Ethyl acetoacetate

Endpoint summary

Description of key information

The test item EAA was tested in two subacute oral toxicity studies over 28 days. Treatment was either by gavage or feeding. Both studies were performed under GLP

having a Klimisch 1 rating. Dose levels were 0, 50, 225 and 1000 mg/kg/day in the gavage study and 0, 100, 300 and 1000 mg/kg/day in the feeding test. In the gavage test, 5 animals per sex and dose group were treated with the test article. In addition, another 5 animals per sex of the control and highest dose group were treated and further observed in a 2-weeks recovery period. For the feeding study, 16 males and 16 females were tested per dose group, without treatment-free recovery period. There were no toxicologically relevant findings observed in both studies. Results were comparable, in both studies the NOEL/NOAEL values were found to be 1000 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 1990 - April 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Batch-no.: 157
- Storage: room temperature

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

the rodent species is acceptable to the regulatory authorities and has documented susceptibility to a wide range of toxic substances. Background data of the strain are available at the testing facility. There are no known contra-indications to its use.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA Credo, L'Arbresle, France
- Age at study initiation: 6 weeks
- Weight at study initiation: males 172-197.9 g / females 141.5-174.2 g
- Housing: gang housing (groups of 5 animals)
- Acclimation period: 7 days

Route of administration:

oral: gavage

Details on route of administration:

ROUTE AND METHOD OF ADMINISTRATION
- Route: oral
- Justification of the route: potential human exposure is by the oral route
- Volume of administration: 10 ml/kg/day
- Individual dose volumes were calculated weekly using the most recent body weight
- Method of administration: gavage, using a metal cannula

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Solution of the test article in the vehicle at the concentrations of 5, 22.5 and 100 mg/ml
- Stabilify of the test article in the vehicle: determined over 24 hours for the lowest and highest concentrations. The analyses were carried out at test lab
- Frequency of preparation: daily
- Storage: at room temperature, protected from light
- Analysis of formulation: a sample from each preparation (including control group) was taken at weeks 1 and 4

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability/homogeinity was determined for the lowest and highest dose concentrations by Gas chromatography (GC):
- Apparatus: Varian 3400 with autosampler
- Detector: FID
- Column: OV101, 15 m x 0.53 mm
- Injection volume: 1 ul
- Gas: nitrogen 10 ml/min
- Temperature: injector: 210°C / detector 260°C / column oven 70°C
- Test sample: dosing solutions were dissolved in acetone

The test item was found to be stable at room temperature over 24 hours.

Duration of treatment / exposure:

Duration:
- males and females killed at week 4 : 29 days
- males and females killed at week 6 : 28 days
For animals killed at week 4, treatment continued until the day before necropsy.

Frequency of treatment:

Once daily

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:

60 animals were used in this study:
- 0 mg/kg/day: 20 (5 males/5 females for treatment period PLUS 5 males/5 females for recovery period)
- 50 mg/kg/day: 10 (5 males/5 females)
- 225 mg/kg/day: 10 (5 males/5 females)
- 1000 mg/kg/day: 20 (5 males/5 females for treatment period PLUS 5 males/5 females for recovery period)

Control animals:

yes

Details on study design:

Dose selection rationale:
The high dose level of 1000 mg/kg/day is the maximal dose recommended by the OECD and EEC guidelines. The low dose level is expected to be a noobservable effect level and the intermediate dose level of 225 mg/kg is close to the geometric mean between the low and high dose levels.

Positive control:

no positive control tested

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed daily, before and at least once after dosing, Full clinical observation was performed weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly

FOOD CONSUMPTION:
- Food consumption was measured weekly for each cage from the first week of treatment

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once pretest and at week 4
- Examination: All animals; Direct and indirect ophthalmoscopy, a mydriatic agent (tropicamidel was instilled in to the eyes before examination

HAEMATOLOGY/CLINICAL CHEMISTRY: Yes
- Animals examined and frequency : 5 animals for each sex/group at week 4
- Samples: blood was withdrawn from the retro-orbital sinus following light ether anaesthesia on overnight fasted animals (about 16 hours). Bone marrow smears were prepared at scheduled necropsy for all animals killed at week 4. In consideration of the results for other parameters examined during the study.

