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Genetic toxicity: in vitro

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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:
12 April - 6 June 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted according to OECD Guideline 471 with minor deviations: temperature of the incubator rose to 38.1 °C overnight, just above the 37 ± 1 °C limit.
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:
yes
Remarks:
temperature of the incubator rose to 38.1 °C overnight, just above the 37 ± 1 °C limit
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No. : 122207
Purity : 98.7%
Name of test material (as cited in study report): NEROL (CAS 106-25-2)
Physical state: colourless - slightly yellow liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 21 July 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: 8.192, 20.48, 51.2, 128, 320 and 800 μg/plate, with S9 mix (preincubation method) and without S9 mix (plate incorporation method) in all 5 strains [2000 μg/plate employed for treatments of all strains in the absence of S9 only; 2500 μg/plate employed for treatments of all strains in the presence of S9 only]
- Experiment 3: 5, 10, 25, 50, 75, 100 and 125 μg/plate, with S9 mix in strain TA 102 (preincubation method)
- Experiment 4: 2.344, 4.688, 9.375, 18.75, 37.5, 75 and 150 μg/plate, with S9 mix in strain TA 1537 (preincubation method)

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
Formulation procedure:
- Test article stock solutions were prepared by formulating Nerol in DMSO under subdued lighting conditions with the aid of vortex mixing (as required) 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 6 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 where 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. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p ≤ 0.01) which was concentration related.
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
Key result
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 in all strains at 1581 and 5000 μg/plate.
- Experiment 2: Toxicity was observed at 51.2 μg/plate and above in strain TA 1537 in the presence of S9; 128 μg/plate and above in strains TA 98, TA 100 and TA 102 in the presence of S9; 320 μg/plate and above in strains TA 98 and TA 1537 in the absence of S9 and strain TA 1535 in the presence of S9; and 800 μg/plate in strains TA 100, TA 1535 and TA 102 in the absence of S9.
- Experiment 3: Toxicity was observed at 125 μg/plate with S9 mix in strain TA 102.
- Experiment 4: Toxicity was observed at 150 μg/plate with S9 mix in strain TA 1537.
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

Concentration (µg/plate)

Mean revertant numbers/plate

 

Experiment 1

Experiment 2

TA 98 -S-9

DMSO

 

23.4

 

20.6

Test item

5

28.3

8.192

24.3

15.81

23.0

20.48

18.3

50

22.0

51.2

22.7

158.1

26.7

128

16.3

500

19.0

320

16.3

1581

8.0

800

17.0

5000

T

2000

5.0

2NF

5

816.3

5

1183.0

TA 98 +S-9

DMSO

 

30.4

 

37.2

Test item

5

31.3

8.192

21.7

15.81

32.0

20.48

23.3

50

36.3

51.2

27.7

158.1

32.0

128

28.3

500

29.3

320

28.3

1581

15.0

800

T

5000

T

2500

T

B[a]P

10

306.3

10

540.3

TA 100 -S-9

DMSO

 

94.8

 

91.6

Test item

5

95.0

8.192

92.0

15.81

108.7

20.48

94.7

50

105.3

51.2

99.7

158.1

100.3

128

89.3

500

91.3

320

95.0

1581

45.0

800

78.7

5000

T

2000

22.7

NaN3

2

670.0

2

828.3

TA 100 +S-9

DMSO

 

100.4

 

97.2

Test item

5

110.3

8.192

101.0

15.81

107.7

20.48

96.0

50

116.3

51.2

97.0

158.1

104.0

128

95.3

500

110.0

320

75.0

1581

66.3

800

T

5000

T

2500

T

AAN

5

667.7

5

1343.0

TA 1535 -S-9

DMSO

 

22.8

 

18.2

Test item

5

21.0

8.192

20.7

15.81

27.3

20.48

23.7

50

27.3

51.2

20.0

158.1

25.7

128

25.0

500

24.0

320

23.7

1581

15.3

800

24.0

5000

T

2000

15.3

NaN3

2

463.7

2

711.3

TA 1535 +S-9

DMSO

 

22.2

 

