Registration Dossier

Registration Dossier

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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100. Escherichia coli WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
S-9 from rat liver homogenate
Test concentrations with justification for top dose:
0, 4, 20, 100, 500, 2500 and 5000 ug/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Rationale for test conditions:
range finding test
Evaluation criteria:
statistically significant dose dependent increase
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
non-toxic up to 5000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

TA100

DOSE

Without Activation

With Activation

ug/Plate

Mean Value

Exp 1

Mean Value

Exp 2

Mean Value

Exp 1

Mean Value

Exp 2

0

168

129

168

148

4

178

137

178

136

20

170

133

170

137

100

169

140

169

146

500

185

139

185

135

2500

154

138

154

133

10000

157

148

157

139

+ ctrl

425

374

546

374

TA1535

DOSE

Without Activation

With Activation

ug/Plate

Mean Value

Exp 1

Mean Value

Exp 2

Mean Value

Exp 1

Mean Value

Exp 2

0

17

10

15

11

4

16

10

14

14

20

17

9

16

12

100

15

11

12

10

500

12

10

14

11

2500

14

9

15

13

10000

18

7

12

8

+ ctrl

350

297

114

102

TA1538

DOSE

Without Activation

With Activation

ug/Plate

Mean Value

Exp 1

Mean Value

Exp 2

Mean Value

Exp 1

Mean Value

Exp 2

0

16

10

19

13

4

16

10

16

15

20

16

11

19

14

100

19

11

19

16

500

14

11

21

12

2500

17

9

19

13

10000

14

9

19

15

+ ctrl

439

376

491

348

TA98

DOSE

Without Activation

With Activation

ug/Plate

Mean Value

Exp 1

Mean Value

Exp 2

Mean Value

Exp 1

Mean Value

Exp 2

0

24

22

30

28

4

22

24

32

28

20

23

21

19

30

100

25

24

32

29

500

23

24

32

31

2500

22

24

30

28

10000

24

24

31

28

+ ctrl

342

402

471

487

TA1537

DOSE

Without Activation

With Activation

ug/Plate

Mean Value

Exp 1

Mean Value

Exp 2

Mean Value

Exp 1

Mean Value

Exp 2

0

11

9

11

12

4

9

8

10

10

20

11

8

10

9

100

11

9

10

10

500

9

8

9

9

2500

11

11

11

9

10000

14

7

10

8

+ ctrl

98

126

98

106

 

WP2uvrA

DOSE

Without Activation

With Activation

ug/Plate

Mean Value

Exp 1

Mean Value

Exp 2

Mean Value

Exp 1

Mean Value

Exp 2

0

72

55

74

57

4

75

54

75

53

20

74

55

77

56

100

69

61

78

55

500

75

55

75

52

2500

75

57

79

52

10000

76

55

75

55

+ ctrl

280

509

216

446

Conclusions:
Not Mutagenic with or without metabolic activation
Executive summary:

Methyl acetate was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence adn in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 micrograms/plate to 5000 micrograms/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxic to the bacterial strains at 5000 micrograms/plate. 5000 micrograms/plate was chosen as the top dose level for the mutagenicity study.

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Methyl acetate did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that Methyl acetate is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Current guideline OECD 476 is from 1997 whereas study was reported in 1987.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
6TG resistance gene
8AG resistance gene
OUR resistance gene
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9mix
Test concentrations with justification for top dose:
15.8, 31.7, 47.4, and 63.3 mg/ml
Vehicle / solvent:
Culture medium "Eagle MEM"
Details on test system and experimental conditions:
The cells were treated with the test sample, exposed to gene mutation for 6 d, and selected.
Evaluation criteria:
In selecting the mutation cells, the number of mutation colonies was counted, with resistance to 8-Azoguanine, 6-Thioguanine and Ouabain.
Statistics:
t-test was performed.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

See "Attached background material"

Conclusions:
Methanol does not increase the frequency of gene mutation caused by drug resistance and no dose-response relationship was observed between the mutation frequency and the doese level.
Executive summary:

Methanol is a hydrolysis product of methyl acetate.