URINALYSIS: Yes
- Collected in metabolism cages (approximately 16 hours) from animals deprived of food and water but receiving 20 ml/kg of mains water by gavage before being placed in the metabolism cages

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At weeks 4 and 6, the animals were necropsied after randomisation. The animals were weighed before necropsy. The animals were killed by carbon dioxide inhalation and exsanguination. All animals were submitted to full necropsy procedures including examination of :
- the external surface
- ail orifices
- the cranial cavity
- the carcass
- the external surface of the brain and of samples of the spinal cord
- the thoracic, abdominal and pelvic cavities and viscera
- the cervical tissues and organs

HISTOPATHOLOGY: Yes
All organs/tissues sampled were fixed and preserved in 10% neutral formalin with the following exception: bone marrow smears fixed in methanol. All sections were stained with haematoxylin and eosin.
Histopathological examinations were performed on all organs/tissues. For all animals in groups 1 (control) and 4 (high dose) at week 4. In each group, histopathological examination was performed for all gross lesions except those for which the diagnosis was judged unnecessary for the outcome of the study by the pathologist.

Other examinations:

none

Statistics:

no data

Clinical signs:

no effects observed

Description (incidence and severity):

In week 2, approximately half the high dose animals (group 4) were salivating immediately after dosing. In weeks 3 and 4, this observation was present in all high dose animals. Salivation is seen occasionally in studies of this type (gavage) and probably represents a local reaction to the test article. It is considered not to represent systemic toxicity.

Mortality:

no mortality observed

Description (incidence):

There were no deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no biologically significant differences between the groups in the amount of body weight gained.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The differences in food consumption between the groups were small and not considered to be treatment-related. Food conversion ratios were similar in all groups.

Food efficiency:

no effects observed

Description (incidence and severity):

No treatment-related effects observed.

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

not specified

Description (incidence and severity):

No treatment-related effects observed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no eye lesions. The data have therefore not been tabulated.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no differences between the groups to indicate an effect of treatment on the haematological parameters.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The clinical chemistry profiles of the treated animals were similar to the controls.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related 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:

effects observed, treatment-related

Description (incidence and severity):

The few microscopic findings were considered to be of agonal or incidental origin (i.e. pulmonary haemorrhage). There were no treatment-related microscopic findings.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related neoplastic histopathological findings noted.

Other effects:

not examined

Details on results:

see above

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Organ:

other: no organ toxicity observed

Conclusions:

The test item did not show any mortality, clinical signs or effects on organs and food consumption. There were also neither abnormalities in the hematological and biochemical parameters nor treatment-related macroscopic and microscopic findings.

Executive summary:

The study was performed 1991 under GLP according to OECD-guideline no. 407. Appropriate dose levels were evaluated in a DRF study. Application route was gavage, in the main study dose levels were 0, 50, 225 and 1000 mg/kg/day. Ten males and ten females were used at 0 and 1000 mg/kg/day, whereas five animals of each sex were killed at the end of treatment and the other five males and females were further observed throughout a two weeks recovery period. At 50 and 225 mg/kg/day, five animals of each sex were used per dose group. No treatment-related effects were observed, therefore the NOEL was found to be 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key - Repeated dose toxicity (28d): oral

The study was performed 1991 under GLP according to OECD-guideline no. 407. Appropriate dose levels were evaluated in a DRF study. Application route was gavage, in the main study dose levels were 0, 50, 225 and 1000 mg/kg/day. Ten males and ten females were used at 0 and 1000 mg/kg/day, whereas five animals of each sex were killed at the end of treatment and the other five males and females were further observed throughout a two weeks recovery period. At 50 and 225 mg/kg/day, five animals of each sex were used per dose group. No treatment-related effects were observed, therefore the NOEL was found to be 1000 mg/kg/day.

