Methyl acetoacetate

Endpoint summary

Description of key information

The key study was performed in 1996 according to OECD 407- and the corresponding GLP-guidelines. The appropriate dose levels were evaluated in a 7 -day-range-finding test. Based on this RF-study, dose levels of 0, 100, 300 and 1000 mg/kg b.w./day were fixed for the main study, including 2-weeks recovery for low and high dose groups. There were neither premature deaths nor clinical signs as a result of treatment with MAA. There were some minor effects observed on body weights, hematology, clinical chemistry, urinalysis, and organ weights. However, all these findings were either incidental or within the normal background range and therefore not considered to be of toxicological significance and treatment-related. There were also necropsy and histological findings. In conclusion, in the absence of any effect, the NOEL and NOAEL were determined to be 1000 mg/kg/day. 
A supporting study was performed in compliance with GLP and according to OECD 422 at dosages of 100, 300 and 1000 mg/kg/day. There were some effects observed on the clinical signs, blood chemistry and some histopathological parameters. The changes were seen in single animals only, without dose dependency. Therefore, these findings were considered to be of incidental nature and not related to treatment with the test article. No effects were reported on the remaining parameters such as body weight, food consumption, hematology. 

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:

April 1995 - July 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed in accordance with the corresponding OECD-/EU-testing guidelines

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

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number of animals: 5 per dose group; plus 5 for control (0 mg/kg/day) and high dose (1000 mg/kg/Day) group as recovery study animals
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: ca. 4 weeks
- Weight at study initiation: males: ca. 83-87 grams / females: ca 58-62 grams
However, on arrival a proportion of the animals weighed 74-84 grams for males and 52-59 grams for females. This minor deviation was not considered to have affected the validty of the study.
- Fasting period before study: not known
- Housing: 1 or 2 rats per cage and dose group
- Diet (e.g. ad libitum): Rat & Mouse (modified) No.1 SQC Expanded (pelleted) Maintenance diet supplied by Special Diets Services Limited, Stepfield, Witham, Essex, England
- Water (e.g. ad libitum): Domestic tap water
- Acclimation period: 13 days before treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C (+/- 2°C)
- Humidity (%): 50& (+/- 15%)
- Air changes (per hr): not mentioned in report
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle (light hours from 07:00-19:00)

IN-LIFE DATES:
- From: 28 August 1995
- To: 09 October 1995

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
A stock solution for the High dose group was formulated weekly using water for irrigation as the vehicle. This was then split into 7 replicates. The replicates were dispensed daily and diluted to prepare the Low and Intermediate dose group formulations. The High dose group formulation was formulated by adding an appropriate volume of vehicle to the test material. It was then mixed by gentle inversion until visibly homogeneous. The High dose group formulation was stored in the dark at ambient temperature prior to daily dispensing.

DOSING:
Orally via a steel dosing cannula at a constant volume of 10 ml dosing solution. To achieve this, animals were weighed each day and the body weight used to derive the correct volume of dosing solution to be administered. Control rats received the vehicle only at the same dose volume.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to study commencement trial formulations of the highest and lowest concentrations were investigated for stability, concentration and homogeneity. Routine analysis of formulated solutions were undertaken for concentration and homogeneity.

One 5 ml sample was taken from each dose formulation, including Control, from which three sub-samples were taken. Samples for analysis were taken on 1 day during Weeks 1 and 4 of dosing.

Analysis was done by GC with flame ionisation detector. Measuring conditions were as follows:
- Column: J&W Scientific DB-5 Megabore, length 15 m, i.d. 0.53 mm, film thickness 1.5 microm
- Carrier gas: Helium
- Injection mode: Splitless (split vent flow ca. 25 ml/min.)
- Head pressure: 5 psi
- Injection volume: 2 microl
- Detection: FID
- Internal standard: Ethyl acetoacetate
- Injector temperature: 150°C
- Column temperature: 60°C (isothermal)
- Detector temperature: 180°C
- Data handling: LabSystems, Multichrom v2.0

Duration of treatment / exposure:

4 weeks for all dose groups plus 2 weeks recovery period for control and high dose group

Frequency of treatment:

Daily; 7 days a week.

