tert-butyl 3-oxobutanoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data from J- check

Data source

Reference

Reference Type:

other: authoritative database

Title:

Combined Repeat Dose and Reproductive / Developmental Toxicity Screening of test material by Oral Administration in Rats

Author:

J-check

Year:

2018

Bibliographic source:

J-check, National Institute of Technology and Evaluation2018

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Combined Repeat Dose and Reproductive / Developmental Toxicity Screening Test of test material was performed on Sprague Dawley rats.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl acetoacetate

EC Number:

203-299-8

EC Name:

Methyl acetoacetate

Cas Number:

105-45-3

Molecular formula:

C5H8O3

IUPAC Name:

Methyl acetoacetate

Details on test material:

- Name of test material (as cited in study report):Methyl acetoacetate- Molecular formula : C5H8O3- Molecular weight :116.115g/mol- Substance type: Organic

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animals and env. conditionsTEST ANIMALS- Source: No data available- Age at study initiation: 10 weeks - Weight at study initiation: 373.2 to 428.2 g for males and 227.7 to 255.3 g for females- Fasting period before study: No data available- Housing: stainless steel hanger cage in a barrier system room- Use of restrainers for preventing ingestion (if dermal): yes/no- Diet (e.g. ad libitum): solid feed (MF, Oriental Yeast Industry ) ad libitum.- Water (e.g. ad libitum): drinking water added sodium hypochlorite (about 2 ppm), ad libitum- Acclimation period: 13 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 24 ± 2°C- Humidity (%):55±10%- Air changes (per hr): 13 to 15 times - Photoperiod (hrs dark / hrs light): illumination 12 hours (7 am to 7 pm)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

water for injection

Details on exposure:

Details on exposurePREPARATION OF DOSING SOLUTIONS:Test material mixed water for injectionDIET PREPARATION- Rate of preparation of diet (frequency):No data available- Mixing appropriate amounts with (Type of food )- Storage temperature of food: No data availableVEHICLE- Justification for use and choice of vehicle (if other than water): Test material mixed with water for injection- Concentration in vehicle: 0,100,300,1000 mg/kg bw/day- Amount of vehicle (if gavage): 10ml/kg- Lot/batch no. (if required): water for injection- Purity: No data availableOther :

Details on mating procedure:

- M/F ratio per cage:1:1 - Length of cohabitation: overnight - Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:the day when the sperm or vaginal plug in the vaginal plaque was confirmed the next day was taken as the 0th gestation - After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. - Further matings after two unsuccessful attempts: [no / yes (explain)] - After successful mating each pregnant female was caged (how): - Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

7 weeks (males for a total of 49 days before the mating and for the 35 days including the mating period, for a total of 49 days in the female, 14 days before the mating in the female, a mating period (maximum 14 days), a period of gestation and a period of 3 days of nursing,)

Frequency of treatment:

once per day

Doses / concentrations

Remarks:

0, 100,300,1000mg/kg bw/day

No. of animals per sex per dose:

Total:960 mg/kg bw/day:12male and 12 female 100 mg/kg bw/day:12male and 12 female300 mg/kg bw/day:12male and 12 female1000mg/kg bw/day:12male and 12 female

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selectionsThe dose was set from the results of the preliminary test. That is, as a result of repeated administration of 100, 300 and 1000 mg / kg of the test substance for 2 weeks, changes in general condition, body weight, food intake, hematology test, blood biochemical examination, autopsy and organ weight by administration of the test substance Was not found. Therefore, for the dose of this study, we set 1000 mg / kg as the critical dose according to the OECD guidelines to high doses, and 300 and 100 mg / kg divided by the common ratio of about 3, respectively, as medium dose and low dose .- Rationale for animal assignment (if not random):the grouping was carried out by stratified continuous randomization method based on the body weight the day before the start of administration.