The test system using Chinese hamster V79 cells (similar to OECD 476 HPRT) detects the change of enzyme activation in the nucleic acid biosynthesis cycle (forward mutation from enzyme-ective to enzyme-damaging property) with the chemical resistance as indicator. Independent of the metabolism activation method, methanol did not induce a resistant colony (mutated colony) and did not show the gene mutation inducing property at a concentration of 31.7 to 63.3 mg/l.

A similar result is expected when testing methyl acetate as methyl acetate is rapidly hydrolysed after intake.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Near guideline studies, published in peer-reviewed literature, adequate for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells grown in Fischer's medium supplemented with 10% horse serum and 0.02% pluronic F-68
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes, at approximately 3 month intervals
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced liver S9 from male Sprague-Dawley rats
Test concentrations with justification for top dose:
0.04-0.3 µg/mL
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- A total of 1.2 x 107 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37± 1°C, washed twice with growth medium, and maintained at 37± 1°C for 48 h in log phase growth to allow recovery and mutant expression.
- Cells in the cultures were adjusted to 3 x 105/mL at 24 h intervals.
- They were then cloned (1 x 106 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar.
- Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. The 100x stock solution of TFT in saline was stored at -70°C and was thawed immediately before use.
- Plates were incubated at 37±1°C in 5% CO2 in air for 10-12 days and then counted with an automated colony counter. Only colonies larger than -0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 106 surviving cells.
Evaluation criteria:
Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
other: Inconclusive With and without S9
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: Inconclusive. With and without metabolic activation
Conclusions:
Inconclusive. With and without metabolic activation.
Acetic anhydride is considered to have no significant mutagenic activity in mammalian cells.
Executive summary:

Acetic anhydride has been examined for mutagenic activity in mammalian cells in vitro using the mouse lymphoma L5178Y assay. Although the data can be formally regarded as equivocal, they do not indicate any significant genotoxic activity for acetic anhydride in mammalian cells in vitro. This evaluation of the data reports the findings as inconclusive.

Acetic anhydride is considered to have no significant mutagenic activity in mammalian cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test) and EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winklemann GmbH
- Age at study initiation: 6-7 weeks (males); 8-10 weeks (females)
- Weight at study initiation: 160-200 g
- Assigned to test groups randomly: yes
- Housing: makrolon cages type 4 (5 animals per cage) on soft wood granulate
- Diet: ad libitum, rat/mice small diet ssniff(r) R/M-H (V 1534)
- Water: ad libitum, tap water inplastic bottles
- Acclimation period: 5 days under study conditions