Remarks:

Doses / Concentrations:

Basis:
nominal in water

No. of animals per sex per dose:

0 mg/kg/day: 5 males & 5 females (plus 5 of each sex as recovery study animals)
100 mg/kg/day: 5 males & 5 females
300 mg/kg/day: 5 males & 5 females
1000 mg/kg/day: 5 males & 5 females (plus 5 of each sex as recovery study animals)

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE:
- Duration of range-finding study: 7 days
- Dose groups: 0, 100, 300 and 1000 mg/kg/day
- Number of animals: 5 males & 5 females
- Strain: Sprague-Dawley rats

Results:
- Mortality: none
- Clinical signs: none
- Body weights: very slight non statistically significant decrease in body weight gain in high dose group males
- Food consumption: high dose males showed a very slight decrease in food consumption
- Organ weights: no effects
- Necropsy findings: none

Positive control:

None.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
No data

DETAILED CLINICAL OBSERVATIONS:
All animals were examined for reaction to treatment during each day. The onset, intensity and duration of these signs were recorded. All animals received a detailed clinical examination once each week, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT:
The weight of each animal was recorded twice during the week before the start of treatment and then daily during the treatment period. This included spare animals during pretrial. During Weeks 5 and6 twice weekly body weights were recorded.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The quantity of food consumed by each cage of animals was recorded twice each week, commencing one week before the start of treatment up until the end of the study. This included spare animals during pretrial.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
Water consumption was monitored by visual inspection on a weekly basis throughout the study.

LABORATORY INVESTIGATIONS
Samples were taken from aU Main Study animals during Week 4, and from all Recovery
Study animals during Week 6.

HAEMATOLOGY:
Blood samples were collected from the orbital sinus under isoflourane anaesthesia without overnight deprivation of food. 0.5 ml of whole blood was taken into EDTA for assessment of haematology parameters; and 0.75 ml of whole blood into sodium citrate for assessment of prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY:
Clinical chemistry parameters were measured on plasma from 2.0 ml of whole blood taken into tubes containing lithium heparin.

URINALYSIS:
Urine samples were collected from animals in metabolism cages. The collection was made over a 4 h period of food and water deprivation.

NEUROBEHAVIOURAL EXAMINATION:
Not required at the date of the study.

Sacrifice and pathology:

GROSS PATHOLOGY:
All surviving animals were killed and necropsied. The method of killing was by carbon dioxide asphyxiation followed by exsanguination. The gross dissection and necropsy were performed under the supervision of a pathologist with the organs/tissues listed in the report being weighed/fixed from allanimals.
Unless otherwise noted samples of the above tissues were taken from all animals and placed in 10% neutral buffered formalin. Carcasses of animalswere discarded immediately following necropsy and fixing of all tissues listed in the report.

HISTOPATHOLOGY:
Processing of Fixed Tissues: Tissues were trimmed to a maximum thickness of 3 mrn for processing. Parenchymal organs, eg liver, were trimmed to allow the largest surface area possible for examination.
Mid-transverse sections through the entire cortex and medulla of each kidney were submitted. Hollow organs were trimmed and blocked to allow a cross section slide from mucosa to serosa. Tissues were cut at 4-6 microm thickness and stained with haematoxylin and eosin (H and E).
The tissues detailed in the examined column of the table on p.23 were processed and examined histologically from all animals in the Control and Highdose groups.

Other examinations:

None.

Statistics:

Body weight, food consumption, haematology, clinical chemistry, urinalysis and organ weight data were statistically analysed for homogeneity of variance using the 'F-max' test. If the group variances appeared homogeneous a parametric ANOVA was used and pairwise comparisons made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a nonparametric test such as Kruskal-Wallis ANOVA was used.
Organ weights were also analysed conditional on body weight ie analysis of covariance (ANCOVA).
Histology data were analysed by Fisher's Exact Probability test.

