Allyl heptanoate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

 Oral (OECD 421), rat: NOAEL fertility ≥ 100 mg/kg bw/day

Link to relevant study records

Reference

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

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

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

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"

Remarks:

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

Remarks:

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

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.

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.

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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.

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'

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'

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

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

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.

Reproductive effects observed:

not specified

- 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.

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No 1907/2006. The extended one-generation reproductive toxicity study set out in Annex IX, 8.7.3, of Regulation (EC) No 1907/2006 does not need to be conducted, since the available repeated dose toxicity studies (e.g. 28-day, 90-day and OECD 421 screening study) indicate no adverse effects on reproductive organs or tissues.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The test substance was tested in a reproduction/developmental toxicity screening study according to OECD Guideline 421 and in compliance with GLP (Hansen, 1983). Three dose levels (10, 30 and 100 mg/kg bw/day) of the test substance 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 females. The body weights of females treated at 100 mg/kg bw/day were slightly increased during the pre-mating and gestation period. In both sexes, macroscopic changes were noted at necropsy. White flocculation in the urinary bladder was noted in 2/10 males and a partly thickened liver (adhered to diaphragm) in 1/10 females at 30 mg/kg bw/day. However, these changes were not considered to be adverse. The liver of 6/10 males was indurated and/or thickened at 100 mg/kg bw/day. Several yellow foci and reticulated yellowish discolourations were noted in 4/10 and 1/10 females, respectively. Additionally, the spleen was enlarged in 5/10 females at 100 mg/kg bw/day.

Therefore, the NOEL for systemic toxicity was considered to be 10 mg/kg bw/day.

In parental animals, no test substance-related influence on reproductive function or performance was noted at any of the dose levels tested. Therefore, a NOAEL for parental fertility of ≥ 100 mg/kg bw/day was derived for male and female rats.

Effects on developmental toxicity

Description of key information

Oral (OECD 414), rat: NOAEL developmental ≥ 77.40 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Nov - 19 Apr 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Government of India, Department of Science and Technology, New Delhi, India

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Advinus Therapeutics Ltd, Bengaluru, India
- Age at study initiation: 14 - 15 weeks
- Weight at study initiation: 251.78 - 252.76 g
- Housing: individually in standard polysulfone rat cages (L 425 x B 266 x H 185 mm) on steam sterilized clean corn cob
- Diet: Teklad Certified (2014CM) Global 14% Protein Rodent Maintenance Diet - Powder (Harlan Laboratories B.V., Venray, The Netherlands), ad libitum
- Water: deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier (Eureka Forbes Ltd., Mumbai, India), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 65 - 67
- Air changes (per hr):12 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: The test substance fortified diet was prepared twice during the conduct of the study and were used within the established stability period of 15 days.
- Mixing appropriate amounts with Teklad powder feed
The required quantities of the test substance (1.001 , 4.003 and 15.004 g for low, mid and high dose) were weighed and mixed with 10 mL of acetone. This was mixed manually with approximately 1.0 kg of powder food in stainless steel drum for 2 minutes and then added in portions to the remaining portion of the food (approximately 9 kg) and was mixed in a ribbon blender for 20 minutes.
- Storage temperature of food: The experimental feed was stored in the polyethylene bags within labeled stainless steel drums in the experimental room.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and active ingredient analysis was carried from the samples of first preparation. For this, samples were taken from top, middle and bottom layers of the fortified diet of each dose group to determine the homogeneity and active ingredient of the test substance in the experimental fortified diet. Further, active ingredient analysis was carried out at termination of treatment period (6 days before termination of treatment for batch I). For this, one composite sample from each dose was taken to determine the active ingredient concentration by GC analysis. For the control group, one composite sample was collected as scheduled above. The results of analysis indicated that the test substance concentrations in the fortified diet samples were found to be in acceptable range, as the percent agreement of the analyzed concentrations were within ± 20% of the nominal dose concentration. Homogeneity of the dose formulation was also considered acceptable as the relative standard deviation from three replicates (1 replicate from top layer, 1 replicate from middle layer and 1 replicate from the bottom layer of the dietary preparation) at each dose level were <15%. The test substance was found to be stable in the diet for 15 days at room temperature.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy

Duration of treatment / exposure:

Day 5 - 20 of gestation

Frequency of treatment:

continuously (via diet)

Duration of test:

20 days

Dose / conc.:

100 ppm

Remarks:

equivalent to 6.95 mg/kg bw/day

Dose / conc.:

400 ppm

Remarks:

equivalent to 25.71 mg/kg bw/day

Dose / conc.:

