Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 June 2017 - 01 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-methylheptyl acrylate
EC Number:
256-005-5
EC Name:
1-methylheptyl acrylate
Cas Number:
42928-85-8
Molecular formula:
C11H20O2
IUPAC Name:
octan-2-yl prop-2-enoate
Test material form:
liquid

Method

Target gene:
Histidine operon
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):
n/a
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
n/a
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route
Test concentrations with justification for top dose:
The highest recommended dose level of 5000 µg/plate was achievable. Thus, the dose levels selected for the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose levels.
Without S9 mix, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose levels equal or above 500 µg/plate in the TA 100 strain, equal or above 1000 µg/plate in the TA 98 strain and at 5000 µg/plate in the TA 102 strain.
With S9 mix, a moderate toxicity was noted at dose levels equal or above 2500 µg/plate in the TA 98 strain and at 5000 µg/plate in the TA 100 strain.

Therfore, the selection of the highest dose level for the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

Without S9 mix:

Selected dose levels were:
- 20.6, 61.7, 185.2, 555.6, 1667 and 5000 µg/plate for the TA 102 strain in the 1st experiment,
- 6.86, 20.6, 61.7, 185.2, 555.6 and 1667 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the 1st experiment,
- 31.3, 62.5, 125, 250, 500 and 1000 µg/plate for the TA 1535, TA 1537, TA 98 and TA 102 strains in the 2nd experiment,
- 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 100 strain in the 2nd experiment.

With S9 mix:

The selected dose levels were:
- 20.6, 61.7, 185.2, 555.6, 1667 and 5000 µg/plate for the 5 strains in the 1st experiment,
- 25, 50, 100, 200, 400 and 800 µg/plate for the TA 102 strain in the 2nd experiment,
- 12.5, 25, 50, 100, 200 and 400 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the 2nd experiment.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO).
- Justification for choice: the test item was soluble in the vehicle at 100 mg/mL.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
see Table 7.6.1/1
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
see Table 7.6.1/1
Positive control substance:
benzo(a)pyrene
other: 2-anthramine
Remarks:
With S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

DURATION
- Preincubation period: 60 minutes, 37°C (for 2nd experiment with S9 mix only).
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn

NUMBER OF REPLICATIONS:
Three plates/dose level.
Evaluation criteria:
Acceptance criteria:
Each main experiment was considered valid if the following criteria are fully met:
-> the mean number of revertants in the vehicle controls is consistent with the historical data, in each strain and test condition (see table 7.6.1/2),
-> at least five analyzable dose levels (i.e. including at least three non-cytotoxic dose levels) are obtained for each strain and test condition,
-> the mean number of revertants in the positive controls is higher than that of the vehicle controls (at least 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or at least 3-fold increase (for the TA 1535 and TA 1537 strains)).
When these criteria were not met for one strain or test condition, the corresponding results were invalidated and the experiment was repeated.

Criteria for assessing mutagenic potential:
In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

->The test item was considered to have shown mutagenic activity in the study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains)
in the mean number of revertants compared with the vehicle controls was observed, in any strain, at any dose level,
- and/or a reproducible dose-response relationship was evidenced.

->The test item was considered to have shown no mutagenic activity in the study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains)
or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, was observed at any of the tested dose levels,
- nor any evidence of a dose-response relationship was noted.
Statistics:
no

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see tables 7.6.1/2 to 7.6.1/5
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the main experiment, no precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose levels.

RANGE-FINDING STUDY:
- No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose levels.
- Without S9 mix, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose levels equal or above to 500 µg/plate in the TA 100 strain, equal or above to 1000 µg/plate in the TA 98 strain and at 5000 µg/plate in the TA 102 strain.
- With S9 mix, a moderate toxicity was noted at dose levels equal or above to 2500 µg/plate in the TA 98 strain and at 5000 µg/plate in the TA 100 strain.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean revertant colony counts for the vehicle controls were within the current historical control range of the laboratory (see table 7.6.1/ 2 in Any Other Information on Materials and methods).

RESULTS OF CYTOTOXICITY and GENOTOXICITY:

Experiments without S9 mix

- A moderate to strong toxicity was noted at dose levels equal or above 250 µg/plate in the TA 1535, TA 1537, TA 98 and TA 102 strains and at dose levels equal or above 125 µg/plate in the TA 100 strain.
- Slight increases in the number of revertants were noted in the TA 1537 strain in the second experiment. Since these increases did not reach the positive threshold of 3-fold the vehicle control value, were not dose-related and since no similar results were observed in the first experiment, they were considered to be non-biologically relevant.
No other noteworthy increase in the number of revertants was observed in any other strains.

