Ethyl acetoacetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Short description of key information:
The study was performed 1999 as GLP-study according to OECD-guideline no. 421. The administration route was oral gavage. Ten animals were treated per sex and dose groups resulting in a total of 80 animals. Dose levels were 0, 50, 225 and 1000 mg/kg/day. There were no effects on clinical signs, mortality, body weights, food consumption. There were minor effects in the highest dose groups on reproduction. However, these findings were considered to be not relevant and therefore, a NOEL of 1000 mg/kg/day was established.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 1998 - June 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test item in the report: Acetoacetic acid ethyl ester
- Appearance: colorless liquid

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sulzfeld/Germany
- Adaptation: 5 days
- Age at study initiation: 68-72 days
- Weight at study initiation: Males: 264-287 g; Females: 195-216 g
- Housing: single housing except mating period, where 1 male & 1 female was placed per cage

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance was suspended in tap water and was administered orally at a constant volume of 10 ml/kg b.w. 7 days per week during the following periods:
- males: 2 weeks prior to mating, during the mating period and approx. 2 weeks post mating at least until the minimum total dosing period of 28 days had been completed (up to and including the day before sacrifice).
- 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.

The control animals received 10 ml tap water/kg b.w./day in the same manner. The test substance preparations were freshly prepared every day. The daily dose of the test substance was adjusted to the animal's body weight daily.

Details on mating procedure:

4 groups of sexually mature male and female rats were randomly paired for mating. Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until pregnancy occurred or two weeks had elapsed. Each morning the females were examined for the presence of sperm. If findings were negative, mating was repeated. The day of conception (day 0 of pregnancy) was considered to be the day on which sperm was found. In case pairing was unsuccessful, re-mating of females with proven males of the same group was considered. 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/homogeinity was analyzed by gas chromatography (GC)
- Intrument: HP 5890 Series II (Hewlett-Packard)
- Autosampler: HP 7673 (Hewlett-Packard)
- Integrator: HP 3396 Series II linked to a host computer with Peak96 software (Hewlett-Packard)
- Column: HP-1 (100% methyl silicone), 25 m, 0.32 mm i.d., 0.52 pm film (Hewlett-Packard)
- Injector: 220°C
- Detector: FID, 280°C
- Oven: hold 50°C (2 min.), heat up to 150°C (20°C/min.), hold 150°C (2 min.)
- Carrier gas: Helium, 2.5 ml/min.
- Split: 51 ml/min.
- Septum purge: 2.6 ml/min.
- Retention times: Ethyl acetoacetate: approx. 4.7 min. / Internal standard: approx. 3.9 min.

The test item was found to be stable in the vehicle at room temperature.

Duration of treatment / exposure:

The test substance was suspended in tap water and administered orally at a constant volume of 10 ml/kg b.w. 7 days per week during the following periods:
- males: 2 weeks prior to mating, during the mating period and approx. 2 weeks post mating at least until the minimum total dosing period of 28 days had been completed (up to and including the day before sacrifice).
- 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.

The control animals received 10 ml tap water/kg b.w./day in the same manner. The test substance preparations were freshly prepared every day. The daily dose of the test substance was adjusted to the animal's body weight daily.

Frequency of treatment:

7 days per week

Details on study schedule:

- Start of the study (adaptation period): November 25th, 1998
- 1st dosing: November 30th, 1998
- Study termination in-life phase males: December 28th, 1998 / females: January 21st, 1999

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

225 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

80 animals were used in this study:
- control group (0 mg/kg/day): 10 animals/sex
- low dose group (50 mg/kg/day): 10 animals/sex
- mid dose group ( 225 mg/kg/day): 10 animals/sex
- high dose group (1000 mg/kg/day): 10 animals/sex

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose selection was based on toxicological data already available for this substance.