1 500 ppm

Remarks:

equivalent to 77.40 mg/kg bw/day

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a dose range-finding study. The study was carried out using seven Day ‘0’ pregnant rats per group at dose levels of 200, 1000 and 5000 ppm. Rats were treated from GD 5 to GD 20 and observed for clinical signs, mortality, body weight changes and food intake. The caesarean section was performed on GD 20. The dams were observed to obtain maternal and litter data and the fetuses were subjected to external examination. No clinical signs, mortality and morbidity were observed at any of the doses tested. The food consumption was significantly lower at 1000 and 5000 ppm with concomitant reduced body weight gains at 5000 ppm mainly due to palatability reasons. The reduced body weights resulted in lower pregnancy rates at 5000 ppm. However there were no effects on uterine/litter parameters at any of the dose levels tested. Based on the results of the study 100, 400 and 1500 ppm were selected for the main study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on holidays)
- Cage side observations included: mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: All females included in the study were weighed on gestation days 0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 20

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: gross evaluation of placenta, pregnancy status

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: approximately half per litter
- Skeletal examinations: Yes: approximately half per litter
- Head examinations: Yes: approximately half per litter

OTHER: total number of fetuses, number of viable fetuses, number of non-viable fetuses, individual fetal body weight, fetus sex, sex ratio

Statistics:

The data on maternal body weight, body weight change, gravid uterine weight, body weight change corrected for gravid uterine weight, maternal food consumption, number of corpora lutea, number of implantations, litter size, litter weight, male and female fetus number and weight were analyzed using ANOVA model, after testing for homogeneity for intra group variance using Levene’s test.
If the intra group variances are heterogeneous ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, if the group differences were found significant.
Incidence of pre-implantation loss, post implantation loss, number of early, late, total resorptions, was analyzed using Kruskal Wallis.
Overall number and percentage of external, visceral and skeletal malformations, sex ratio, number of dams with any resorptions were analyzed using 2 X 2 Contingency Table.

Indices:

Embryonic resorption index (%) = (No. of early resorptions / No. of implantations) x 100
Fetal resorption index (%) = (No. of late resorptions / No. of implantations) x 100
Pre-implantation loss per group (%) = (No. of corpora lutea - No. of implantations / No. of corpora lutea) x 100
Post-implantation loss per group (%) = (No. of (early + late) resorptions / Total No. of implantations) x 100
Implantation index (%) = (No. of implantations sites / No. of corpora lutea) x 100
Percentage of live fetuses per group (Live fetus index) = (No. of live fetuses / Total No. of fetuses) x 100
Percentage of dead fetuses per group (Dead fetus index) = (No. of dead fetuses / Total No. of fetuses) x 100

Historical control data:

The results of the study will be discussed taking into consideration the historical control data of this lab. Historical control data was obtained from 10 prenatal developmental toxicity studies conducted from 2011 until 2015. Studies were performed in female Wistar rats at the age of 11 -15 weeks.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs in any of the rats at the tested dose levels.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities in any of the rats at the tested dose levels.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weights were not affected by the treatment at the doses of 100 and 400 ppm.
At 1500 ppm, the group means of maternal body weights were significantly reduced on GD 8, 14, 17 and 20. However, these reductions in body weights at 1500 ppm were less than 10% as compared to control group and hence not considered to be adverse.
There was significant reduction in intermittent body weight gains during GD 5 - 8 at 400 ppm, and during GD 5 - 8, 11 - 14, 14 - 17 and 17 - 20 at 1500 ppm as compared to control group.
The maternal body weight gain was significantly reduced during treatment period (GD 5 - 20) and throughout gestation (GD 0 - 20) at 1500 ppm as compared to control group (reduced up to 39% and 34%, respectively).
The corrected body weight gain was also significantly lower at 400 and 1500 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The significant increase in food intake during GD 11 - 14 at 100 ppm was considered as an incidental finding as the maternal body weights were unaffected at this dose.
The food intake was significantly reduced during GD 5 - 8 and 17 - 20 at 400 ppm and during GD 5 - 8, 8 - 11, 11 - 14, 14 - 17 and 17 - 20 at 1500 ppm compared to the vehicle control group.
The food intake was significantly reduced during treatment period (GD 5 - 20) and throughout gestation (GD 0 - 20) at 1500 ppm as compared to control group.
Reduction in food intake was mainly due to palatability reasons and this was associated with reduced body weights in the respective dose groups.

Food efficiency:

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:

no effects observed

Description (incidence and severity):

Mean gravid uterine weights were statistically comparable to the control group at all dose levels and were not affected by the treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological findings in any of the rats at the tested dose levels.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre- and post-implantation loss rates were statistically comparable to the control group at all dose levels and were not affected by treatment with the test substance.

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Description (incidence and severity):

Early and late resorptions were statistically comparable to the control group at all dose levels and were not affected by treatment with the test substance.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses in any of the dose groups.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

All 24 dams of each dose group were sacrificed at cesarean section on GD 20.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

2 rats each of the control and high dose group as well as 1 rat each of the low and mid dose group were non-pregnant at cesarean section on GD 20.