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/_ 3 C
- Humidity (%): 50 +/- 20%
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: October 13, 1998 To: December 15, 1998
Route of administration:
inhalation
Vehicle:
None
Details on exposure:
nose -only
Duration of treatment / exposure:
28 days, 20 exposures
Frequency of treatment:
6 hours/day 5 days a week
Post exposure period:
24 h
Dose / conc.:
0.227 mg/L air
Dose / conc.:
1.057 mg/L air
Dose / conc.:
6.04 mg/L air
No. of animals per sex per dose:
5 male / 5 female
Control animals:
yes, concurrent no treatment
Positive control(s):
Endoxan (r)
cyclophosphamide
C7H15Cl2N2P ~ H2O
50-18-0
ASTA Medica AG, Weismullerstr.45, D-60314 Frankfurt Germany
bathc Number: 603575B
Administered once orally by gavage at a dose of 25 mg/kg body weight 24 hours before killing.
Tissues and cell types examined:
Erythrocytes from femora bone marrow
Details of tissue and slide preparation:
Animals of the negative and postive control groups were killed by dissection of the vena cava cranialis in deep narcosis and exsanguinated. the animals of the positive control group were killed by carbon dioxide asphyxiation. For each animal, about 6 ml fetal bovine serum was poured into a centrifuge tube. one femora was removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. the mixture was then centifuged for 5 minutes at approx. 1200 rpm, after which almost all of the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours. Staining was performed as follows:
-5 min in methanol
- 5 min in May-Grunwald's solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2
-rinsing in distilled water
-drying
-coating with Entellan(r)
Evaluation criteria:
1000 polychromatic and 1000 normochromatic erythrocytes were counted for each animal. The number of cells with the micronuclei was recorded, no the number of individual micronuclei. in addition, the ratio of polychromatic erythrocytes to 1000 normochromatic erythrocytes were determined. Main parameter for the the statistical anaylsis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 1000 counted erythyrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
The test substance is considered as positive if there is a significant dose-related increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control gruop. A test substance producing no significant dose -related increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in the test system.
Statistics:
A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).
Based on a monotone-dose-relationship one-sided Wilcoxon test were performed starting with the highest dose group. These tests were performed with a multiple level of significance of 5%. Tests on lower dose groups were only performed if all higher dose groups were significantly different from the control.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: systemic, probably secondary effects on a very low level.
Additional information on results:
Due to the rapid cleavage of the ester by hydrolases it was not possible to detect methyl acetat in the blood of rats (detection limit 5ppm)

Sex

Dose

Sample

Time

Number of Animals

Poly mean

Normo

mean

Erythrocytes

Eyrthrocytes with Micronuclei

Mean

P/N SD

Poly

mean

Mut.I

Normo

Mean

Mut. I.

No

%

SD

No

%

SD

Pooled

(-) Ctrl

24H

10

1000

1000

0.7

0.18

2.3

0.2

0.13

1.0

0.7

0.1

0.07

1.0

 

75

24H

10

1000

1000

0.6

0.18

1.8

0.2

0.1

1.8

0.2

0.1

0.04

0.3

 

350

24H

10

1000

1000

0.7

0.16

2.4

0.2

0.11

1.0

1.2

0.1

0.06

1.7

 

2000

24H

10

1000

1000

0.6

0.13

1.6

0.2

0.1

0.7

0.8

0.1

0.08

1.1

 

(+) ctrl

24H

10

1000

1000

0.7

0.16

17.9

1.8

0.45*

7.8

0.7

0.1

0.05

1.0

Sex

Dose

Sample

Time

Number of Animals

Poly mean

Normo

mean

Erythrocytes

Eyrthrocytes with Micronuclei

Mean

P/N SD

Poly

mean

Mut.I

Normo

Mean

Mut. I.

No

%

SD

No

%

SD

Male

(-) Ctrl

24H

5

1000

1000

0.5

0.13

2.2

0.2

0.15

1.0

0.8

0.1

0.08

1.0

 

75

24H

5

1000

1000

0.4

0.06

1.8

0.2

0.08

0.8

0.4

0.0

0.05

0.5

 

350

24H

5

1000

1000

0.6

0.11

1.8

0.2

0.08

0.8

1.4

0.1

0.05

1.8

 

2000

24H

5

1000

1000

0.6

0.11

1.6

0.2

0.05

0.7

1.2

0.1

0.08

1.5

 

(+) ctrl

24H

5

1000

1000

0.7

0.20

19.2

1.9

0.61

8.7

0.8

0.1

0.04

1.0

Female

(-) Ctrl

24H

5

1000

1000

0.8

0.09

2.4

0.2

0.11

1.0

0.6

0.1

0.05

1.0

 

75

24H

5

1000

1000

0.7

0.17

1.8

0.2

0.13

0.8

0.0

0.0

0.0

0.0

 

350

24H

5

1000

1000

0.8

0.16

3.0

0.3

0.10

1.3

1.0

0.1

0.07

1.7

 

