Benzene, mono C10-13-alkyl derivs, dist residues, sulfonated

Genetic toxicity: in vitro

Administrative data

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:

2010-01-14 to 2010-03-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Benzene, mono C10-13-alkyl derivs, distn. residues, sulfonated

IUPAC Name:

Benzene, mono C10-13-alkyl derivs, distn. residues, sulfonated

Details on test material:

- Name of test material (as cited in study report): Benzene, mono C10-13-alkyl derivs, distn. resiudes, sulfonated
- Substance type: product
- Physical state: dark brown, viscous liquid
- Storage condition of test material: storage at room temperature

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 activated liver homogenate from male Sprague-Dawley rats

Test concentrations with justification for top dose:

- 1st experiment: 5, 1.581, 0.5, 0.158 and 0.05 mg/plate with and without metabolic activation
- 2nd experiment: 5, 2.5, 1.25, 0.63, 0.31 and 0.16 mg/plate with metabolic activation, with strain TA 1537 in addition 0.08 mg/plate with metabolic activation; resp. 1, 0.5, 0.25, 0.13 0.06 and 0.03 mg/plate without metabolic activation
- 3rd experiment: 0.86, 0.72, 0.60, 0.50, 0.42, 0.35 and 0.29 mg/plate without metabolic activation , only TA 1535
All concentrations were applied corresponding to the sulphonated material

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not given

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

sterile deionized water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 50 µg/mL 2-Nitrofluorne with TA 98; 10 µg/mL Sodiumazide with TA 100 and TA 1535; 1.5 µL/mL Cumene hydroperoxide (ca. 80%) with TA 102; 1.5 mg/mL 9-Aminoacridine with TA 1537

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

sterile deionized water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 100 µg/mL with all tester strains; additionally 0.5 mg/mL Benz-a-pyrene with TA 98

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: growth of micro-colonies in the background in comparison to the negative control by estimating the diameter and number of the micro-colonies under the microscope

OTHER EXAMINATIONS:
- Counting of revertants: with a semi-automatic colony counter (Ernst Schütt, jun., Göttingen, Germany)
- Titre of bacteria in the overnight culture: determined by counting colonies formed upon plating an aliquot of a 10E6-fold dilution with NaCl-solution and confirming the concentration by correlating the turbidity of the culture measured photometrically

OTHER:
- Qualitiy assurance of the Salmonella strains: Every half year the bacteria were checked concerning their genotype (sensitivity to crystal violet, sensitivity to ultraviolet light, testing for ampicillin resistance).

Evaluation criteria:

Criteria for validity of the test:
- the titre of the bacterial culture is sufficient (about 10E9 bacteria/mL)
- the positive control reach at least a doubling of the revertants in comparison to the negative controls
- the numbers of revertants in the negative controls are within the historical ranges (according to Venitt et al. (1984) and laboratory specific historical min-max data)
Criteria for a positive result:
A sample is judged to be mutagenic
- if there is a concentration-related increase over the range tested
- if there is a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation
- if a statistically significant difference between the mean values can be demonstrated in the case of a reproducible increase of the number of revertants
- if at least a doubling of revertant colonies with the sample compared to the control can be observed (Induction rate (IR) >= 2).

Statistics:

Calculation of the mean value and standard deviation of the replicates (Microsoft Excel), t-test with Bonferri adjustment and Wilcoxon rang sum test.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see tables below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA100 and TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

exception: cytotoxicity observed at the highest concentration with TA 98 only in the first experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the first experiment with the stain TA 1535 a maximum induction rate of 2.1 was obtained with 0.5 mg/plate. In the second experiment, which was performed independently, this result was confirmed. All other concentrations tested were inconspicuous. In both studies this increase was not statistically significant and the increase did not follow any dose-response curve. To confirm this ambiguous result a third test with a very narrow concentration range was performed. In this experiment no increase of revertant colonies at all could be observed.

In the experiment without metabolic acitvation the test item was toxic for the bacteria at higher concentrations. Strain TA 102 turned out to be most sensitive for cytotoxicity. Here up to 250 µg/plate the test item was toxic.