Positive control:

No data available

Examinations

Parental animals: Observations and examinations:

Parental animals observation and examinationsCAGE SIDE OBSERVATIONS: yes DETAILED CLINICAL OBSERVATIONS: twice a dayTime schedule: BODY WEIGHT: YesTime schedule for examination male was measured twice a week throughout the administration period. Females are given twicea week during the administration period and the mating period before mating, pregnancy 0 (pregnancyconfirmation date), 4, 7, 10, 14, 17 and 21 days during pregnancy, nursing 0 during delivery Day) and 4 days.FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes males were measured twice a week during the administration period excluding the matingperiod. Females were measured twice a week for the administration period before mating, 1, 4, 7, 10, 14, 17 and 21 days pregnancy during pregnancy, and on nursing days 1 and 4 during the feeding period.Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: no Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data availableWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data Time schedule for examinations: OTHER: Hematology examination, Blood biochemical examination

Oestrous cyclicity (parental animals):

Estrous cyclicity was observed

Sperm parameters (parental animals):

No data available

Litter observations:

STANDARDISATION OF LITTERS- Performed on day 4 postpartum: [yes/no]- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded. PARAMETERS EXAMINEDThe following parameters were examined in [F1 ] offspring:number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalitiesASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:No data availableASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:No data available

Postmortem examinations (parental animals):

SACRIFICE - Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.] - Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.] GROSS NECROPSY: yes - Gross necropsy consisted of external and internal examinations HISTOPATHOLOGY / ORGAN WEIGHTS: yes The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE On the fourth day of nursing, all the newborns were exsanguinated underether anesthesia, - These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: GROSS NECROPSY: yes - Gross necropsy consisted of external examinations ,the organ and tissue were macroscopically observed HISTOPATHOLOGY / ORGAN WEIGTHS The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Statistics:

Weight, food consumption, hematology examination, blood biochemical examination, number of days required for copulation, sexual cycle examination (estrus frequency, estrus cycle), organ weight, pregnancy period, luteinum number, implantation number, total number of births, For the number and neonatal weight, average value and standard deviation were determined for each group, and the homogeneity of dispersion was first tested by the Bartlett method between the control group and the test substance administration group. When the variance was uniform, comparison with the control group was performed using Dunnett's multiple comparison test, and when the variance was not uniform, comparison with the control group was performed using Steel's multiple comparison test. With respect to the mating rate, receiving (a) germination rate, birth rate and neonatal sex ratio, by the χ ^ 2 test, Wilcoxon rank sum test for the stillbirth rate, birth rate and 4th day survival rate of newborn babies, pathological examination , Comparison between the control group and each administration group was conducted by Mann-Whitney's U test. In both cases the significance levels were set at 1 and 5%. The measured values for newborn babies were processed in units of stomach.

Reproductive indices:

yes

Offspring viability indices:

yes

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No change was observed in general condition in male and female

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In males, there was no death in each group.In females, one patient in the 300 mg / kg group had decreased activity, slow respiration, decreased body temperature and prone posture before the 2 nd day feeding and died on the 3rd day of nursing. No deaths occurred in the 100 and 1000 mg / kg groups,

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no difference between in body weight of the control group and the sex of each treated group throughout the administration period.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In males, lymphocyte infiltration of the left ventricular endocardium was observed in one patient in the 1000 mg / kg group. In addition, in one case of macroscopically enlarged spleen, 100 mg / kg group, the following findings were observed histologically, so it was judged as myeloid leukemia. That is, in this example, the femoral bone marrow is occupied by tumor cells with poor cytoplasm and relatively bright circular to elliptical nucleus, similar tumor cells are found around the portal vein of the liver, and also in the sinuses of the lobules It was diffusely diffuse. Furthermore, in the spleen, tumor cells invaded and proliferated in the whole area, replacing the normal structure of the spleen. No neoplastic changes were observed in the mesenteric lymph nodes.In females, no change was found in any organs in each group.In one case of the 300 mg / kg group who died, hepatic focal necrosis, lung thrombus, necrosis of proximal renal tubular epithelial cells of the kidneys and bleeding into tubules were observed, and ulcers in the forestomach, Erosion of the glandular stomach, atrophy of the thymus, hypertrophy of the spherical zone and bundle of the adrenal glands were also observed.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

there was no difference in the number of estrus and estrus cycle with the control group in each group

