Ethyl 2-methylbutyrate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Combined repeated dose toxicity study with the reproductive/developmental toxicity screening test (OECD 422)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2-methylbutyrate

EC Number:

231-225-4

EC Name:

Ethyl 2-methylbutyrate

Cas Number:

7452-79-1

Molecular formula:

C7H14O2

IUPAC Name:

ethyl 2-methylbutanoate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): EMB
- Analytical purity: >99.5%
- Lot/batch No.: Batch Number: H3-H-5
- Expiration date of the lot/batch: 04 Aug 2014
- Storage condition of test material: Immediately upon receipt, the test item was registered, then stored at room temperature in accordance with the Sponsors instructions. The complete description of the chemical and physical properties of the test item including stability is the responsibility of the Sponsor.
- Other:
- Supplier: Toyo Gosei Co., Ltd.
- Intended use: Ind. Chemical
- Test item named EMB corresponds to Ethyl 2-methylbutyrate.
- On 12 Mar 2012, four flasks of test item were received labelled “Ethyl 2-methylbutyrate (EMB) Batch No.H3-H-5”. The study monitor confirmed that this test item corresponds to EMB Batch No. H3-H-5”, the name used in the study report.
- Handling instructions for test item: General safety procedures as appropriate for handling of chemicals of unknown hazard potential were applied. For further details about safety, see the material safety data sheet supplied with the test item by the Sponsor.
- Remaining test item: The remaining test item, except the sample to be archived, will be sent to Laboservice, route de la Centrale, 69702, Givors Cedex, where it will be destroyed by incineration, under the responsibility of CERB.
- The certificate of analysis of test item is presented in Appendix A, page 181 of the attached report.

Test animals

Species:

rat

Strain:

other: SPF (Specific Pathogen Free) Sprague-Dawley - Crl: OFA (SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Origin: Charles River Laboratories France, Domaine des Oncins, 69210 L’ARBRESLE Cedex, France.
- Age at study initiation: Age: 10-11 weeks on the day of mating i.e. 8-9 weeks on the day of the first administration.
- Weight at study initiation: Weight: The weight variation of animals used were minimal and did not exceed ± 20% of the mean weight of each sex.
- Housing: Daily observations were undertaken at the time of delivery of the animals and during the period of acclimatisation. Animals were housed individually in cages of standard dimensions with sawdust bedding. During the mating, one male was housed with one female. The animals were placed in an air-conditioned (20-24°C) animal house kept at relative humidity between 45% and 65% (except during the cleaning slot) in which non-recycled filtered air was changed approximately 10 times per hour. No deviations outside of the temperature or hygrometry ranges were reported by the Study Director according to the SOP 5.36. The artificial day/night cycle involved 12 hours light and 12 hours darkness with light on at 7.30 am.
- Diet (e.g. ad libitum): Feeding: RM1 (E)-SQC SDS/DIETEX feed (quality controlled/radiation sterilised) was available ad libitum, except during the fasting experimental period. For pregnant females, RM3 (E)-SQC SDS/DIETEX feed (quality controlled/radiation sterilised) was available ad libitum, except during the fasting experimental period. The certificates of analysis concerning this feed product are included hi Appendix B, page 183 of the attached report. The criteria for acceptable levels of contaminants hi the feed supply were within the limits of the analytical specifications established by the diet manufacturer.
- Water (e.g. ad libitum): Drinking water: Dunking water was available ad libitum in polycarbonate bottles with a stainless steel nipple. A specimen of water is obtained approximately every 6 months and sent to the LAEASE Région Sud Est - 5, avenue Achille Maureau - B .P. 95 - 84703 Sorgues Cedex - France, for analysis. The certificates of analysis are included in Appendix C, page 190 of the attached report. The criteria for acceptable levels of contaminants in the water supplied were within the limits of the analytical specifications.
- Acclimation period: Acclimatisation: A minimum of five days in the laboratory animal house where the experiment took place. Only animals without any visible sign of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): air-conditioned (20-24°C)
- Humidity (%): relative humidity between 45% and 65% (except during the cleaning slot)
- Air changes (per hr): non-recycled filtered air was changed approximately 10 times per hour.
- Photoperiod (hrs dark / hrs light): The artificial day/night cycle involved 12 hours light and 12 hours darkness with light on at 7.30 am.
(No deviations outside of the temperature or hygrometry ranges were reported by the Study Director according to the SOP 5.36).

