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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-isobutyl-4-methyltetrahydro-2H-pyran-4-yl acetate
EC Number:
942-380-9
Cas Number:
131796-64-0
Molecular formula:
C12H22O3
IUPAC Name:
2-isobutyl-4-methyltetrahydro-2H-pyran-4-yl acetate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ZH 291 A1 Fr.2-4
- Date of production of the lot/batch: 2016-08

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2600, and 5200 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with S9-mix
Positive control substance:
other: 2-aminoanthracene
Remarks:
2.5 μg/plate, TA 1535, TA 100, TA 1537, TA 98. 60 μg/plate, Escherichia coli WP2 uvrA
Untreated negative controls:
yes
Remarks:
Sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
5 μg/plate, TA 1535, TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
10 μg/plate, TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
9-aminoacridine
Remarks:
100 μg/plate, TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
5 μg/plate, E. coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation test

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method:
decrease in the number of revertants (factor ≤ 0.6)
clearing or diminution of the background lawn (= reduced his- or trp- background growth)

Evaluation criteria:
Acceptance criteria:
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10 to the power of 9 cells per mL were used.

Assessment criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 100 µg/plate with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 333 µg/plat with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 1000 µg/plate with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 1000 µg/plate, with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 1000 µg/plate with and without S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Experiment 1 (SPT)

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and Escherichia coli

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

 

 

 

Results with S9

DMSO

14.3

8.0

31.0

117.3

19.7

33

11.7

10.3

34.0

123.3

17.3

100

8.0

8.3

30.3

124.0

18.0

333

8.7

8.3

28.0

107.0

17.7

1000

6.7

7.3

32.0

112.7

17.3

2600

2.3

5.3

22.0

91.0

22.0

5200

0.0

1.7

9.7

0.0

18.7

positive control

141.0

164.7

1543.7

2151.7

97.0

 

 

 

Results without S9

DMSO

13.3

6.3

25.7

97.7

20.3

33

15.0

7.7

22.0

107.3

21.0

100

12.3

5.3

22.0

117.0

24.3

333

9.7

6.3

22.7

107.0

19.3

1000

11.7

6.3

19.7

108.0

21.7

2600

6.0

5.3

8.7

92.3

17.7

5200

3.3

0.0

0.0

0.0

24.0

positive control

3923.0

1032.0

714.0

3595.7

1391.3

 

 

 

 

 

 

Experiment 2 (PIT)

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and Escherichia coli

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

 

 

 

Results with S9

DMSO

9.7

6.7

24.7

89.7

28.7

33

8.7

8.3

25.7

83.3

21.3

100

9.3

7.0

20.3

97.3

22.7

333

8.0

8.0

28.0

100.0

26.3

1000

8.7

3.0

27.7

83.0

20.0

2600

2.3

2.7

6.7

28.3

24.0

5200

0.0

0.0

0.0

0.0

18.3

positive control

241.0

160.3

2192.7

2388.3

94.3

 

 

 

Results without S9

DMSO

8.3

7.0

18.3

89.0

20.3

10

12.3

9.3

25.3

91.0

26.0

33

10.0

8.3

20.0

96.7

23.3

100

8.0

8.7

18.7

102.3

25.0

333

5.0

6.0

13.3

79.7

22.3

1000

5.3

4.7

12.3

87.3

19.7

2600

7.7

3.7

14.7

68.7

17.3

positive control

3376.0

1214.0

555.3

2661.0

467.0

 

Applicant's summary and conclusion

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