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

In reproductive ability tests, all but one group of 100 mg / kg group was mated, and fertility was confirmed in both cases. Therefore, in the control group, 100, 300 and 1000 mg / kg group, the mating rates were 100, 91.67, 100 and 100%, respectively, the conception rates were all 100%, no difference was observed between the control group and each group It was. Also in the number of days required for copulation, no difference was observed between the control group and each group. No abnormalities were found in the autopsy of the unexposed cases and histopathological examination of the ovaries.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

Remarks on result:

other: Overall no toxic effects observed

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

there was no difference between the survival rate of the control group and each treated group

Body weight and weight changes:

no effects observed

Description (incidence and severity):

there was no difference between the body weight of the neonates on the 4th day of nursing in the neonates from the control group

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

no abnormality was observed in the neonatal exterior examination in each group

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: overall no effects on developmental parameters

Target system / organ toxicity (F1)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Table: Reproductive performance of rats treated orally with test material in the combined repeat dose and reproductive / developmental toxicity screening test

Dose (mg/ kg)	0	100	300	1000
No. of females examined	12	12	12	12
Count of estrus	3.42 ±0.51	3.83 ±0.39	3.50 ±0.52	3.75 ±0.45
Estrous cycle	3.97 ±0.10	4.00± 0.00	4.13± 0.31	4.04± 0.14
No . of mated
Male	12	12	12	12
Female	12	12	12	12
No of copulated
Male	12(100)	11(91.67)	12(100)	12(100)
Female	12(100)	11(91.67)	12(100)	12(100)
No of impregnated	12(100)	11(100)	12(100)	12(100)
No of pregnant	12(100)	11(100)	12(100)	12(100)
Duration of mating	2.08 ± 1.16	2.91 ±1.45	2.25± 1.29	3.17 ±1.59

 

a) Values are mean ±SD.

b) Values are mean: ±S.D.(day)

c) Values in parentheses represent percentages to the number of mated animals.

d) Values in par-theses represent percentages to the number of copulated animals.

 

Table: Findings of delivery of Fo dams treated orally with methyl acetoacetate and observations on their offspring in the combined repeat dose and reproductive/ developmental toxicity screening test

 

Dose (mg/ kg)	0	100	300	1000
No. of dams	12	12	12	12
Gestational days	22.42 ±0.51	22.36 ± 0.50	22.42 ± 0.51	22.33 ±0.49
No. of corpora lutea	204 (17.00 ± 0.85)	192 (17.45 ± 1.21)	214 (17.83 ±1.11)	212(17.67± 1.78)
No. of implantations	185(15.42± 1.73)	180(16.36± 1.21)	209 (17.42±1.16)*	199(16.58 ±3.15)
No. of litter	173(14.42± 1.31)	161 (14.64± 2.11)	194(16.17 ±1.27)*	185(15.42 ± 2.91)
Gestation index	100	100	100	100
No. of stillborns
Male	1	1	1	1
Female	1	1	2	2
Total	2(1.16)	2(1.24)	3(1.55)	3(1.62)
No. of live newborns	171(14.25± 1.22)	159(14.45±2.07)	191(15.92 ±1.31)	182 (15.17 ±2.98)
Birth index	92.43	88.33	91.39	91.46
Sex ratio of live newborns	1.11(90/81)	0.69(65/94)*	1.27(107/84)	1.36(106/77)
Body weight of live newborns (g)
Male On day 0	6.3 ±0.5	6.4 ±0.6	6.2 ±0.3	6.2 ±0.5
day 4	9.6 ±1.0	9.5 ±1.2	9.2 ±0.6	9.4± 1.1
Female On day 0	6.0 ±0.5	6.0 ±0.6	5.8± 0.4	5.7 ±0.4
Female On day 4	9.1 ±1.0	9.0 ±1.0	8.5 ±0.8	8.6 ±0.7
Viability index	98.83	98.74	89.01	95.05
No. of external anomalies	0	0	0	0

*P<0.05(significantly different from control)

a) Values are mean: ±S.D.

b) Values in parentheses represent mean: ±S. D.

c) Gestation index=( Number of females with live newborns/ N umber of pregnant females) x100.

d) Values in parentheses represent percentages to the number of litters.

e) Birth index=( Number of live newborns/number of implantations)x100

f )Values in parentheses represent the number of male/female live newborns.

g) Values are mean: ±S.D.

h) Viability index=(Number of live newborns on day 4 after birth/Number of live newborns) X100.

i )n=11.