Experiments with S9 mix

- When using the direct plate incorporation method (i.e. first experiment), a moderate to strong toxicity was noted at 5000 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains. When using the pre-incubation method (i.e. second experiment), a moderate to strong toxicity was noted at dose levels equal or above 200 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and at 800 µg/plate in the TA 102 strain. In the TA 1537 and TA 100 strains, the strong toxicity induced at 400 µg/plate prevented the scoring of revertant colonies (presence of microcolonies).
- The test item did not induce any noteworthy increase in the number of revertants, in any strains and test conditions.

Any other information on results incl. tables

7.6.1/2: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation in the first test (direct plate incorporation method)

Strain

Compound

Dose-level (µg/plate)

With or without S9 mix

Number of revertant per plate

Mean revertant colony count (SD)

TA1535

NAN3

1

-

537-496-546

526.3 (26.7)

TEST ITEM

1667

-

7St-9St-4St

6.7 (2.5)

555.6

-

7Mt-3Mt-4Mt

4.7 (2.1)

185.2

-

10-8-9

9.0 (1.0)

61.7

-

6-6-10

7.3 (2.3)

20.6

-

7-4-6

5.7 (1.5)

6.86

-

8-7-10

8.3 (1.5)

DMSO

 

-

12-8-9

9.7 (2.1)

TA1537

9 AA

50

-

88-51-75

71.3 (18.8)

TEST ITEM

1667

-

2St-2St-3St

2.3 (0.6)

555.6

-

3Mt-4Mt-4Mt

3.7 (0.6)

185.2

-

4-4-6

4.7 (1.2)

61.7

-

3-4-3

3.3 (0.6)

20.6

-

6-7-4

5.7 (1.5)

6.86

-

7-3-9

6.3 (3.1)

DMSO

 

-

9-4-9

7.3 (2.9)

TA 98

2 NF

0.5

-

107-112-86

101.7 (13.8)

TEST ITEM

1667

-

11St-12St-13St

12 (1.0)

555.6

-

11Mt-12Mt-10Mt

11 (1.0)

185.2

-

11-19-12

14.0 (4.4)

61.7

-

13-19-15

15.7 (3.1)

20.6

-

21-18-21

20 (1.7)

6.86

-

29-11-10

16.7 (10.7)

DMSO

 

-

18-10-21

16.3 (5.7)

TA 100

NAN3

1

-

484-473-433

463.3 (26.8)

TEST ITEM

1667

-

60St-48St-95St

67.7 (24.4)

555.6

-

76St-87St-85St

82.7 (5.9)

185.2

-

79Mt-78Mt-113Mt

90 (19.9)

61.7

-

85-72-70

75.7 (8.1)

20.6

-

73-77-81

77 (4.0)

6.86

-

76-79-60

71.7 (10.2)

DMSO

 

-

72-102-85

86.3 (15.0)

TA102

MMC

0.5

-

1869-1985-2004

1952.7 (73.1)

TEST ITEM

5000

-

182St-177St-188St

182.3 (5.5)

1667

-

184St-151St-149ST

161.3 (19.7)

555.6

-

201St-179St-140St

173.3 (30.9)

185.2

-

341-318-369

342.7 (25.5)

61.7

-

362-348-321

343.7 (20.8)

20.6

-

411-445-417

424.3 (18.1)

DMSO

 

-

445-409-399

417.7 (24.2)

7.6.1/3: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation in the first test (direct plate incorporation method) 

Strain

Compound

Dose-level (µg/plate)

With or without S9 mix

Number of revertant per plate

Mean revertant colony count (SD)

TA1535

2AM

2

+

 138 -153 -135

142.0 (9.6)

TEST ITEM

5000

+

8Mt-16Mt-11Mt

11.7 (4.0)

1667

+

6-8-11

8.3 (2.5)

555.6

+

9-16-13

12.7 (3.5)

185.2

+

12-6-13

10.3 (3.8)

61.7

+

10-12-8

10.0 (2.0)

20.6

+

4-7-8

6.3 (2.1)

DMSO

 

+

11-10-11

10.7 (0.6)

TA1537

2AM

2

+

78-87-82

82.3 (4.5)

TEST ITEM

5000

+

7Mt-4Mt-7Mt

6.0 (1.7)

1667

+

4-3-7

4.7 (2.1)

555.6

+

10-8-9

9.0 (1.0)

185.2

+

8-6-10

8.0 (2.0)

61.7

+

23-6-7

12.0 (9.5)