Details on maternal toxic effects:

The maternal uterine parameters which includes mean gravid uterine weights, mean numbers of corpora lutea, implantations, early and late resorptions, pre and post-implantation loss rates and were statistically comparable to the control group at all dose levels and were not affected by treatment with the test substance.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 500 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

necropsy findings

organ weights and organ / body weight ratios

pre and post implantation loss

Key result

Dose descriptor:

NOAEL

Effect level:

>= 77.4 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

necropsy findings

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean fetal weights of male fetuses of the high dose group were significantly lower, however the decrease was within the historical control range and hence considered incidental.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Number of live fetuses was statistically comparable to control group at all the tested doses.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was statistically comparable to control group at all the tested doses.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean litter size was statistically comparable to control group at all the tested doses.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no observations in the control and treatment groups considering normal variants, minor anomalies and major malformations.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Incidences of normal variants were comparable between the control and the treatment groups. However, statistically significant increase in the percent incidences of delayed skeletal ossification and incomplete/poor ossification in some of the bone components was observed in the treatment groups. These findings were randomly distributed across treatment groups and were not dose-dependent. Further, these normal variants are commonly observed in rat fetuses (comparable with historical control data) and were not considered as biological significant.
There was significant increase in dumbbell thoracic vertebral centra 1/13 at 100 and 400 ppm as compared to control group. This finding was considered not treatment-related as the values were comparable to historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were 2 incidences of median lobe extra lobation in liver at 400 ppm and a single incidence of slight kidney renal pelvis dilation at 100 ppm. These variations were commonly observed in colonies of rats from the laboratory and hence were not attributed to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Key result

Dose descriptor:

NOAEL

Effect level:

>= 77.4 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Based on the results of this study, the NOAEL for maternal and fetal developmental toxicity was set at ≥ 1500 ppm (equivalent to 77.40 mg/kg bw/day).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

77.4 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The test substance was tested in an oral feeding developmental toxicity study in Wistar rats according to OECD Guideline 414 and in compliance with GLP (Ramesh, 2016). The dose levels for the main study were selected based on the results of a dose range-finding study performed in Wistar rats. The study was carried out using 7 pregnant females per dose group at dose levels of 200, 1000 and 5000 ppm. Rats were treated from GD 5 to GD 20 and observed for clinical signs, mortality, body weight changes and food intake. The caesarean section was performed on GD 20. No clinical signs, mortality and morbidity were observed at any of the doses tested. The food consumption was significantly lower at 1000 and 5000 ppm with concomitant reduced body weight gains at 5000 ppm mainly due to palatability reasons. The reduced body weights resulted in lower pregnancy rates at 5000 ppm. However there were no effects on uterine/litter parameters at any of the dose levels tested. Based on the results of the study 100, 400 and 1500 ppm were selected for the main study.

In the main study, 24 pregnant Wistar rats per dose group were treated via diet with the test substance from GD 5 to GD 20. The control group received the plain diet. The caesarean section was performed for all rats on GD 20. No mortality and clinical signs were observed at any of the dose levels tested. Maternal body weights were not affected by the treatment at the dose level of 100 and 400 ppm. At 1500 ppm, the group means of maternal body weights were significantly reduced on GD 8, 14, 17 and 20. However, these reductions in body weights at 1500 ppm were less than 10% as compared to control group and hence not considered to be adverse. The maternal body weight gain was significantly reduced during treatment period (GD 5-20) and throughout gestation (GD 0-20) at 1500 ppm as compared to control group and it was 39% and 34% lower, respectively. The corrected body weight gain was also significantly lower at 400 and 1500 ppm. The food intake was significantly reduced during treatment period (GD 5-20) and throughout gestation (GD 0-20) at 1500 ppm as compared to control group. Reduction in food intake was mainly due to palatability reasons and this was associated with reduced body weights in the respective dose groups. Gross pathological examination revealed no abnormalities and no effects were observed on mean gravid uterine weights. Mean numbers of corpora lutea, implantations, early and late resorptions, pre and post-implantation loss rates and were statistically comparable to the control group at all dose levels and were not affected by the treatment with the test substance. The litter parameters which includes total number of fetuses, number of live fetuses, mean litter size, mean fetal weights of female fetuses and sex ratio and were statistically comparable to control group at all the tested doses. Fetal examination revealed significant reduced mean fetal weights of male fetuses. However, the decrease was within the historical control range and hence considered incidental. The fetal external, visceral and skeletal examination revealed no treatment-related signs of teratogenicity or developmental toxicity up to and including 1500 ppm, the highest dose tested.

Based on the results of this study, the NOAEL for maternal and developmental toxicity/teratogenicity was considered to be ≥ 1500 ppm (equivalent to 77.4 mg/kg bw/day).

Justification for classification or non-classification

The available data on toxicity to reproduction and developmental toxicity / teratogenicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.