.3

2.7

2.5

2.1

2.6

N

20

20

20

20

20

20

750

Mean

24

25

19**

23*

25**

23**

SD

2.8

2.9

2.6

3.3

3.3

4.6

N

20

20

20

20

20

20

 

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.

Wi: Treated groups compared with Control using Williams’ test.

* p<0.05; ** p<0.01

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 300 mg/kg bw/day. The NOAEL for embryo-fetal development was considered to be 750 mg/kg bw/day in rats.
Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, (2E)-ETHYL-4-(2,2,3-TRIMETHYLCYCLOPENT-3-EN-1-YL) BUT-2-EN-1-OL was administered by oral (gavage) to groups of mated female Crl:CD(SD) rats (20/dose) at the dose levels of 0, 100, 300 and 750 mg/kg bw/day from Days 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. 

Oral administration of test substance to pregnant CD rats from Day 6 to Day 19 of gestation at doses up to 750 mg/kg bw/day was well tolerated and there were no mortalities. Signs in relation to dose administration were limited to the 750 mg/kg bw/day group where a low incidence of piloerection and underactive behaviour were observed 1-2 hours after dose administration but on some occasions the signs persisted to the end of the working day; two other females showed hunched posture on a single occasion. At routine physical examination, treatment-related signs were limited to a single female receiving 750 mg/kg bw/day which showed signs of piloerection and reduced body tone immediately prior to despatch to necropsy for scheduled termination on Day 20 of gestation. The mean food intake of females receiving 750 mg/kg bw/day was slightly but statistically significantly lower than Control throughout the treatment period and was associated with lower mean body weight during the same period, occasional periods of slightly reduced mean body weight gain, and mean body weight loss following the administration of the first dose. Overall mean weight gain during gestation was statistically significantly lower (-13 %) than Control, mean gravid uterine weight was 7 % lower than Control and net weight gain when adjusted for the contribution of the gravid uterus was 36 % lower than Control. There was no effect of treatment with test substance on the body weight gain, gravid uterine weight or food intake or liver weight of females receiving 100 or 300 mg/kg bw/day. There were no treatment-related macroscopic abnormalities detected at scheduled termination on Day 20 of gestation.

 

Litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- or post-implantation losses and sex ratio were unaffected by maternal treatment. In all treated groups mean fetal weights were marginally low when compared with Controls, with statistical significance attained in the 300 and 750 mg/kg bw/day groups. Mean placental weights were unaffected by maternal treatment at all dose levels investigated.

 

There was considered to be no adverse effect of maternal treatment on embryo-fetal development, and the incidence of major and minor visceral abnormalities showed no relationship to maternal treatment. In all treated groups there was a slightly higher incidence of incompletely ossified sternebrae, sacrocaudal vertebral arches and pelvic bones compared to concurrent control, which was considered likely to relate to marginally low mean fetal weight. The mean percentage of fetuses per litter with affected sternebrae in the 100 and 750 mg/kg bw/day groups (73.7 % and 85.2 %, respectively) exceeded the upper limit of the Historical Control Data (HCD) of 69.2 %. In addition, the fetal incidence of incomplete ossification of the pelvic bones in the 750 mg/kg bw/day exceeded the upper limit of the HCD range (12 fetuses in 10 litters). These minor skeletal variants were considered to reflect a slight delay in development, indicated by the marginally decreased mean fetal body weight in the treated groups. A slight increase in the incidence of 13/14 or 14/14 lumbar ribs was also observed across all treated groups compared to concurrent control; the fetal incidence in the 300 and 750 mg/kg bw/day groups slightly exceeded the upper limit of the HCD range (18 fetuses in 10 litters).

 

Therefore, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 300 mg/kg bw/day. The NOAEL for embryo-fetal development was considered to be 750 mg/kg bw/day in rats.