Positive control:

no positive control tested

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: At first day of dosing, weekly thereafter and at study termination.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily monotoring of drinking water bottles

Individual animals were observed at least once daily for any signs of behavioural
changes, reaction to treatment or illness. In addition, the nature of the faeces was a checked daily.
Immediately after administration any signs of illness or reaction to treatment were
recorded. In case of changes the animals were observed until the symptoms disappeared.
In addition, animals were checked regularly throughout the working day
from 7.30 a.m. to 4.30 p.m..
On Saturdays and Sundays animals were checked regularly from 8.00 a.m. to 12.00
a.m. with a final check performed at approximately 4.00 p.m..
Dated and signed records of appearance, change and disappearance of clinical signs
were maintained on clinical history sheets for individual animals.

Oestrous cyclicity (parental animals):

no details reported

Sperm parameters (parental animals):

no data

Litter observations:

The duration of gestation of the female rats was recorded and was calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups) and the presence of gross abnormalities.
Live pups were counted and the sex was determined. The pups were weighed on days 1 and 4 post-partum. Any abnormal behaviour of the pups was recorded.

Postmortem examinations (parental animals):

The male animals were sacrificed after a minimum total dosing period of 28 days if no longer needed for further mating. Dams with offspring were sacrificed on day 4 post-partum, or shortly thereafter. Females showing no evidence of copulation were sacrificed 24-26 days after the last day of the mating period. a At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the reproductive organs. The number of corpora lutea and implantation sites were recorded.
The testes and epididymides of all male adult animals were weighed. Dead pups and pups killed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities. The ovaries, testes, epididymides, accessory sex organs and all organs showing macroscopic lesions of all adult animals were preserved. The testes and epididymides were preserved in Bouin's fixative; the remaining tissues were preserved in 7%
buffered formalin.

Postmortem examinations (offspring):

see above

Statistics:

For all numerical values homogeneity of variances was tested using the Bartlett chisquare test. When 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 the FISHER's exact test (n < 100) or chi 2-test with Yates' correction for continuity (n >= 100) (p <= 0.05 and p <= 0.01) were employed.

Reproductive indices:

For each group the following reproductive indices were determined: gestation index, fertility index females and males, pre-implantation loss, post-implantation loss. For each litter and group the following indices were determined: birth index, live birth index, viability index

Offspring viability indices:

Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals. This would have allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Clinical signs:

no effects observed

Description (incidence and severity):

No substance-related behavioural changes were observed at 50, 225 or 1000 mg/kg b.w.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Oral study performed

Mortality:

no mortality observed

Description (incidence):

None of the low-, intermediate- and high-dosed male and female parent animals died prematurely.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight remained within the normal range of the control animals at 50, 225 and 1000 mg/kg b.w.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No substance-related influence was noted on absolute and relative food consumption compared to the control at 50, 225 or 1000 mg/kg b.w.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Drinking water consumption was not influenced.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Was restricted to testicle, epididymis and ovary of the animals of the control group and the high dose level group (1000 mg/kg b.w.). The microscopic examination revealed no substance- related pathological findings.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

In this particular case, one female treated with 1000 mg!kg bw had very poor reproductive performances: low number of corpora lutea and high postimplantation loss (44%). For all these reasons: 1) marginal effects not supported by statistical significance; 2) values in the
range of historical controls; 3) one female in the high dose group with poor reproductive performances, I suggest to reconsider the NOEL of this experiment. In conclusion, In conclusion, on the basis of the study, there no scientific reason to fix the NOEL for reproductive toxicity of Ethyl acetoacetate at 225 mg!kg bw.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

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

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

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:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Remarks on result:

not measured/tested

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

0 or 225 mg/kg b.w. did not influence the reproduction parameters of the rats. The post-implantation loss and viability index were similar to those of the control. The number of pups born was not effected, the pups developed normally. Body weight and sex ratio of the pups were not influenced. The pups did not show any substance-related pathological findings at external observation on lactation day 4.
At 1000 mg/kg b. w. the number of pups at birth and during the 4-day lactation period was slightly decreased compared to the control correlating with the reduced number of implants. Hence the mean post-implantation loss was increased to 13.2% (control: 5.9%) and the mean birth index and live birth index (both: 86.8%) were decreased compared to the control indices (both: 94.1 %). However, the mean viability index (97 .6%) was in the range of the control (93. 7%). Further differences observed in comparison to the control are without any biological relevance.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