Applicant's summary and conclusion

Conclusions:

No Observed Adversed Effect Level (NOAEL) was considered to be 1000mg/kg bw/day for reproductive and developmental toxicity. When male and female Sprague Dawley rats were treated with test material orally.

Executive summary:

Reproductive and developmental toxicity study of test material was performed in male and female Sprague Dawley rats. The test material was dissolved in water for injection and administered in dose concentration0 (Vehicle), 100, 300, and 1000 mg/kg-day via oral gavage route.The dose volume was 10 mL / kg, the males were treated for a total of 49 days before the mating and for the 35 days

including the mating period, for a total of 49 days in the female, 14 days before the mating in the female, a mating period (maximum 14 days), a period of gestation and a period of 3 days of nursing, The number of animals per group was 12 male and 12 female, and the grouping was carried out by stratified continuous randomization method based on the body weight the day before the start of administration. Mating was carried out by a method in which both male and female (12 weeks old) live together overnight, and the day when the sperm or vaginal plug in the vaginal plaque was confirmed the next day was taken as the 0^thgestation. For both males and females, observation of general condition and confirmation of life and death were conducted twice a day or more for all cases. For body weight, male was measured twice a week throughout the administration period. Females are given twice a week during the administration period and the mating period before mating, pregnancy 0 (pregnancy

confirmation date), 4, 7, 10, 14, 17 and 21 days during pregnancy, nursing 0 during delivery Day) and 4 days. Regarding food intake, males were measured twice a week during the administration period excluding the mating period. Females were measured twice a week for the administration period before mating, 1, 4, 7, 10, 14, 17 and 21 days pregnancy during pregnancy, and on nursing days 1 and 4 during the feeding period. In males, there was no death in each group and no change was observed in general condition. In females, one in the 300 mg / kg group had decreased activity, slow respiration, decreased body temperature and prone posture before the 2^ndday feeding and died on the 3rd day of nursing. No deaths occurred in the 100 and 1000 mg / kg groups, and no change was observed in the general condition. There was no difference between the control group and the sex of each group throughout the administration period. A decrease in total cholesterol was observed in the 100 mg / kg group, and a decrease in γ-GTP was observed in the case of 300 mg / kg. In male, absolute and relative weight increase of spleen was observed in 300 mg / kg group. In females, no change was observed in each group. In the sexual cycle examination, there was no difference in the number of estrus and estrus cycle with the control group in each group. The conception rates were all 100%, no difference was observed between the control group and each group It was. Also in the number of days required for copulation, no difference was observed between the control group and each group. No abnormalities were found in the autopsy of the unexposed cases and histopathological examination of the ovaries. The number of female neonates was large in the 100 mg / kg group, and the sex ratio was biased. In the 300 mg / kg group, the number of implantation traces, the total number of births and the number of neonates increased. However, in each group, there was no difference between the pregnancy period, the corpus luteum number, the birth rate, the birth rate, the newborn baby weight and the stillbirth rate with the control group, and no abnormality was observed in the neonatal exterior examination in each group. In the nursing examination, there was no difference between the survival rate and the body weight of the neonates on the 4th day of nursing in the neonates from the control group. In each group, there was no effect of administration of the test substance on pregnancy period, birth rate, birth rate, newborn baby weight and stillbirth rate, and there was no expression of external table abnormality. HenceNo Observed Adverse Effect Level (NOAEL) was considered to be 1000mg/kg bw/day for reproductive and developmental toxicity.When male and femaleSprague Dawley rats were treated withtest materialorally.