The numbers of the revertants in the negative controls were within the historical ranges.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mean values of the number of revertants and the induction factors obtained in the 1st experiment
Concentration	S9 mix	TA 98			TA 100			TA 102			TA 1535			TA 1537
		mean	RSD [%]	IR	mean	RSD [%]	IR	mean	RSD [%]	IR	mean	RSD [%]	IR	mean	RSD {%]	IR
Negative control	-	20	26.5	-	77	8.3	-	206	12	-	15	19.9	-	7	24.2	-
Solvent control	-	21	10.4	-	82	13.7	-	194	6.7	-	10	29.2	-	6	37	-
Positive control	-	159	7.3	7.7	347	7.4	4.2	1071	4.6	5.5	180	31.1	18.0	289	2	105.2
5 mg/plate	-	3 *	57.3	0.1	44 *	8.2	0.5	6 *	115.5	0	3	21.7	0.3	2 *	0	0.4
1.581 mg/plate	-	11 *	20.4	0.5	64	0.9	0.8	67 *	39.6	0.3	9	34.4	0.9	3	0	0.5
0.5 mg/plate	-	15	10	0.7	75	4.7	0.9	123 *	12	0.6	21	45.9	2.1	7	14.3	1.3
0.158 mg/plate	-	23	2.5	1.1	76	6.2	0.9	134 (*)	11.9	0.7	11	27	1.1	6	27	1.0
0.05 mg/plate	-	24	15	1.2	71	1.6	0.9	143	15.8	0.7	12	8.3	1.2	7	37.7	1.2
Negative control	+	23	15.4	-	92	8.3	-	223	6.2	-	11	19.3	-	6	20.4	-
Solvent control	+	21	27	-	84	13.4	-	188	16.3	-	13	19.9	-	7	39.8	-
Positive control (2-AA)	+	605	16.6	28.3	864	0.7	10.3	427	2.6	2.3	177	27.2	14.0	589	2	81.9
Positive control (B-a-P)	+	223	25.5	10.4
5 mg/plate	+	17 (*)	15.6	0.8	54	12	0.6	235	5.6	1.3	8	32.8	0.6	2	65.5	0.3
1.581 mg/plate	+	23	22.6	1.1	86	8.7	1.0	336	7.6	1.8	9	13.3	0.7	5	43.3	0.7
0.5 mg/plate	+	23	17.3	1.1	79	10.6	0.9	338	7.5	1.8	11	47.2	0.8	6	48.2	0.9
0.158 mg/plate	+	26	9.6	1.2	76	12.2	0.9	203	3.1	1.6	9	11.1	0.7	10	6	1.3
0.05 mg/plate	+	27	22.1	1.3	83	8.5	1.0	248	7.4	1.3	12	33.8	1.0	11	21.7	1.5
RSD = relative standard deviation   IR = Induction Rate   * = cytotoxic effects (background growth)
Table 2: Mean values of the number of revertants and the induction factors obtained in the 2nd experiment
Concentration	S9 mix	TA 98			TA 100			TA 102			TA 1535			TA 1537
		mean	RSD [%]	IR	mean	RSD [%]	IR	mean	RSD [%]	IR	mean	RSD [%]	IR	mean	RSD {%]	IR
Negative control	-	25	20.4	-	102	14.3	-	261	4.2	-	13	37.2	-	7	24.2	-
Solvent control	-	35	15.5	-	76	8.5	-	175	25.3	-	8	31.9	-	8	48	-
Positive control	-	166	11.3	4.8	174	3.7	2.3	521	6.0	3.0	104	16.7	13.0	638	44.7	83.9
1 mg/plate	-	10 (*)	15.8	0.3	46 (*)	32.3	0.6	49 (*)	47.6	0.3	9	75.1	1.1	5	24.7	0.6
0.5 mg/plate	-	13 (*)	23.1	0.4	68	9.7	0.9	63 (*)	14.5	0.4	17	10.2	2.1	4	86.6	0.5
0.25 mg/plate	-	14	33	0.4	64	11.1	0.8	126 (*)	22.5	0.7	7	47.9	0.9	11	42.3	1.4
0.13 mg/plate	-	23	22.2	0.6	61	9.1	0.8	120	2.9	0.7	8	6.9	1.0	7	34.3	1.0
0.06 mg/plate	-	22	15.7	0.6	65	12.3	0.9	142	12	0.8	8	6.89	1.0	12	22	1.6
0.03 mg/plate	-	26	9.8	0.7	71	11.4	0.9	171	3.2	1.0	11	10.8	1.3	13	7.7	1.7
Negative control	+	27	20	-	74	4.1	-	272	4.3	-	10	24	-	8	15	-
Solvent control	+	22	16	-	72	9.5	-	224	15	-	10	11	-	10	39.4	-
Positive control (2-AA)	+	127	77.1	5.7	628	7.1	8.7	483	2.5	2.2	118	4.7	11.3	228	12.7	23.8
Positive control (B-a-P)	+	625	1.3	27.5
5 mg/plate	+	16	22.5	0.7	46	7	0.6	164	18.1	0.7	8	74.2	0.7	4	31.5	0.4
2.5 mg/plate	+	34	28.1	1.5	53	5.4	0.7	307	8.3	1.4	7	15.7	0.7	6	0	0.6
1.25 mg/plate	+	25	14.4	1.1	61	12.7	0.8	318	6.9	1.4	8	50	0.8	5	20	0.5
0.63 mg/plate	+	26	17.6	1.2	75	21.4	1.0	290	3.8	1.3	7	7.9	0.7	9	11.1	0.9
0.31 mg/plate	+	27	15.2	1.2	67	2.3	0.9	296	3.3	1.3	8	15.1	0.7	9	34.4	1.0
0.16 mg/plate	+	36	26	1.6	70	5.2	1.0	301	3	1.3	8	37.5	0.8	8	12.5	0.8
0.08 mg/plate	+													12	17.8	1.2
RSD = relative standard deviation   IR = Induction Rate   * = cytotoxic effects (background growth)
Table 3: Mean values of the number of revertants and the induction factors obtained in the 3rd experiment
Concentration	S9 mix	TA 1535
		mean	RSD	IR
Negative control	-	12	38.9	-
Solvent control	-	12	31.2	-
Positive control	-	182	7.6	15.2
0.86 mg/plate	-	9	41.6	0.7
0.72 mg/plate	-	8	42.2	0.6
0.60 mg/plate	-	8	36.7	0.7
0.50 mg/plate	-	10	19.9	0.9
0.42 mg/plate	-	10	33	0.9
0.35 mg/plate	-	10	14.5	0.9
0.29 mg/plate	-	11	13.7	0.9

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

According to the results achieved the test item Benzene, mono C10-13-alkyl derivs, distn. residues, sulfonated was evaluated as not mutagneic under the test conditions applied up to a test concentration of 5 mg per plate (related to the sulfonated material of 73.2%).

Executive summary:

This Bacterial Reverse Mutation Test (Ames test) with S. typhimurium strains TA 98, TA 100, TA 102 , TA 1535 and TA 1537 was carried out according to OECD Guideline 471 (July 1997).

The test item Benzene, mono C10 -13 -alkyl derivs, distn. residues, sulfonated was evaluated as not mutagenic under the test conditions applied up to a test concentration of 5 mg per plate (related to the sulfonated material of 73.2%) which is the highest test concentration required according to the guideline.