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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source and target chemicals are analagous

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source and target chemicals are both pure with low impurity.

3. ANALOGUE APPROACH JUSTIFICATION
In light of the structural and physico-chemical similarities between EMB, EMV and IMB it is our consideration that experimental testing for an acute dermal 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
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:
Butanoic acid, 2-methyl-, ethyl ester
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)
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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
SIGMA, Batch No. MKBH4894V, Expiry date: May 2017 and Jul 2017
Details on mating procedure:
one male with one female for up to one week or until mating confirmed. If after one week, pairing was unsuccessful, the female was placed with a proven male of the same group for up to 7 days.
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
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 250, 500, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
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). The general disposition, behaviour and activity were observed (see in Section 1.14, page 37 of the attached report).

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
• on D0pp or D1pp and D4pp for each pup
• on the day of necropsy (not exsanguinated), this is the reference weight used in the calculation of relative organ weights

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption and fasted periods: Food consumption was measured weekly during the whole study except during the mating period. Animals were fasted during the night before scheduled blood sampling for haematology/coagulation parameters, clinical chemistry and dining urine collection for urinalysis.

HAEMATOLOGY:
Haematology, coagulation parameters: Haematology and coagulation parameters are presented in Table 1.2, page 29 of the attached report.

Coagulation parameters were assessed using the following method:
• Prothrombin time: sec
DIAGNOSTICA STAGO reagent.
Automatic method of prothrombin time
• Activated partial thromboplastin time: sec
DIAGNOSTICA STAGO reagent.
Automatic method of activated partial thromboplastin time

CLINICAL CHEMISTRY:
Clinical laboratory determination: Haematology, coagulation parameters and blood chemistry analysis were performed by CERB.

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.

Sample volumes and anticoagulant were as follow: 0.2 mL into EDTA tubes (haematology), 1 mL into citrated tubes (coagulation parameters) and 1 mL into lithium heparin tubes (blood chemistry). Tubes were kept at room temperature, until the time of the analysis (for a maximum of 8 hours). Labels on the tubes were marked in water proof ink with Testing Facility, Animal Number, Study Number, Sampling Day, Test item, Dose or Group, Sex and Kind of Sampling.

Blood chemistry analysis: Blood chemistry parameters are presented in Table 1.3, page 29 of the attached report.

Clinical chemistry analysis was performed by using the following methods:
• Calibration: Multical - Horiba Medical
• Control: Control N - Horiba Medical
• ALT and AST: UV test according to IFCC recommendations
• ALP: kinetic photometric test according to IFCC recommendations
• Urea: UV enzyme test using urease and glutamate dehydrogenase
• Creatinine: colourimetric test according to JAFFE reaction
• Albumin: colourmetric test with bromocresol green
• Total Bilirubin: colourimetric test with 2,4 - dichloroaniline (DCA)
• Total proteins: Biuret reaction
• Glucose: colourimetric test according to Tinder method
• Total cholesterol: Photometric enzyme test (CHOD - PAP)
• Chloride - Potassium - Sodium: Direct Ion Selective Electrode

In general, Horiba Medical reagents were used.

URINALYSIS: At the end of the dosing period shortly before scheduled necropsy, mine 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. Urinalysis parameters are presented Table 1.4, page 30 of the attached report.

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.

[1] Council Directive 86/609/EEC. Of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes. November 24, 1986

OTHER:
- Examination: Mortality - Morbidity: Mortality was recorded twice a day, i.e. in the morning and at the end of the working day. Female No. 1202509 found in a moribund condition and showing enduring signs of severe distress was euthanased on D1pp by subtotal exsanguination under anaesthesia by isoflurane inhalation.
- Observations of pups: Each litter was examined as soon as possible after delivery to establish the stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities. On D0pp or D1pp and on D4pp, live pups were counted and sexed. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Examinations

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals 14 days after the mating period.
- Maternal animals: All surviving animals on day 5 post partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
From 5 males and 5 females per group:
The following tissues were weighed: liver, epididymides, thymus, adrenals, brain, heart, kidneys, testes, spleen. Testes and epididymides were weighed from all males.
The following tissues were prepared for microscopic examination: stomach, liver, caecum, jejunum (with Peyer's patches), colon, duodenum, ileum, rectum, heart, trachea, lung, urinary bladder, uterus, epididymides, vagina, kidneys, testes, oviducts, prostate and seminal vesicles, ureters, ovaries, bone marrow (sternum), thymus, lymph nodes (mesenteric and sub-maxillary), spleen, adrenals, thyroids/parathyroids, brain, spinal cord, peripheral nerve, gross lesions.
Testes were examined for missing stages (with particular emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).
Postmortem examinations (offspring):
Dead pups and pups killed on day 4 post partum were carefully examined externally for gross abnormalities
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.)
Reproductive indices:
Gestation length, number of implantation sites and corpora lutea, number in litter (dead or alive), pup sex and weight.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on fertility or reproductive performance (gestation, litter size, pup sex, survival or body weight to day 4 post partum)
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No evidence of systemic toxicity (clinical signs, body weight, haematology, clinical chemistry, neurobehaviour, organ weights or histopathology)

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 examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on body weight, clinical condition, survival or gross pathology to day 4 post partum

Overall reproductive toxicity

Reproductive 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.

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