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Genetic toxicity: in vitro

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Administrative data

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:
5 April - 1 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD Guideline No 471 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name of the test item (as cited in the study report): NOPYL ACETATE
IUPAC Name of the test item: (1R,5S)-2-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl) ethyl acetate
Synonym: (-) Nopyl Acetate; 6,6-dimethyl -(1R,5S)-bicyclo[3.1.1]hept-2-ene-2-ethanol-2-acetate
Substance type: monoconstituent
Batch No.: 117042
Purity: 99.3%
This composition is within the specifications of the substance identity profile agreed within the SIEF.
Colour: colourless – slightly amber
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 10 April 2013

Method

Target gene:
Histidine gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: all strains were checked for characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline).
Metabolic activation:
with and without
Metabolic activation system:
10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Mutagenicity tests:
- Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, with and without S9 mix in all 5 strains (plate incorporation method)
- Experiment 2: 4.096, 10.24, 25.6, 64, 160, 400 and 1000 μg/plate, with S9 mix (preincubation method) and without S9 mix (plate incorporation method) in all 5 strains
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)

Formulation procedure:
Test item solutions were prepared by formulating nopyl acetate in DMSO under subdued lighting conditions immediately prior to assay to give the maximum required treatment solution concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 5 h of initial formulation.
Volume addition: 0.1 mL volume additions of vehicle/test article solution were used for all plate-incorporation treatments, 0.05 mL volume additions of vehicle/test article solution were used for pre-incubation treatments.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2- Aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM
Strains TA 98, TA 1535 and TA 1537 were originally obtained from the UK NCTC.
Strains TA 100 and TA 102 were derived from cultures originally obtained from Covance Laboratories Inc., USA.

METHOD OF APPLICATION
In agar (plate incorporation and preincubation method)

DURATION
Preincubation period: 20 minutes at 37 ± 1 °C
Incubation period: plates were inverted and incubated at 37 ± 1 °C in the dark for 3 days.

NUMBER OF REPLICATIONS
Treatment and positive control groups: 3 plates/dose
Vehicle control group: 5 plates/dose

DETERMINATION OF CYTOTOXICITY
Method: Background lawn was inspected for signs of toxicity.

OTHER:
Colony counting: colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually when confounding factors, such as bubbles or splits in the agar, affected the accuracy of the automated counter.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
1. an increase in revertant numbers gave a significant response (p ≤ 0.01) which was concentration related (when assessed using Dunnett's test).
2. the positive trend/effects described above were reproducible.

Test article was considered positive in this assay if all of the above criteria were met.
Test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
Mean vehicle control counts fell within the normal historical ranges (historical control data of February 2008 - July 2009).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: toxicity was observed at 500 µg/plate and above in all strains in the absence and presence of S9 except for the TA1537 in the absence of S9, where toxicity (decrease of the mean revertant number) was observed from 158.1 μg/plate.
- Experiment 2: toxicity was observed at 160 µg/plate and above in all strains in the absence and in the presence of S9.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/2: Results of mutagenicity

Group

Concentration (µg/plate)

Mean revertant numbers/plate

Standard Deviation

Concentration (µg/plate)

Mean revertant numbers/plate

Standard Deviation

Experiment 1

Experiment 2

TA 98 -S-9

DMSO

 

17.0

6.8

 

18.2

7.5

Nopyl acetate

5

22.3

5.8

4.096

18.7

5.5

15.81

18.7

6.4

10.24

17.7

2.9

50

19.7

10.8

25.6

21.3

5.1

158.1

14.3

2.1

64

22.3

4.6

500

11.7

2.1

160

17.7

10.7

1581

9.0

3.6

400

21.3

3.2

5000

17.0

5.2

1000

19.3

11.4

2NF

5

801.7

31.6

5

975.7

143.0

TA 98 +S-9

DMSO

 

31.4

6.5

 

30.8

7.7

Nopyl acetate

5

23.7

1.5

4.096

16.3

6.4

15.81

33.0

1.7

10.24

28.7

9.1

50

23.7

7.5

25.6

25.3

1.2

158.1

21.3

0.6

64

18.3

4.9

500

19.3

5.8

160

29.0

11.1

1581

18.0

7.9

400

24.0

7.0

5000

5.3

4.2

1000

16.7

3.8

B[a]P

10

339.3

45.6

10

442.7

51.1

TA 100 -S-9

DMSO

 

97.0

6.0

 

98.8

10.8

Nopyl acetate

5

74.3

15.7

4.096

84.7

4.2

15.81

84.3

10.4

10.24

91.3

4.2

50

76.0

2.6

25.6

82.3

7.1

158.1

59.3

4.9

64

80.7

4.0

500

61.0

1.0

160

68.3

7.0

1581

53.3

6.0

400

69.7

7.1

5000

48.3

12.1

1000

60.7

5.7

NaN3

2

675.0

8.2

2

696.0

82.4

TA 100 +S-9

DMSO

 

94.8

9.1

 