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):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

At 1000 mg!kg the mean number of corpora lutea, implantation sites, and pups at birth was marginally decreased in comparison to control. For this reason the NOEL was considered 225 mg!kglbw. It must be stressed that this value of NOEL is not statistically supported, as no statistically significant differences were observed in comparison to control for any of these parameters. That means that this value of NOEL has to be considered very conservative. The most relevant effect observed in this study was the reduced number of born pups in the high dose group. Such effect is due to a sum of events: reduced number of ovulated ova (i.e. n. of copora lutea), increased preimplantation loss, decreased number of implantations, increased postimplantation loss. Each of these events showed a marginal deviation from the control values, but the sum of them produced a more evident effect. It is important to signal that all these values are in the range of the historical controls of the laboratory which performed the experiment. In conclusion, on the basis of the data of the report, there no scientific reason to fix the NOEL for reproductive toxicity of Ethyl acetoacetate at 225 mg!kg bw.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Remarks on result:

not measured/tested

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

none

Conclusions:

Under the present test conditions, the NOEL (no-observed-effect level) of Ethyl acetoacetate was found to be 1000 mg/kg b.w./day.

Executive summary:

The study was performed 1999 as GLP-study according to OECD-guideline no. 421. The administration route was oral gavage. Ten animals were treated per sex and dose groups resulting in a total of 80 animals. Dose levels were 0, 50, 225 and 1000 mg/kg/day. There were no effects on clinical signs, mortality, body weights, food consumption. There were minor effects in the highest dose groups on reproduction. However, these findings were considered to be not relevant and therefore, a NOEL of 1000 mg/kg/day was established.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Justification for selection of Effect on fertility via oral route: Guideline study; Klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The developmental toxicity study requirement according to REACH Annex IX was waived based on a weight of evidence approach, based on the following information:

1. a reproduction/developmental toxicity screening test of Ethyl acetoacetate according to OECD 421 by oral administration to Sprague-Dawley rats did not reveal significant substance-related effects. There were minor findings in the highest dose groups on reproduction only, however, these findings were considered to be not relevant and therefore, a NOEL of 1000 mg/kg/day was established in this study

2. read-across to Ethyl acetate, a chemical with similar chemical structure as Ethyl acetoacetate. A review of the available toxicity and toxicokinetic data showed, that both substances have a comparable toxicity profile. In a guideline repeated dose inhalation study with histopathology of reproductive organs and evaluation of sperm parameters, rats were exposed to doses of 750 and 1500 ppm. Analysis of sperm parameters and histopathological evaluation of male and female reproductive organs was included in a subchronic inhalation toxicity study of ethyl acetate. 13 -week exposures up to 1500 ppm ethyl acetate had no effect on sperm number, motility or morphology; and there were no test substance-related pathology finding in reproductive tissues examined microscopically. The NOAEL was considered to be 1500 ppm.

3. read-across to Methyl acetate, a chemical with similar chemical structure as Methyl acetoacetate. A review of the available toxicity and toxicokinetic data showed, that both substances have a comparable toxicity profile. Toxicity data for Methyl acetate were also previously reviewed by the European Chemicals Bureau and reported in the "European Union Risk Assessment Report: Methyl acetate / Vol. 34". According to a comprehensive compilation of data from several experimental studies after peer-review, it was concluded, that Methyl acetate is not considered to produce developmental toxicity / teratogenicity.

References:

- European Union Risk Assessment Report: Methyl acetate (CAS No: 79 -20 -9 / EINECS No: 201 -185 -2), Vol. 34, 2003

- ECHA-CHEM (http://echa.europa.eu/web/guest/information-on-chemicals/registered-substances): Ethyl acetate (EC No. 205-500-4 / CAS-No 141-78-6)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Species:

rat

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available (further information necessary)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data available the substance is not classified or labeled according to Directive 67/548/EEC (DSD) or Regulation 1272/2008/EC (CLP).