Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

24 November 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

None

Molecular formula:

C10-13H21-27-C6H4-SO2-O-CH2-C(H)OH-CH2-CO2-C9H19

Test material form:

liquid

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

Method

Target gene:

Reversion to histidine / tryptohan independence

Species / strain

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.