Other:
- Choice of species: The rat was chosen because of its acceptance as a predictor of toxic effects of drugs in man and the recognition by regulatory authorities that this species is suitable for toxicity studies.
- Sex: Male and female. Females were nulliparous and non-gravid at the beginning of the study.
- Identification: Animals were identified individually, using labelling by ear clips. Pups were identified individually by marker pen.
- Number: 80 animals.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

SIGMA, Batch No. MKBH4894V, Expiry date: May 2017 and Jul 2017

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis was performed on one formulation prepared during the first and on the last week of treatment at each concentation. The concentrations were within +/- 10% of intended concentration except for the last formulation at 100 mg/mL which was +10.7% of intended.

Details on mating procedure:

- Impregnation procedure: cohoused
- 1 M/1 F ratio per cage:
- Length of cohabitation: 7 days
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post coitum

Duration of treatment / exposure:

28-41 days in males, 40-51 days in females

Frequency of treatment:

Once daily

Duration of test:

28-41 days in males, 40-51 days in females

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
General observations: Animals were observed in the home cage before the first dosing and at least once a day during the treatment period. The time of observation was between 1 hour post dose (± 30 min). T

BODY WEIGHT:
Weighing: Animals were weighed on the following days:
• on the day of randomisation
• Before the first administration (on D-1 or D1)
• once a week
• on D0pc, D7pc, D14pc, D20pc (gestation period), D26pc (for females showing no evidence of copulation) and within 24 hours of parturition (D0pp or D1pp) and D4pp for each female

HAEMATOLOGY:
Haematology, coagulation parameters: Prothrombin time, Activated partial thromboplastin time

CLINICAL CHEMISTRY:
Blood sampling: At the end of the dosing period shortly before scheduled necropsy, blood was taken from 5 males and 5 females of each group (randomly selected) and just before euthanasia for moribund animals. Blood samples were taken from the retro-orbital sinus (or abdominal aorta for Female No. 1202509) from animals under isoflurane anaesthesia.

Blood chemistry parameters: ALT, AST, ALP, Urea, Creatinine, Albumin, Total Bilirubin, Total proteins, Glucose, Total cholesterol, Chloride, Potassium, Sodium.

URINALYSIS: At the end of the dosing period shortly before scheduled necropsy, urine was collected from 5 males and 5 females per group (randomly selected). Urine collection was performed individually in metabolism cages for a period of about 16 hours. Food and water were withheld during collection. Animals were given 20 mL/kg of tap water before transferring to metabolism cages. Clinitek Advantus was used for semi-quantitative estimation of pH, protein, glucose, ketones, urobilinogen, bilirubin, specific gravity, blood, leukocytes and colour. Volume was noted. Quantitative estimation of Na, K and Creatinine were performed.

NEUROBEHAVIOURAL EXAMINATION:
Functional and neurobehavioural tests: Once before the first dosing and once a week during the whole study, all animals were observed according to a standardised observation battery for neurobehavioural, neurovegetative or psychotropic signs or neurotoxic effects. Functional and neurobehavioural tests were not performed in females during the gestation period. During the lactation period, in order to avoid hyperthermia of pups, females were removed from the pups for not more than 30-40 minutes. The method is based on an lrwin screen [1] modified by suppressing the graduation of intensity of clinical signs. Animals were observed individually in a cage without sawdust in a quiet room. Clinical signs were observed according to Table 1.6, page 38 of the attached report. The time of observation was at 60 min post dose (± 30 min). The rectal temperature was measured at the end of each observation. The room temperature was between 19°C and 24°C and was recorded at the beginning and at the end of all observations.

