Allyl heptanoate

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-03 to 2010-02-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-11-12

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Allyl heptanoate

EC Number:

205-527-1

EC Name:

Allyl heptanoate

Cas Number:

142-19-8

Molecular formula:

C10H18O2

IUPAC Name:

allyl heptanoate

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: (P) 9-10 wks
- Weight at study initiation: (P) Males: 311.0-443.8 g; Females: 215.6-275.5 g
- Housing: Except during the mating period, the parental males and females were kept singly in MAKROLON cages (type III) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 15 cm.
- Diet: Commercial ssniff® R-Z V1324; ad libitum. Food residue was removed and weighed.
- Water: Tap water (in drinking bottles) was offered ad libitum.
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C (maximum range)
- Humidity (%): 55% ± 15%
- Photoperiod: 12 hours dark/light cycle

IN-LIFE DATES:
- Termination of the in-life phase: 2010-01-01

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was diluted in the vehicle to the appropriate concentrations and was administered orally (5 mL/kg b.w./day) once daily. The test item-vehicle mixtures were freshly prepared every day.

VEHICLE
- Volume of vehicle (if gavage): 5mL/kg body weight/day
- Lot/batch no. (if required): 90903229

Details on mating procedure:

- M/F ratio per cage: monogamous (1 male and 1 female rat per cage); 4 groups of sexually mature male and female rats were randomly paired for mating.
- Length of cohabitation: until copulation occurred or 2 weeks had elapsed
- Proof of pregnancy: Each morning, females were examined for presence of sperm in the vaginal lavage or for the presence of a vaginal plug referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing: replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
This procedure was repeated until at least 8 pregnant dams were available for each group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and concentration were analysed immediately after preparation of the mixture as well as 8 and 24 hours after storage of the test item preparations at room temperature and during the last administration of the test item to the group, always before administration to the last animal/dose level group.

Duration of treatment / exposure:

Males: 2 weeks prior to mating, during the mating period and approximately 2 weeks post mating up to and including the day before sacrifice. Treatment of the males was conducted over a period of 29 days.
Females: Throughout the study beginning 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.

Frequency of treatment:

once daily

Details on study schedule:

indicated under "duration of treatment"

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
10 mg/kg body weight/day
Basis:
nominal conc.
(low dose)

Doses / concentrations 2

Remarks:

Doses / Concentrations:
30 mg/kg body weight/day
Basis:
nominal conc.
(intermediate dose)

Doses / concentrations 3

Remarks:

Doses / Concentrations:
100 mg/kg body weight/day
Basis:
nominal conc.
(high dose)

No. of animals per sex per dose:

80 animals (40 males and 40 females), a sufficient number in order to grant at least 8 pregnant females per group.
10 male and 10 female rats were assigned to one of the 4 test groups, respectively.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for this study were selected based on the results of a 7-day dose-range finding study.

Positive control:

No positive control substance was tested.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed for clinical signs at least once daily throughout the test period. The frequency was increased when signs of toxicity were observed. Relevant behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality, were recorded. Animals which died or were sacrificed prematurely during the study would have been necropsied as soon as possible after exitus. However, no parental animal died prematurely or were prematurely sacrificed.

BODY WEIGHT: Yes
- Time schedule for examinations: Parental male and female animals were weighed on the first day of dosing, weekly thereafter and at study termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum during lactation. Body weights were recorded individually for each adult animal.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption of each animal was recorded weekly during the pre-mating period, daily during gestation and on day 4 post-partum. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Drinking water consumption was monitored daily by visual appraisal throughout the study.