20.6

+

6-8-8

7.3 (1.2)

DMSO

 

+

9-8-11

9.3 (1.5)

TA 98

2 AM

2

+

817-981-888

895.3 (82.2)

TEST ITEM

5000

+

12Mt-20Mt-9Mt

13.7 (5.7)

1667

+

15-17-19

17 (2.0)

555.6

+

21-17-22

20.0 (2.6)

185.2

+

19-19-26

21.3 (4.0)

61.7

+

12-21-23

18.7(5.9)

20.6

+

19-22-25

22.0 (3.0)

DMSO

 

+

17-18-23

19.3 (3.2)

TA 100

BaP

5

+

1083-1085-1082

1083.3 (1.5)

TEST ITEM

5000

+

85St-87St-104St

92.0 (10.4)

1667

+

98-100-114

104.0 (8.7)

555.6

+

108-104-103

105.0 (2.6)

185.2

+

105-125-107

112.3 (11.0)

61.7

+

102-124-108

111.3 (11.4)

20.6

+

103-111-105

106.3 (4.2)

DMSO

 

+

113-143-98

118.0 (22.9)

TA102

2AM

20

+

2633-2639-3372

2881.3 (424.9)

TEST ITEM

5000

+

436-361-351

382.7 (46.5)

1667

+

503-437-457

465.7(33.8)

555.6

+

464-391-433

429.3 (36.6)

185.2

+

448-479-423

450.0 (28.1)

61.7

+

447-463-443

451.0 (10.6)

20.6

+

489-504-470

487.7 (17.0)

DMSO

 

+

499-514-441

484.7 (38.6)

 

 

7.6.1/4: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation in the second test (direct plate incorporation method)

 

Strain

Compound

Dose-level (µg/plate)

With or without S9 mix

Number of revertant per plate

Mean revertant colony count (SD)

TA1535

NAN3

1

-

636-666-690

664.0 (27.1)

TEST ITEM

1000

-

15Mt-11Mt-9Mt

11.7 (3.1)

500

-

9Mt-13Mt-4Mt

8.7 (4.5)

250

-

8Mt-7Mt-9Mt

8.0 (1.0)

125

-

13-16-2

10.3 (7.4)

62.5

-

9-6-13

9.3 (3.5)

31.3

-

11-8-11

10.0 (1.7)

DMSO

 

-

4-7-15

8.7 (5.7)

TA1537

9AA

50

-

210-292-287

263.0 (46.0)

TEST ITEM

1000

-

4Mt-4Mt-6Mt

4.7 (1.2)

500

-

8Mt-3Mt-4Mt

5.0 (2.6)

250

-

9Mt-7Mt-3Mt

6.3 (3.1)

125

-

3-1-9

4.3 (4.2)

62.5

-

7-3-6

5.3 (2.1)

31.3

-

9-8-6

7.7 (1.5)

DMSO

 

-

2-3-3

2.7 (0.6)

TA 98

2 NF

0.5

-

151-139-143

144.3 (6.1)

TEST ITEM

1000

-

20Mt-12Mt-17Mt

16.3 (4.0)

500

-

20Mt-15Mt-17Mt

17.3 (2.5)

250

-

12Mt-17Mt-17Mt

15.3 (2.9)

125

-

7-17-16

13.3 (5.5)

62.5

-

12-12-18

14.0 (3.5)

31.3

-

13-15-11

13.0 (2.0)

DMSO

 

-

17-18-17

17.3 (0.6)

TA 100

NAN3

1

-

559-568-595

574.0 (18.7)

TEST ITEM

500

-

100Mt-115Mt-89Mt

101.3 (13.1)

250

-

79Mt-97Mt-104Mt

93.3 (12.9)

125

-

93Mt-79Mt-93Mt

88.3 (8.1)

62.5

-

91-78-107

92.0 (14.5)

31.3

-

88-97-105

96.7 (8.5)

15.6

-

101-77-69

82.3 (16.7)

DMSO

 

-

96-83-78

85.7 (9.3)

TA102

MMC

0.5

-

2462-2453-2235

2383.3 (128.5)

TEST ITEM

1000

-

237St-219St-172St

209.3 (33.6)

500

-

283Mt-335Mt-309Mt

309.0 (26.0)

250

-

278Mt-332Mt-344Mt

318.0 (35.2)

125

-

323-325-296

314.7 (16.2)

62.5

-

379-342-381

367.3 (22.0)

31.3

-

385-353-373

370.3 (16.2)

DMSO

 

-

343-339-357

346.3 (9.5)