2000

24H

5

1000

1000

0.7

0.13

1.6

0.2

0.13

0.7

0.4

0.0

0.05

0.7

 

(+) ctrl

24H

5

1000

1000

0.6

0.10

16.6

1.7

0.23

6.9

0.6

0.1

0.05

1.0

Conclusions:
Interpretation of results (migrated information): negative
The results lead to the conclusion that methyl acetate did not lead to a substantial increase of micronucleated polychromatic erythyrocytes and is not mutagenic in the micronucleus test.
Executive summary:

The micronucleus test was carried out with methyl acetate to evaluate potential for cytogenetic damage. The animals were administered with the test compound in a rat subacute inhalation toxicity study (20 exposures within 28 days, 6 hours/day, report no. 99.0011) at exposure concentrations of 75, 350 and 2000 ppm (226.5, 1057.0 and 6040.0 mg/m3). According to the test procedure the animals were killed 24 hours after the last administration.

Endoxan(r) (cyclophosphamide) was used as positive control substance and was administered once orally at a dose of 25 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to normochromatic erythrocytes in both and female animals remained unaffected by the treatment with Methyl acetate adn was not less than 20% of the control value.

Endoxan(r) induced a marked statistically significant increase in the number of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that Methyl acetate is not genotixic in the micronucleus test.

Additional information

In vitro

Methyl acetate was tested for its mutagenic potential in strains of S. typhimurium (Ames test) and E. Coli (Muller, 1988). The test was conducted according to OECD Guideline 471 (Bacterial Reverse Mutation Assay). The following strains were used: TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 micrograms/plate to 5000 micrograms/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxic to the bacterial strains at 5000 micrograms/plate. 5000 micrograms/plate was chosen as the top dose level for the mutagenicity study.

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Methyl acetate did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that Methyl acetate is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated. This result is supported by a study by Zeiger et al. (1992).

In this study methyl acetate was tested a 0, 100, 333, 1000, 3333 and 10000 ug/plate in Salmonella typhimurium strains TA 97, TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation. Metabolic activation consisted of 10% and 30% Hamster liver derived S-9 as well as 10% and 30% rat liver derived S-9 for all strains but TA 1537 which was only tested with 30% hamster- and 30% rat liver derived S-9. Testing was negative with and without metabolic activation at all test levels and strains and variations of metabolic activation. 

There is no study available in which methyl acetate was tested for mammalian cell gene mutagenicity according to OECD guideline 476. Methyl acetate is rapidly hydrolysed into methanol and acetic acid after intake. Read-across with methanol and acetic acid is therefore scientifically justified.

Methanol was tested for mammalian cell gene mutation in Chinese hamster lung fibroblasts (V79) (NEDO, 1987). This study was similar to OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test) (NEDO, 1987).

Independent of the metabolism activation method, methanol did not induce a resistant colony (mutated colony) and did not show the gene mutation inducing property at a concentration of 31.7 to 63.3 mg/l. No increases in mutant frequency in gene mutation to drug resistance vs. neg. control was observed, whereas the pos. control DMN produced increases in dose-related manner.

Only few studies are available on the mutagenicity of acetic acid (EU-RAR, 2003). Acetic acid was negative in a bacterial gene mutation test (Ames test; von der Hude et al., 1988). EFSA (2008a) concluded in the DAR for acetic acid that "Long term toxicity/carcinogenicity studies in animals with oral exposure are not necessary, considering that humans are exposed to orally ingested acetic acid from various food sources and there is no evidence that such exposure is causally related to toxic effects and an increased cancer incidence."

Acetic anhydride has been examined for mutagenic activity in mammalian cells in vitro using the mouse lymphoma L5178Y assay similar to OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test. Although the data can be formally regarded as equivocal, they do not indicate any significant genotoxic activity for acetic anhydride in mammalian cells in vitro. This evaluation of the data reports the findings as inconclusive.