OTHER: Yes
The females were allowed to litter normally. The day on which parturition commenced was designated as day 0 of lactation. The pre-coital time, the number of pregnancies, and the duration of gestation in days were evaluated.
Evaluation/parameters:
- Corpora lutea: number per dam, absolute number per group, mean per group
- Implantation sites: number per dam, distribution in the uterine horns, absolute number per group, mean per group
- Number of pups absolute: at birth (alive and dead), after 4 days of life
- Number of pups per dam: at birth
- Number of male and female pups: at birth, after 4 days of life
- Offspring sex ratio
- Number of stillbirths: absolute, per dam

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Number and sex of pups: Live pups were counted and the sex was determined.
- Stillbirths, live births, postnatal mortality, presence of gross anomalies: Each litter was examined as soon as possible after delivery to establish the number of stillbirth, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities.
- Weight gain: Live pups were weighed individually within 24 hours of parturition (day 0 or 1 postpartum) and on day 4 post-partum during lactation.
- Physical or behavioural abnormalities: Any abnormal behaviour of the pups was recorded.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after a minimum total dosing period of 28 days.
- Maternal animals: Dams with offspring were sacrificed on day 4 post-partum, or shortly thereafter. Females which did not show any evidence of copulation were sacrificed 24 days after the last day of mating.

GROSS NECROPSY
- All animals were examined macroscopically for any abnormalities or pathological changes.
- Male animals: Testes and epididymides of all male adult animals were weighed. Testes, epididymides, accessory sex organs (coagulating gland, prepuital gland, prostate, seminal vesicle) and all organs showing macroscopic lesions of all adult males were preserved. The testes and epididymides were preserved in Bouin’s fixative; the remaining tissues were preserved in 7% buffered formalin.
- Female animals: The number of corpora lutea and implantation sites was recorded in the female adult animals. Special attention was paid to the organs of the reproductive system. Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI. The ovaries, accessory sex organs and all organs showing macroscopic lesions of all adult femals were preserved in 7% buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histopathologic examination was performed on the following organs and tissues of the animals of group 4 (high dose) and group 1 (control) following H&E and PAS staining:
- ovaries
- testes
- epididymides

Postmortem examinations (offspring):

SACRIFICE
- Dead pups and pups killed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined

Statistics:

For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out, limit of significance was p ≤ 0.01.
For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or chi2-test with Yates' correction for continuity, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01) were employed.

Reproductive indices:

For each group the following indices were determined:
a) Fertility Index Female = number of pregnancies/number of matings x 100;
b) Gestation Index = number of litters with live pups/number of pregnancies x 100.

Offspring viability indices:

For each litter and group the following indices were determined:
a) Live birth Index = number of pups born alive/total number of pups born x 100;
b) Sex Ratio = number of males/number of females;
c) Percentage by Sex = number of males (females)/total number of animals x 100;
d) Viability Index = number of pups alive on day 4/number of pups alive and kept on day 0 x 100;
e) Post-implantation loss = implantations - no. pups born alive/implantations x 100.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No test item-related mortality was noted. Treatment with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day led to salivation in 3/10 female rats on a few test days during the pre-mating, mating and/or gestation period.
One female animal (female no. 31) treated with 10 mg Allyl heptanoate (Sym09/611041)/kg b.w./day died prematurely and was found dead in the morning of test day 16, i.e. one day after start of the mating period. At necropsy, dark red discoloured lungs were noted in this female rat, hence, the death is considered to be possibly caused by misgavage. Additionally, the death is not regarded to be test item related as none of the intermediate- or high-dosed animals died prematurely during the course of the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males: No test item-related changed were noted.
Females: Treatment with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day led to a slightly increased mean body weight in the female animals starting in test week 2 of the pre-mating period lasting until the end of the gestation period (statistically significant on gestation days 3 to 9 and 12 to 15).