20.4

Test item

5

22.3

8.192

21.3

15.81

22.7

20.48

23.3

50

25.0

51.2

17.7

158.1

29.0

128

12.3

500

26.7

320

11.3

1581

13.0

800

T

5000

T

2500

T

AAN

5

276.7

5

223.3

TA 1537 -S-9

DMSO

 

12.0

 

10.8

Test item

5

18.3

8.192

9.3

15.81

11.3

20.48

9.7

50

15.0

51.2

12.0

158.1

9.0

128

14.0

500

8.0

320

8.3

1581

3.0

800

5.0

5000

T

2000

T

AAC

50

201.7

50

55.3

TA 1537 +S-9

DMSO

 

12.6

 

17.2

Test item

5

15.0

8.192

16.3

15.81

18.0

20.48

12.0

50

17.3

51.2

12.3

158.1

15.0

128

13.0

500

16.7

320

7.7

1581

6.7

800

T

5000

T

2500

T

AAN

5

184.7

5

95.3

TA 102 -S-9

DMSO

 

265.8

 

241.2

Test item

5

232.3

8.192

241.7

15.81

278.3

20.48

257.7

50

274.7

51.2

248.0

158.1

277.0

128

244.0

500

259.7

320

232.3

1581

71.3

800

149.3

5000

T

2000

10.7

MMC

0.2

743.0

0.2

794.7

TA 102 +S-9

DMSO

 

231.0

 

239.8

Test item

5

266.3*

8.192

256.7

15.81

235.3

20.48

282.0*

50

261.3

51.2

296.0**

158.1

222.0

128

231.3

500

198.3

320

217.3

1581

202.7

800

T

5000

T

2500

T

AAN

20

1108.0

20

1042.0

 

Experiment 3 (TA 102 +S-9)

Experiment 4 (TA 1537 +S-9)

DMSO

 

233.6

 

10.8

Test item

5

212.7

2.344

8.0

10

239.3

4.688

8.0

25

242.7

9.375

7.3

50

242.0

18.75

8.7

75

242.0

37.5

5.0

100

218.7

75

7.7

125

222.3

150

7.0

AAN

20

1012.3

5

103.3

T: Toxic, no revertant colonies; * p ≤ 0.05; ** p ≤ 0.01

Applicant's summary and conclusion

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

Nerol 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 nerol 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: 8.192, 20.48, 51.2, 128, 320 and 800 μg/plate, with S9 mix (preincubation method) and without S9 mix (plate incorporation method) in all 5 strains [2000 μg/plate employed for treatments of all strains in the absence of S9 only; 2500 μg/plate employed for treatments of all strains in the presence of S9 only]

- Experiment 3: 5, 10, 25, 50, 75, 100 and 125 μg/plate, with S9 mix in strain TA 102 (preincubation method)

- Experiment 4: 2.344, 4.688, 9.375, 18.75, 37.5, 75 and 150 μg/plate, with S9 mix in strain TA 1537 (preincubation method)

Metabolic activation system used in this test was 10% of 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 in all strains at 1581 and 5000 μg/plate. In Experiment 2, toxicity was observed at 51.2 μg/plate and above in strain TA 1537 in the presence of S9; 128 μg/plate and above in strains TA 98, TA 100 and TA 102 in the presence of S9; 320 μg/plate and above in strains TA 98 and TA 1537 in the absence of S9 and strain TA 1535 in the presence of S9; and 800 μg/plate in strains TA 100, TA 1535 and TA 102 in the absence of S9. In Experiment 3, toxicity was observed at 125 μg/plate with S9 mix in strain TA 102. In Experiment 4, toxicity was observed at 150 μg/plate with S9 mix in strain TA 1537. The positive and vehicle controls induced the appropriate responses in the corresponding strains. Statistically significant increase in revertant numbers was observed in experiment 2 following Nerol treatments of strain TA102 at 51.2 μg/plate in the presence of S9 (Dunnett's Test, 1 % level), however this was not concentration-related or reproducible (Experiment 3) and was of insufficient magnitude to be considered as clear evidence of mutagenic activity in this assay system.

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