[No public or meaningful name is available]

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:

17 August 2020 - 7 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 June 2020

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:

Benzenesulfonic acid, mono- and dialkylation products with C16-20 (even numbered, branched and linear) olefins

Molecular formula:

Cannot be adequately determined for this UVCB substance

IUPAC Name:

Benzenesulfonic acid, mono- and dialkylation products with C16-20 (even numbered, branched and linear) olefins

Test material form:

liquid

Details on test material:

Physical Description: Brown liquid
Storage Conditions: At room temperature

Specific details on test material used for the study:

Physical Description: Brown liquid
Storage Conditions: At room temperature

Method

Target gene:

Histidine locus and tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

Histidine mutations: TA1537 hisC3076; TA98 hisD3052/R-factor; TA1535 hisG46; TA100 hisG46/R-factor

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH, Giessen, Germany
- method of preparation of S9 mix: rat liver microsomal enzymes (S9 homogenate) prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight). S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL or 5.0 mL Milli-Q
water (first or second experiment respectively); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 μm)-sterilized.
- concentration of S9 mix: 5% (v/v) S9-fraction (first experiment); 10% (v/v) S9-fraction (second experiment)

Test concentrations with justification for top dose:

Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
First mutation experiment: 17, 52, 164, 512, 1600 and 5000 μg/plate
Second mutation experiment: 10, 50, 100, 500, 1000, and 5000 μg/plate

Vehicle / solvent:

- Vehicle used: DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9-mix

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 ± 4 h at 37.0 ± 1.0 °C
- Harvest time after the end of treatment: Not reported

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

Evaluation criteria:

No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow-up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Absence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Absence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Absence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Absence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

First mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

First mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

First mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With and without S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

First mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With and without S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

First experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With and without S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose-range Finding test/First Mutation Experiment
- Precipitation of the test item on the plates was observed in tester strains TA1535, TA1537 and TA98 in the absence of S9-mix.
- Cytotoxicity, as evidenced by a decrease in the number of revertants and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strains WP2uvrA and TA1537 (presence of S9-mix).
- No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Second mutation experiment
- In tester strain TA1537, the test item precipitated on the plates at the highest tested dose level in the presence of S9-mix.
- In the second mutation assay, cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in the absence of S9-mix in tester strains TA1535, TA98, TA100 and WP2uvrA, and in the presence of S9-mix in tester strains TA1537 and TA100.
- In the second mutation assay, no increase in the number of revertants was observed upon treatment with X-21415 under all conditions tested.

Any other information on results incl. tables

Table 1 Dose-Range Finding Test: Mutagenic Response of X-21415 in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	1668	±	62		428	±	5
Solvent control	97	±	6		21	±	3
1.7	109	±	15		23	±	3
5.4	105	±	20		19	±	1
17	98	±	9		24	±	4
52	100	±	6		20	±	5
164	125	±	14		15	±	1
512	117	±	19		16	±	10
1600	71	±	5		9	±	6
5000	6	±	4	^n NP	11	±	5	^n NP

 

With S9-mix¹

 

Positive control	830	±	24		1192	±	127
Solvent control	85	±	19		16	±	1
1.7	94	±	6		15	±	5
5.4	96	±	22		14	±	4
17	93	±	6		12	±	3
52	100	±	14		14	±	9
164	51	±	1	^n	13	±	2
512	12	±	5	^s	15	±	7
1600				^e MC	13	±	2
5000				^e NP  MC	15	±	1	^n NP

 

^1	Plate incorporation assay (5% S9)
MC	Microcolonies
NP	No precipitate
e	Bacterial background lawn extremely reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

 

Table 2. Experiment 1: Mutagenic Response of X-21415 in the Salmonella typhimurium Reverse Mutation Assay 

 

 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium.
	TA1535	TA1537	TA98

 

Without S9-mix

 

Positive control	767	±	5		1009	±	101		1290	±	138
Solvent control	10	±	2		3	±	2		15	±	5
17	7	±	3		5	±	2		16	±	7
52	8	±	3		2	±	2		16	±	3
164	7	±	3		0	±	1		13	±	7
512	3	±	1		1	±	0		11	±	3
1600	0	±	1	^NP	1	±	1	^NP	4	±	3	^NP
5000	0	±	1	^n SP	1	±	1	^n SP	0	±	0	^n SP

 

With S9-mix¹

 

Positive control	320	±	24		251	±	14		1035	±	45
Solvent control	6	±	3		5	±	2		18	±	5
17	8	±	1		3	±	2		21	±	4
52	7	±	5		4	±	1		19	±	3
164	10	±	2		6	±	3		15	±	1
512	7	±	3		5	±	5		23	±	1
1000	6	±	5		2	±	2		16	±	7
5000	2	±	2	^n NP	11	±	1	^n NP	2	±	2	^n NP

 

^1	Plate incorporation assay (5% S9)
NP	No precipitate
SP	Slight Precipitate
n	Normal bacterial background lawn

 

Table 3. Experiment 2: Mutagenic Response of X-21415 in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.
	TA1535	TA1537	TA98	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	913	±	42		956	±	80		1485	±	83		609	±	15		925	±	50
Solvent control	5	±	3		4	±	3		7	±	0		85	±	8		17	±	2
10	10	±	4		3	±	2		6	±	3		67	±	10		14	±	5
50	5	±	5		3	±	2		10	±	6		71	±	17		14	±	2
100	7	±	3		4	±	1		6	±	5		68	±	9		18	±	3
500	2	±	1		4	±	5		7	±	4		9	±	9		12	±	2
1000	3	±	2		3	±	4		4	±	1	^n	2	±	1	^n	16	±	7
5000	0	±	0	^n NP	4	±	3	^n NP				^e NP  MC				^e NP  MC	9	±	2	^n NP

 

 

With S9-mix¹

 

Positive control	246	±	12		254	±	46		666	±	105		1327	±	255		195	±	27
Solvent control	8	±	2		4	±	1		17	±	6		73	±	6		20	±	3
10	10	±	3		4	±	1		14	±	6		62	±	3		23	±	8
50	8	±	2		3	±	2		18	±	2		78	±	2		20	±	3
100	8	±	6		2	±	1		13	±	3		80	±	19		25	±	12
500	9	±	2		4	±	2		13	±	3		79	±	5		15	±	1
1000	8	±	3		2	±	2	^NP	19	±	5		81	±	15		19	±	6
5000	5	±	2	^n NP	0	±	0	^n SP	9	±	6	^n NP	45	±	7	^n NP	19	±	4	^n NP

 

 

^1	Plate incorporation assay (10% S9)
MC	Microcolonies
NP	No precipitate
SP	Slight Precipitate
e	Bacterial background lawn extremely reduced
n	Normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:

In an Ames test, performed according to OECD guideline 471 and in accordance with GLP principles, X-21415 was found to be not mutagenic with or without metabolic activation.

Executive summary:

An Ames test was performed according to OECD guideline 471 and in accordance with GLP principles. The test item did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate, and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that X-21415 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation. Since all acceptibility criteria were met, the study was considered valid.