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
No influence was noted on the food intake of males and females during the pre-mating period and of females during the pre-mating, gestation and lactation periods at any of the tested dose levels.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
no data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Examination of female fertility: No test item-related influence was noted at any of the tested dose level.
Evaluation of the pre-coital time: No test item-related influence was noted.
Evaluation of female reproduction parameters: There were no test item-related differences in any of the reproduction parameters examined between the control group and the animals treated with 10, 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No test item-related influence was noted for the relative and absolute epididymides and testes weights of the male animals treated with 10, 30 or 100 mg Allyl heptanoate/kg b.w./day.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic post mortem findings: Treatment with 30 mg Allyl heptanoate (Sym09/611041)/kg b.w./day led to a white flocculation in the urinary bladder in 2/10 male rats and a partly thickened liver (adhered to diaphragm) in 1/10 female rats. A white flocculation in the urinary bladder was noted in 4/10 males treated with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day. The liver of 6/10 high dosed females was indurated and/or thickened. Several yellow foci were noted in five of these females, reticulated yellowish discolourations were recorded in one female animal.
The spleen was enlarged in 5/10 high-dosed females.
No test item-related influence was noted on the absolute and relative epididymides and testes weights of male animals.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The histomorphological examination of ovaries, testes and epididymides of the parental animals treated with 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day did not reveal any microscopical changes.

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEL

Remarks:

reproductive

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

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 specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Details on results (F1)

VIABILITY (OFFSPRING)
No test item-related mortality occurred.

CLINICAL SIGNS (OFFSPRING)
No abnormal behaviour was noted.

BODY WEIGHT (OFFSPRING)
No test item-related influence was noted on the mean and total litter weight at any dose level.

SEXUAL MATURATION (OFFSPRING)
no data

ORGAN WEIGHTS (OFFSPRING)
no data

GROSS PATHOLOGY (OFFSPRING)
External examinations at dissection revealed no external abnormalities in any of the pups examined.

HISTOPATHOLOGY (OFFSPRING)
no data

Effect levels (F1)

Dose descriptor:

NOEL

Remarks:

developmental

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item-related influence was noted at any of the tested dose levels on the growth and development of the offspring from conception to day 4 post-partum.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

- Dose verification (content of Allyl heptanoate (Sym09/611041) in samples in the concentration range of 20 to 200 mg/mL) was successful.

- Homogeneity of samples during the application period was given.

- Stability of the test item Allyl heptanoate (Sym09/611041) over a period of 113 days at -20 ± 2 °C was confirmed (sampling at LPT Laboratory of Pharmacology and Toxicology until further sample dilution and analysis at the test facility).

For further information please refer to the appendix of this robust study summary.

Applicant's summary and conclusion

Conclusions:

Under the present test conditions the following effect levels were noted for the test item Allyl Heptanoate (Sym09/611041) administered during the pre-mating and mating periods to parental males, during the pre-mating, mating, gestation and lactation periods until day 3 post-partum to parental female animals.
- Parental generation: The no-observed-effect level (NOEL) was 10 mg/kg b.w./day, p.o.
- Effects on the development of the conceptus and the F1 offspring (pups): The no-observed-effect level (NOEL) was above 100 mg/kg b.w./day, p.o.

Executive summary:

The results indicate that the content of Allyl heptanoate in the dosing suspensions corresponded reasonably well with the nominal concentrations in the concentration range of 6 to 20 g/L, and were shown to be stable and homogeneous during the application period.

Three dose levels (10, 30 and 100 mg/kg b.w./day) of the test item Allyl Heptanoate (Sym09/611041) were administered during the pre-mating and mating periods to parental males, and during the pre-mating, mating, gestation and lactation periods until day 3 post-partum to parental female animals.

In parental animals, macroscopic changes were noted in the urinary bladder (males) or in the liver and spleen (females) of the animals treated with 30 or 100 mg Allyl heptanoate (Sym09/611041)/kg b.w./day. The body weight of the high-dosed female rats (100 mg/kg) was slightly increased during the pre-mating and gestation period. Hence, the no-observed-effect level (NOEL) was 10 mg/kg b.w./day, p.o.

No test item-related influence was noted at any of the tested dose levels on the growth and development of the offspring from conception to day 4 post-partum. Hence, the no-observed-effect level (NOEL) was above 100 mg/kg b.w./day, p.o.