Isopropyl 2-methylbutyrate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June to 03 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

HYPOTHESIS FOR THE ANALOGUE APPROACH
The target and source chemicals are of similar structure in the same family.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

3. ANALOGUE APPROACH JUSTIFICATION
All three substances are Cramer class 1 (simple chemical structure with low order oral toxicity with a toxicological threshold of 1800µg/person/day or 30µg/kg bw/day) and have similar chemical and physico-chemical properties, being short chain aliphatic esters. Each is rapidly hydrolysed by esterases to form constituent alcohols and carboxylic acids which become substrates for β-oxidation. For each substance, toxicological endpoint classification is identical for acute oral toxicity, skin irritation and mutagenicity (point mutation, gene mutation and structural chromosomal aberrations).

In light of the structural and physico-chemical similarities between EMB, EMV and IMB it is our consideration that experimental testing for an in vivo repeated dose toxicity study using IMB can be waived, as study data from the structurally related esters EMB and EMV can be used to read-across and predict the toxicity of IMB.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Minor deviations which did not affect the integrity of the study.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Toyo Gosei; H3-H-5
- Expiration date of the lot/batch: 04 August 2014
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: no data

Species:

rat

Strain:

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

Sex:

male/female

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.

Route of administration:

oral: unspecified

Vehicle:

corn oil

Remarks:

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

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly

VEHICLE
- Justification for use and choice of vehicle (if other than water): Palatability
- Concentration in vehicle: 10 to 200 mg/l
- Amount of vehicle (if gavage): n/a
- Lot/batch no. (if required): (SIGMA, Batch No. MKBH4894V
- Purity: no data

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.

Duration of treatment / exposure:

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

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

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).

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.

Sacrifice and pathology:

- Euthanasia of animals: On the day of necropsy and after overnight (about 16 hours) fast, all surviving animals (adults) were euthanased by subtotal exsanguination following anaesthesia by isoflurane inhalation. Females with offspring were euthanased on D5pp. Males were euthanased 14 days after the mating period, on D15. Moribund Female No.1202509 was euthanased on D1pp in the same way in agreement with the Study Director and by the veterinary staff.
                            
Necropsy: sampling, macroscopic examination and fixation: Female euthanased in a moribund condition was necropsied as quickly as possible and specimens as required by the study plan obtained whenever practically possible. All animals were submitted to gross necropsy procedures including an examination of
• the external surface
• all orifices
• the cranial cavity
• the external surface of the brain and samples of the spinal cord
• the thoracic and abdominal cavities and organs
• the cervical tissues and organs
• the carcass
 
Special attention was paid to the organs of the reproductive system. The number of implantation sites and the corpora lutea were recorded. The ovaries, testes, epididymides and accessory sex organs were collected in all animals. Preputial and Cowpers glands were fixed and preserved in 4% buffered formalin. Paired organs were weighed together. Organs were weighed after dissection of fat and other contiguous tissues at the discretion of the prosector. The organs/tissues sampled were fixed and preserved in 4% buffered formalin with the following exception: testes and epididymides were fixed in alcoholic Bouin’s fluid (about 5 days), then transferred to ethanol 95%.
 
Dead pups and pups euthanased on day 4 post-partum were carefully examined externally for gross abnormalities. Organs for organ weight determination and for histopathological examination from 5 males and 5 females, randomly selected, from each group are presented in Table 1.5, page 32 of the attached report. Testes and epididymides were weighed from all males.
 
Testicular staging: Testis of males were sampled. Subjective scrutiny for missing stages (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure), with a report on the stages was performed. The sections were stained PAS and haematoxylin.
 

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.)S

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

Under the experimental conditions adopted, 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 Sprague Dawley rat. 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.

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.