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:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

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

Other effects:

not examined

Details on results:

There were some minor effects observed on body weights, hematology, clinical chemistry, urinalysis, and organ weights. However, all these findings were either incidental or within the normal background range and therefore not considered to be of toxicological significance and treatment-related.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

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

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

none

Conclusions:

There were no adverse effects of treatment with MAA observed at any of the dose levels used under the conditions of this study. The no effect level (NOEL) is therefore at least 1000 mg/kg/day.

Executive summary:

The study was performed in 1996 according to OECD 407- and the corresponding GLP-guidelines. The appropriate doese levels were evaluated in a 7 -day-range-finding test. Based on this RF-study, dose levels of 0, 100, 300 and 1000 mg/kg b.w./day were fixed for the main study. There were neither premature deaths nor clinical signs as a result of treatment with Methyl Acetoacetate. There were some minor effects observed on body weights, hematology, clinical chemistry, urinalysis, and organ weights. However, all these findings were either incidental or within the normal background range and therefore not considered to be of toxicological significance and treatment-related. There were also necropsy and histological findings. In conclusion, in the absence of any effect, the NOEL was determined 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

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 in 1996 according to OECD 407- and the corresponding GLP-guidelines. The appropriate dose levels were evaluated in a 7 -day-range-finding test. Based on this RF-study, dose levels of 0, 100, 300 and 1000 mg/kg b.w./day were fixed for the main study. There were neither premature deaths nor clinical signs as a result of treatment with Methyl Acetoacetate. There were some minor effects observed on body weights, hematology, clinical chemistry, urinalysis, and organ weights. However, all these findings were either incidental or within the normal background range and therefore not considered to be of toxicological significance and treatment-related. There were also necropsy and histological findings. In conclusion, in the absence of any effect, the NOEL was determined to be 1000 mg/kg/day.

Supporting - Repeated dose toxicity (28d): oral

The study was performed in Japan according to OECD-guideline no. 422 as GLP-study and summarized in the JETOC Information sheet No. 43 of April 2000 - Special Issue no. 6. MAA at dosages of 0 (vehicle control), 100, 300 and 1000 mg/kg/day was orally administered to Crj:CD(SD) male and female rats (12 animals/sex/group) from 14 days before mating through the mating period and up to the day before necropsy (Total: 35 days) in males and 14 days prior to mating through the gestation period up to day 3 of delivery in females to investigate the effect on the repeat dose and reproductive toxicity for the parent animals and the development for the offspring. The no observed effect level (NOEL) under this study conditions was assumed to be 1000 mg/kg/day for repeat dose toxicity in both males and females, and also to be 1000 mg/kg/day for reproductive/ developmental toxicity in parent animals and offspring.

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 MAA, the selected structure show similar physical-chemical, toxicological and ecotoxicological properties. The available data on MAA 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 Methyl acetate. Based on the similar structure and properties, the substance Methyl acetate was selected as representative model structure for read-across. Compared to MAA, the selected structure show similar physical-chemical, toxicological and ecotoxicological properties. A data matrix covering all available endpoints was previously published in the EU-Risk assessment Report Vol. 34 of 2003.

The subacute 28-day inhalation toxicity on rats with the model structure Methyl acetate showed the following effects: Repeated exposure of Sprague Dawley rats to methyl acetate at the concentration of 2000 ppm cause impaired body weight gain, decreased food consumption and pathological changes of the olfactory epithelium. The toxicological relevance of the clinical chemistry findings remain unclear. The same applies to the hematological findings, for which an explanation could be hemoconcentration or oxygen deficit. Adrenal weights were increased in both sexes, possible reflecting stress of the animals caused by exposure to an irritant concentration of the test compound. Thymus weights were decreased in females. However, there were no changes observed by histopatholgical examination of adrenals and thymus. At the concentration of 350 ppm, slight increases in adrenal weights and slight decreases in thymus weights were observed in females. As there were no histopathological changes in these organs even at the much higher concentration of 2000 ppm, these findings are considered not to be of toxicological relevance. With regard to the present study, the No Observed Adverse Effect Level (NOAEL) is 350 ppm methyl acetate for male and female Sprague-Dawley rats, equivalent to a concentration of 1057 mg/m3 air.

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).