 

7.6.1/5: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation in the second test (preincubation method) 

Strain

Compound

Dose-level (µg/plate)

With or without S9 mix

Number of revertant per plate

Mean revertant colony count (SD)

TA1535

2AM

2

+

187-154-163

168.0 (17.1)

TEST ITEM

400

+

6st-13St-3St

7.3 (5.1)

200

+

12Mt-11Mt-7Mt

10.0 (2.6)

100

+

11-12-10

11.0 (1.0)

50

+

10-10-18

12.7 (4.6)

25

+

13-16-12

13.7 (2.1)

12.5

+

15-13-9

12.3 (3.1)

DMSO

 

+

17-15-11

14.3 (3.1)

TA1537

2AM

2

+

63-65-70

66.0 (3.6)

TEST ITEM

400

+

StUpMc-StUpMc-StUpMc

 

200

+

6St-6St-8St

6.7 (1.2)

100

+

6-7-6

6.3 (0.6)

50

+

8-7-13

9.3 (3.2)

25

+

17-16-12

15.0 (2.6)

12.5

+

10-10-8

9.3 (1.2)

DMSO

 

+

12-15-7

11.3 (4.0)

TA 98

2 AM

2

+

151-139-143

144.3 (6.1)

TEST ITEM

400

+

16St-9St-10St

11.7 (3.8)

200

+

17st-29St-21St

22.3 (6.1)

100

+

25-25-27

25.7 (1.2)

50

+

27-31-27

28.3 (2.3)

25

+

23-38-16

25.7(11.2)

12.5

+

26-21-23

23.3 (2.5)

DMSO

 

+

27-20-21

22.7 (3.8)

TA 100

BaP

5

+

647-858-816

773.7 (111.7)

TEST ITEM

400

+

StUpMc-StUpMc-StUpMc

 

200

+

104St-93St-107St

101.3 (7.4)

100

+

129-141-124

131.3 (8.7)

50

+

149-149-144

147.3 (2.9)

25

+

160-130-143

144.3 (15.0)

12.5

+

133-142-133

136.0 (5.2)

DMSO

 

+

102-119-126

115.7 (12.3)

TA102

2AM

20

+

1629-1883-1477

1663.0 (205.1)

TEST ITEM

800

+

155St-219St-176St

183.3 (32.6)

400

+

310-358-329

332.3 (24.2)

200

+

423-378-391

397.3 (23.2)

100

+

397-369-424

396.7 (27.5)

50

+

461-433-377

423.7 (42.8)

25

+

328-448-451

409.0 (70.2)

DMSO

 

+

423-388-436

415.7 (24.8)


 

 


Applicant's summary and conclusion

Conclusions:
The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.
Executive summary:

The objective of the study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium. The study was performed according to OECD No. 471 guideline and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose levels of the test item, dissolved in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes). Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to six dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose levels for each strain and test condition. The study was therefore considered to be valid. The selection of the highest dose level for the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose levels.

 

Without S9 mix, the selected dose levels were:

- 20.6, 61.7, 185.2, 555.6, 1667 and 5000 µg/plate for the TA 102 strain in the first experiment,

6.86, 20.6, 61.7, 185.2, 555.6 and 1667 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the first experiment,

31.3, 62.5, 125, 250, 500 and 1000 µg/plate for the TA 1535, TA 1537, TA 98 and TA 102 strains in the second experiment,

15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 100 strain in the second experiment.

 

A moderate to strong toxicity was noted at dose levels equal or above 250 µg/plate in the TA 1535, TA 1537, TA 98 and TA 102 strains and equal or above 125 µg/plate in the TA 100 strain. No increase in the number of revertants which could be considered as biologically relevant was observed in any tested strains.

With S9 mix, the selected dose levels were:

- 20.6, 61.7, 185.2, 555.6, 1667 and 5000 µg/plate for the five strains in the first experiment,

25, 50, 100, 200, 400 and 800 µg/plate for the TA 102 strain in the second experiment,

- 12.5, 25, 50, 100, 200 and 400 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the second experiment.

When using the direct plate incorporation method (i.e. first experiment), a moderate to strong toxicity was noted at 5000 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains. When using the pre-incubation method (i.e. second experiment), a moderate to strong toxicity was noted at dose levels equal or above 200 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and at 800 µg/plate in the TA 102 strain. In the TA 1537 and TA 100 strains, the strong toxicity induced at 400 µg/plate prevented the scoring of revertant colonies.The test item did not induce any noteworthy increase in the number of revertants, in any strains and test conditions.

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.

Categories Display