97.2

9.5

Nopyl acetate

5

85.0

5.0

4.096

105.7

12.7

15.81

95.0

11.3

10.24

90.0

9.2

50

86.0

13.9

25.6

95.3

7.0

158.1

106.3

6.0

64

98.7

7.6

500

85.0

6.6

160

85.3

6.1

1581

63.0

6.1

400

78.3

2.1

5000

T

1000

53.5

9.2

AAN

5

754.0

75.3

5

1086.3

91.5

TA 1535 -S-9

DMSO

 

13.8

1.6

 

15.4

6.3

Nopyl acetate

5

16.7

5.0

4.096

16.7

8.0

15.81

13.7

1.5

10.24

15.3

5.0

50

16.0

9.8

25.6

23.7

7.2

158.1

12.7

2.1

64

20.7

3.5

500

9.3

4.9

160

12.3

2.5

1581

8.7

2.5

400

7.7

3.1

5000

6.0

1.7

1000

10.7

5.0

NaN3

2

612.7

29.3

2

787.3

73.5

TA 1535 +S-9

DMSO

 

14.0

2.5

 

15.0

5.1

Nopyl acetate

5

20.0

3.6

4.096

15.0

1.7

15.81

17.0

4.4

10.24

21.0

4.0

50

18.7

4.7

25.6

22.0

5.3

158.1

12.3

2.5

64

19.7

5.5

500

13.0

2.6

160

20.7

6.0

1581

11.0

3.6

400

12.7

1.2

5000

4.7

0.6

1000

10.3

3.5

AAN

5

249.7

6.7

5

227.3

25.1

TA 1537 -S-9

DMSO

 

11.2

5.7

 

9.4

3.4

Nopyl acetate

5

8.0

3.6

4.096

15.0

6.6

15.81

9.0

5.3

10.24

14.3

2.1

50

8.7

7.2

25.6

15.7

3.5

158.1

2.7

2.1

64

11.0

6.0

500

4.3

0.6

160

7.0

3.0

1581

2.7

1.2

400

4.3

2.1

5000

T

1000

4.3

2.5

AAC

50

277.0

78.1

50

119.3

31.2

TA 1537 +S-9

DMSO

 

10.6

4.0

 

14.0

3.7

Nopyl acetate

5

11.7

2.9

4.096

16.7

3.8

15.81

13.3

6.0

10.24

14.7

4.0

50

18.3

6.7

25.6

17.7

7.4

158.1

14.3

2.1

64

13.3

5.9

500

11.0

7.2

160

17.0

8.5

1581

9.3

4.0

400

7.0

4.4

5000

T

1000

7.0

2.6

AAN

5

184.0

6.2

5

213.3

30.4

TA 102 -S-9

DMSO

 

234.4

7.7

 

254.8

19.8

Nopyl acetate

5

220.0

21.3

4.096

274.0

15.7

15.81

230.3

15.2

10.24

248.3

17.5

50

197.7

13.3

25.6

256.3

17.2

158.1

189.0

7.0

64

218.0

28.2

500

174.7

12.4

160

185.0

2.6

1581

138.0

3.5

400

200.0

21.3

5000

109.7

1.5

1000

210.3

24.2

MMC

0.2

715.7

7.4

0.2

727.3

111.0

TA 102 +S-9

DMSO

 

212.2

22.1

 

218.4

9.9

Nopyl acetate

5

222.3

33.5

4.096

248.3

17.7

15.81

212.0

15.4

10.24

208.3

24.7

50

194.0

24.8

25.6

221.7

17.6

158.1

218.0

20.8

64

235.7

29.3

500

184.0

18.7

160

217.3

36.3

1581

94.3

13.3

400

190.3

15.3

5000

T

1000

205.3

2.9

AAN

20

836.3

99.3

20

1339.0

85.6

T: Toxic, no revertant colonies

Applicant's summary and conclusion

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

Under the test conditions, nopyl acetate is not considered as mutagenic in S. typhimurium strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102).
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to nopyl acetate at the following concentrations:

- Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, with and without S9 mix in all 5 strains (plate incorporation method)

- Experiment 2: 4.096, 10.24, 25.6, 64, 160, 400 and 1000 μg/plate, with S9 mix (preincubation method) and without S9 mix (plate incorporation method) in all 5 strains.

Metabolic activation system used in this test was 10% S9 mix. S9 fraction was prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254. Vehicle control and positive control groups were also included in mutagenicity tests.

In Experiment 1, toxicity was observed at 500 µg/plate and above in all strains in the absence and presence of S9 except for the TA1537 in the absence of S9, where toxicity was observed from 158.1 μg/plate. In Experiment 2, toxicity was observed at 160 µg/plate and above in all strains in the absence and in the presence of S9. The positive and vehicle controls induced the appropriate responses in the corresponding strains. Test item did not induce statistically significant increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of metabolic activation.

Therefore, nopyl acetate is not considered as mutagenic in this bacterial system.