Acetic anhydride is considered to have no significant mutagenic activity in mammalian cells. Other acetates did not show mutagenic properties in the Ames assay, too; ethyl acetate (OECD SIDS, 2007a) as well as n-butyl acetate (OECD SIDS, 2008), isobutyl acetate (OECD SIDS, 2007b) and n-propyl acetate (OECD, 2009) proofed to be not mutagenic.

 

In vivo

The test by Stammberger (1999) was performed according to OECD guideline 474. Methyl acetate was administered by inhalation (20 exposures within 28 days, 6 hours/day) to the test animals at doses of 75, 350 and 2000 ppm. The animals of the negative control group were treated with air only. The study included a concurrent positive control, which was administered once orally by gavage at a dose of 25 mg per kg body weight 24 hours before sacrifice. Following dosing, the animals were examined regularly for mortality and clinical signs of toxicity. According to the test procedure the animals were sacrificed 24 hours after the last administration.

The incidence of micronucleated polychromatic erythrocytes in the dose groups of methyl acetate was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes was
observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and was not less than 20% of the control values. The positive control cyclophosphamide induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.


Conclusion

Methyl acetate was negative in bacterial reverse mutation tests and in an in-vivo study according to OECD guideline 474. Furthermore, the hydrolysis products methanol and acetic acid did not reveal evidence for a mutagenic or clastogenic potential. There is no concern and no further testing needed with respect to genotoxicity.

Based on the fact that methyl acetate was not mutagenic in the Ames assay and not genotoxic in the in vivo micronucleus test it is not expected that methyl acetate alters genetic material and leads to germ cell mutagenicity. The negative data is supported by data on methanol and acetic acid.

Literature:

EU-RAR (2003): EU Risk Assessment Report - Methyl Acetate, Final Report 2003

von der Hude W, Behm C, Gürtler R, Basler A (1988): Evaluation of the SOS chromo-test. Mutation Research 203, 181-194.

Morita T, Takeda K, Okumura K (1990): Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells. Mutation Research 240, 195-202.

OECD SIDS (2007a): SIDS Dossier for ethyl acetate. OECD HPV Chemical Programme, SIDS Dossier, apprioved at SIAM 14, revised May 2007.

OECD SIDS (2008): SIDS Dossier for n-butyl acetate. OECD HPV Chemical Programme, SIDS Dossier, approved at SIAM 13, revised 2008.

OECD SIDS (2007b): SIDS Dossier for isobutyl acetate. OECD HPV Chemical Programme, SIDS Dossier, apprioved at SIAM 17, dated November 2007.

OECD SIDS (2009): SIDS Dossier for n-propyl acetate. OECD HPV Chemical Programme, SIDS Dossier, apprioved at SIAM 27, dated May 2009.

EFSA (European Food Safety Authority), 2008a. Draft Assessment Report (DAR): Acetic Acid. Vol. 3, Annex B, part 2, B.6 (Aug, 2008).


Short description of key information:
Methyl acetate is not mutagenic in the Ames-test bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated. Other acetates (ethyl acetate, n-butyl acetate, isobutyl acetate, n-propyl acetate) were also not mutagenic in the Ames assay.
An in-vivo-test according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) showed no genetic toxicity of methyl acetate.
Methyl acetate is rapidly hydrolysed into methanol and acetic acid after intake. Read-across with methanol and acetic acid is is therefore justified. Methanol did not show genetic toxicity when tested in the bacterial reverse mutation assay and according to equivalent to OECD guideline 476 HPRT. There is no concern with respect to mutagenicity for acetic acid.
Methyl acetate is not considered as a mutagen.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Methyl acetate is negative in a bacterial mutation test and in an in-vivo study according to OECD guideline 474.Furthermore, the hydrolysis products methanol and acetic acid do not reveal evidence for a mutagenic potential. There is no concern with respect to mutagenicity. Methyl acetate should not be classified as a mutagen.

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