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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test is available for the submission substance [reaction product of 2,3-epoxypropyl neodecanoate and benzenesulfonic acid, C10-13-sec-alkyl derivatives].

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 July - 15 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
24 November 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Identification: Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives
Batch: P718261998
Physical state/Appearance: Brown liquid
Purity: 97.7%
Expiry Date: 01 June 2018
Storage Conditions: Room temperature in the dark
Target gene:
Reversion to histidine / tryptohan independence
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 Microsomal fractions
Test concentrations with justification for top dose:
Up to 5000 μg/plate (limit concentration)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 4-Nitroquinoline-1-oxide (4NQO) / 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
Experiment 1 - Plate Incorporation Method
The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. In the assay without metabolic activation, 0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates. In the assay with metabolic activation, the same procedure was used except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer. All plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to the revertant colonies spreading slightly or for colonies on the edge of the plates, thus distorting the actual plate count.

Experiment 2 – Pre-Incubation Method
Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation. The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate. Eight test item dose levels per bacterial strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation. In the assay without metabolic activation, 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing was performed in triplicate. In the assay with metabolic activation, the same procedure was used except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate. All plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to the revertant colonies spreading slightly, thus distorting the actual plate count.
Evaluation criteria:
The assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are:

TA1535: 7-40
TA100: 60-200
TA1537: 2-30
TA98: 8-60
WP2uvrA: 10-60

All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL. Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. There should be a minimum of four non-toxic test item dose levels. There should be no evidence of excessive contamination.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile or were accompanied by weakened bacterial background lawns were not reported.
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
E. coli WP2 uvr A
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
Additional information on results:
The vehicle (DMSO) control plates gave counts of revertant colonies within the normal historical range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation

Mean revertant counts: Experiment 1

 

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

DMSO

86

86

21

23

27

26

32

38

16

17

1.5 µg

92

91

21

22

26

31

32

36

13

18

5 µg

91

89

22

21

29

29

30

37

15

16

15 µg

90

80

21

22

22

31

32

40

12

17

50 µg

90

84

21

21

24

26

33

39

13

19

150 µg

92

90

22

13

26

30

353

38

14

17

500 µg

76

76

23

21

28

24

24

34

14

16

1500 µg

33

93

21

26

22

25

26

29

7

15

5000 µg

11

16

31

33

17

16

9

10

5

6

ENNG

518

 

549

 

829

 

 

 

 

 

4NQO

 

 

 

 

 

 

205

 

 

 

9AA

 

 

 

 

 

 

 

 

287

 

2AA

 

1615

 

309

 

275

 

 

 

451

B(a)P

 

 

 

 

 

 

 

214

 

 

Mean revertant counts: Experiment 2

 

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

DMSO

110

76

13

21

19

29

32

31

14

15

1.5 µg

119

98

14

24

22

25

36

34

18

16

5 µg

109

112

14

23

19

25

29

28

17

16

15 µg

102

107

12

19

24

22

39

31

8

15

50 µg

138

93

13

22

19

25

40

28/

12

11

150 µg

122

97

13

20

16

29

39

18

9

12

500 µg

94

99

14

24

23

19

42

14

6

13

1500 µg

38

85

23

31

21

27

20

16

6

9

5000 µg

17

21

23

42

22

20

8

4

8

4

ENNG

1149

 

1135

 

764

 

 

 

 

 

4NQO

 

 

 

 

 

 

308

 

 

 

9AA

 

 

 

 

 

 

 

 

266

 

2AA

 

 

 

238

 

442

 

 

 

533

B(a)P

 

 

 

 

 

 

 

233

 

 

Conclusions:
Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives was considered to be non-mutagenic under the conditions of this Ames test.
Executive summary:

The mutagenicity of the submission substance [reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives] was investigated in a GLP- and guideline-compliant Ames test. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using the plate incorporation and pre-incubation methods at eight concentrations, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The concentration range for Experiment 1 was 1.5 to 5000 μg/plate. The experiment was repeated (Experiment 2) on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The concentration range was the same as Experiment 1 (1.5 to 5000 μg/plate). Eight concentrations per bacterial strain were selected in Experiment 2. The vehicle (DMSO) control plates gave counts of revertant colonies within the normal historical range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. In Experiment 1, the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains from 1500 μg/plate in both the presence and absence of S9-mix. The test material induced a toxic response in Experiment 2, with weakened bacterial background lawns noted to all of the tester strains in the absence of S9-mix from 1500 μg/plate. In the presence of S9-mix, weakened bacterial background lawns were noted from 1500 μg/plate to TA100, TA1535 and TA98. Weakened lawns were not observed to TA1537 (presence of S9), however substantial reductions in revertant colony frequency were noted at 5000 μg/plate. No precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any concentration, either with or without metabolic activation (S9-mix) in Experiment 1 or Experiment 2. [Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives] was therefore considered to be non-mutagenic under the conditions of this Ames test.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The mutagenicity of the submission substance [reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives] was investigated in a GLP- and guideline-compliant Ames test. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using the plate incorporation and pre-incubation methods at eight concentrations, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The concentration range for Experiment 1 was 1.5 to 5000 μg/plate. The experiment was repeated (Experiment 2) on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The concentration range was the same as Experiment 1 (1.5 to 5000 μg/plate). Eight concentrations per bacterial strain were selected in Experiment 2. The vehicle (DMSO) control plates gave counts of revertant colonies within the normal historical range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. In Experiment 1, the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains from 1500 μg/plate in both the presence and absence of S9-mix. The test material induced a toxic response in Experiment 2, with weakened bacterial background lawns noted to all of the tester strains in the absence of S9-mix from 1500 μg/plate. In the presence of S9-mix, weakened bacterial background lawns were noted from 1500 μg/plate to TA100, TA1535 and TA98. Weakened lawns were not observed to TA1537 (presence of S9), however substantial reductions in revertant colony frequency were noted at 5000 μg/plate. No precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any concentration, either with or without metabolic activation (S9-mix) in Experiment 1 or Experiment 2. [Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives] was therefore considered to be non-mutagenic under the conditions of this Ames test.

Justification for classification or non-classification

CLP classification for germ cell mutagenicity is not required for the substance reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives based on the negative result obtained in an Ames test.