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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
In vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA (OECD 471) (Huntingdon, 2014).
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-06-10 to 2014-08-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 2012-11-20/22, Date of signature: 2014-04-14
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon (Salmonella strains)
tryptophan operon (E.coli strain)
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/ 5,6-benzoflavone activated rat liver S9
Test concentrations with justification for top dose:
Test 1 : 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Test 2 : 50, 150, 500, 1500, 5000 µg/plate
Test 2 repeat [TA98]: 15, 50, 150, 500, 1500, 5000 µg/plate
Test 2 repeat [TA1537]: 50, 150, 500, 1500, 5000 µg/plate
Test 2 repeat [WP2 uvrA]: 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Test 3 [TA1535]: 50, 150, 500, 1500, 3000, 4000, 5000 µg/plate
Test 4 [TA1535]: 5, 15, 50, 150, 500, 1500, 3000, 4000, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: the test item was fully miscible in dimethyl sulphoxide at 50 mg/ml.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 μg/plate for strains TA98 and TA1537, with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
5 μg/plate for strains TA100 and TA1535; 10 μg/plate for strain WP2 uvrA (pKM101), with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
2 μg/plate for strain WP2 uvrA (pKM101), without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
2 μg/plate for strain TA98, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
50 μg/plate for strain TA1537, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
2 μg/plate for strains TA100 and TA1535, without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours

SELECTION AGENT (mutation assays): histidine or tryptophan deficient agar


NUMBER OF REPLICATIONS: triplicate plates, initial plate incorporation experiment repeated using preincubation


DETERMINATION OF CYTOTOXICITY
- Method other: condition of bacterial lawn, reduction in number of revertants

ACTIVATION

The S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water.
0.5 ml of S9 mix was added to 2ml of agar, giving a final concentration of approximately 2%.
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
Statistics:
No statistical analysis was performed.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experiment 1 Plate incorporation without metabolic activation revertants per plate (mean of three plates)

 

Ta 98

TA100

TA1535

TA1537

WP2 uvrA

Solv cnt

48.0

182.7

46.7

17.3

108.7

5 µg

47.7

238.3

38.7

14.0

103.7

15 µg

42.3

201.7

44.3

19.3

98.0

50 µg

37.7

221.0

38.3

18.3

105.3

150 µg

36.3

206.7

40.0

12.7

92.3

500 µg

51.7

246.7

42.0

15.3

105.0

1500 µg

46.0

235.7

66.0

16.3

127.3

5000 µg

40.7

230.3

63.0

18.3

79.0

Pos cont

335.0

534.7

939.7

326.0

1397.7

 

 

 

Experiment 1 Plate incorporation with metabolic activation revertants per plate (mean of three plates)

 

Ta 98

TA100

TA1535

TA1537

WP2 uvrA

Solv cnt

62.3

203.3

23.0

34.3

120.7

5 µg

50.3

198.0

23.3

38.0

98.0

15 µg

49.7

201.0

17.3

43.3

101.7

50 µg

50.7

208.7

14.3

32.7

117.0

150 µg

47.0

190.7

20.7

32.7

123.3

500 µg

66.7

189.0

17.0

33.7

130.0

1500 µg

58.3

210.7

21.0

38.0

113.3

5000 µg

56.3

102.3

20.0

47.7

84.3

Pos cont

319.7

1258.0

390.3

186.0

421.0

 

 

 

Experiment 2 pre incubation without metabolic activation revertants per plate (mean of three plates)

 

Ta 98

TA100

TA1535

TA1537

WP2 uvrA

Solv cnt

63.7

142.0

24.3

49.0

133.7

50 µg

70.7

184.7

26.3

31.7

142.7

150 µg

67.3

191.0

18.0

37.3

151.0

500 µg

60.3

168.7

17.0

36.3

170.0

1500 µg

32.0

166.0

13.7

48.0

76.7

5000 µg

26.3

31.7

78.0

101.0

131.3

Pos cont

532.0

396.0

384.7

1204.0

2254.3

 

 

 

 

Experiment 2 pre incubation with metabolic activation revertants per plate (mean of three plates)

 

Ta 98

TA100

TA1535

TA1537

WP2 uvrA

Solv cnt

70.0

205.0

34.0

20.3

73.3

50 µg

55.7

140.3

20.7

31.3

110.7

150 µg

51.3

175.0

25.7

28.7

133.0

500 µg

54.3

199.7

30.7

27.7

37.3

1500 µg

56.7

185.0

31.7

24.7

38.3

5000 µg

58.7

162.3

50.3

18.7

41.7

Pos cont

394.3

702.7

193.0

293.3

510.0

 

Pre incubation, revertants per plate (mean of three plates)- Experiment 2 repeat, experiment 3 and experiment 4 

 

Test 2 repeat

Test 3

Test 4

 

Without metabolic activation

 

With metabolic activation

 

Without metabolic activation

 

Without metabolic activation

 

 

TA98

TA1537

WP2 uvrA

TA1535

TA1535

Solv cnt

47.0

26.3

202.7

21.0

24.3

5 µg

 

 

208.0

 

19.3

15 µg

47.3

 

221.3

 

23.0

50 µg

52.3

20.0

210.7

21.7

24.3

150 µg

41.7

25.3

199.3

19.0

19.3

500 µg

48.0

25.3

193.3

29.7

30.7

1000 µg

 

 

 

 

15.7

1500 µg

37.0

17.7

199.7

3.3

0.0

3000 µg

 

 

 

0.7

0.0

4000 µg

 

 

 

0.0

0.0

5000 µg

20.0

0.0

118.0

1.3

0.0

Pos cont

447.0

893.0

507.3

1085.3

873.3

 

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

α-Methylenebenzyl acetate has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, and in compliance with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in the initial or the repeat experiments up to limit concentrations. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that α-methylenebenzyl acetate is negative for mutagenicity to bacteria under the conditions of the test.
Executive summary:

α-Methylenebenzyl acetate has been tested for mutagenicity to bacteria, in a study which was conducted according to OECD TG 471, compliant with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in the initial or the repeat experiments up to limit concentrations using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

 

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

Additional information

Additional information from genetic toxicity in vitro:

α-Methylenebenzyl acetate has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, compliant with GLP (Huntingdon, 2014). No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in the initial or the repeat experiments up to limit concentrations using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.


Justification for selection of genetic toxicity endpoint
The study was conducted in accordance with appropriate guidelines and in compliance with GLP

Justification for classification or non-classification

Based on the available in vitro data, α-methylenebenzyl acetate does not require classification for mutagenicity according to Regulation (EC) No. 1272/2008.