Statistics:

Results of functional and neurobehavioural tests, general observations and mortality were expressed as incidence of the various clinical signs within each group and were compared with those of the vehicle using a Fisher’s test at each measurement time.
- Results of body weight and body temperature were expressed as absolute values and as percentage of variation calculated in relation to predose values. Homogeneity of predose values was tested by an analysis of variance. The effects of EMB on body weight and body temperature changes were compared with those of the vehicle using an analysis of variance for repeated measurements with a Dunnetts’ test in case of significance (P≤0.05).
- Results of food consumption were expressed as mean values and in g/animal/day andwere compared with those of the vehicle using an analysis of variance with a Dunnett’s test in case of significance (P≤0.05).
- The mean number of corpora lutea, implantation sites, live pups per group and per female were compared with those of the vehicle using Mann-Whitney test. The mean body weight per litter and per group of live pups was also indicated.
- Organ weights were expressed as absolute values (g) and relative values (g per 100 g of body weight measured on the day of necropsy and g per 100 g of brain weight). Organ weights, quantitative urinalysis and mean clinical pathology results (haematology and blood chemistry) were compared with those of the vehicle using an analysis of variance with a Dunnett’s test in case of significance (P≤0.05).
Statistical analysis was performed separately for each sex.
Statistical tests were processed using RS/1 software (Release 6.3, APPLIED MATERIALS.)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects on litter size, sex, litter weight, pup weight at birth, pup survival or body weight to day 4 post partum.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

EMB at 250, 500 and 1000 mg/kg induced no signs of toxicity on reproduction performance such as mating performance, fertility, gestation length, conception, parturition and survival and development of conceptus up to D4 post partum. Therefore, the No-Observed Adverse Effect Level (NOAEL) of EMB in the rat corresponds to at least 1000 mg/kg.

Executive summary:

Groups of 10 male and 10 female rats received 0, 250, 500 or 1000 mg /kg bw/day EMB in corm oil by the oral route for a maximum of 51 days at a volume of 10 mL/kg.- Morbidity/mortality checks were performed twice daily. Clinical observations were performed before the first dosing and daily. Functional and neurobehavioural tests were performed before the first dosing and once a week during the whole study except during the gestation period in females. Body weight was recorded at predose and once a week. Body weight of pups was recorded on D0pp or D1pp (post partum) and D4pp. Food consumption was measured weekly except during the mating period. Blood samples for haematology, coagulation parameters and clinical chemistry analysis were collected at the end of the dosing period from 5 males and 5 females. Males were killed 14 days after the mating period. Females with offspring were killed on D5pp. Selected organs were weighed, fixed and preserved at necropsy and examined histopathologically.

With the exception of 1 female at 1000 mg/kg killed on day 2 post partum due to dystocia, there was no mortality in males and females whatever the group during the study. There was no clinical sign related to the test item. There was no change in body temperature. There was no change in body weight gain in males and females treated with EMB in comparison with control group at the pre-mating, mating, post-mating, gestation or lactation period. There was no change in food consumption in males and females treated with EMB whatever the dose when compared to the control group at the pre-mating, mating, post-mating, gestation or lactation period. There was no marked change in haematology and coagulation parameters. There was no change in blood chemistry parameters whatever the group. There was no change in urinary parameters whatever the group. In males 14 days after the mating period or in females on D5pp, there was no major difference between the groups. In males and females, there was no specific pathological change related to the treatment. There were no changes related to test item on testicular stages.

The total number of pregnancies in each treated group was 10/10 females compared with 9/10 females in the control group. The gestation length for the majority of females for which mating was confirmed was 21 days. Gestation length was not determined in few animals (1 to 3 animals) of each group that were pregnant although evidence of copulation was not seen. There were no differences in the number of corpora lutea and in the implantation sites between control and treated groups.

There were no differences in body weight gain in pups from parents treated with EMB when compared with the control group. Litter weights were similar in control and treated groups on D1pp and D4pp. Live litter size remained similar in all groups between D1pp and D4pp. The numbers of males and female pups per litter showed intra group variation for control and treated groups on D1pp and on D4pp but there was no indication of any effect of parental treatment with EMB. There were no gross abnormalities on D1pp and D4pp.

There were no mortality and no major systemic signs of toxicity attributed to EMB at 250, 500 and 1000 mg/kg administered daily by the oral route for a maximum of 51 days in male and female rats. Moreover EMB at 250, 500 and 1000 mg/kg induced no signs of toxicity on reproduction performance such as mating performance, fertility, gestation length, conception, parturition and survival and development of conceptus up to D4 post partum. Therefore, the No-Observed Adverse Effect Level (NOAEL) of EMB in the rat corresponds to at least 1000 mg/kg.