Supporting - Repeated dose toxicity (28d): oral

In another study from 1988, performed according to OECD-guideline no. 407, EAA was administered by diet. Dose levels were 0, 50, 300 and 1000 mg/kg/day. Sixteen animals/sex/dose group were used. Neither treatment-related effects nor toxicologically significant findings were observed, therefore the NOEL and NOAEL were both found to be 1000 mg/kg/day..

Weight-of-evidence (waiver) - Repeated dose toxicity (90d): oral

The subchronic toxicity study requirement according to REACH Annex IX was waived based on a weight of evidence approach and read-across to Ethyl acetate. Based on the similar structure and properties, the substance Ethyl acetate was selected as representative model structure for read-across. Compared to EAA, the selected structure show similar physical-chemical, toxicological and ecotoxicological properties. The available data on EAA and selected model constituent revealed similar properties. Two subchronic toxicity studies with Ethyl acetate were taken into account:

1. In an inhalative guideline and GLP study on Ethyl acetate, rats exposed to concentrations of 350, 750, or 1500 ppm (1.28, 2.75, 5.49 mg/L) in air for 94 days showed no significant toxic effects. In conclusion, the NOAEL for systemic toxicity in this study is considered to be 350 ppm (1.28 mg/L) and the LOAEL 750ppm (2.75 mg/L). The LOAEL for respiratory irritant effects (nasal toxicity) is 350 ppm (1.28 mg/L).

2. In a subchronic oral toxicity of ethyl acetate was evaluated in rats by US EPA. Groups of 30 male and 30 female rats received daily doses of 0, 300, 900 or 3600 mg/kg bw/day by gavage. Some minor effects were noted in the mid and high dose groups. Based on these findings the subchronic oral NOAEL for ethyl acetate in rats is considered to be 900 mg/kg bw/day.

Weight-of-evidence (waiver) - Repeated dose toxicity (28d): inhalation

The subacute inhalation toxicity study requirement according to REACH Annex IX was waived based on a weight of evidence approach and read-across to Ethyl acetate. Based on the similar structure and properties, the substance Ethyl acetate was selected as representative model structure for read-across. Compared to EAA, the selected structure show similar physical-chemical, toxicological and ecotoxicological properties. A In a guideline and GLP study, rats exposed to concentrations of 350, 750, or 1500 ppm (1.28, 2.75, 5.49 mg/L) Ethyl acetate in air for 94 days showed no significant toxic effects. Diminished response to delivery of an alerting stimuli was noted during exposure at the 750 and 1500 ppm levels. This transient diminished response was confined to the exposure period and was attributed to acute sedative properties of ethyl acetate. Other findings at the 750 and 1500 ppm levels were limited to reduced food consumption and body weight gain, and lower levels of serum triglycerides. No other changes were seen. Some evidence of nasal mucosa degeneration was seen at 350 ppm. The NOAEL for systemic toxicity in this study is considered to be 350 ppm (1.28 mg/L) and the LOAEL 750ppm (2.75 mg/L). The LOAEL for respiratory irritant effects (nasal toxicity) is 350 ppm (1.28 mg/L).

Weight-of-evidence (waiver) - Repeated dose toxicity (28d): dermal

According to REACH Annex VIII column 1 and 2 section 8.6.1 repeated dose toxicity testing via the dermal route was waived. Instead data on repeated dose toxicity testing (28d) via the oral route is presented. A NOAEL long-term, dermal of 1000 mg/kg bw/day was calculated from the NOAEL long-term, oral of 1000 mg/kg bw/day, assuming 100 % resorption through the skin. The results of laboratory animal studies with Methyl acetoacetate show low acute dermal toxicity. In the 28 - days repeated dose study via oral gavage administration does not exacerbate systemic toxicity effects, which suggest bioavailability is low, thereby there is low toxicity potential. This intrinsic property/toxicity potential can be extrapolated to repeated dermal route administration.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Guideline study; Klimisch 1

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).