1,1'-ethan-1,2-diylbisbenzene, brominated

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:

February 9th, 2009 - February 23rd, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): SH-1
- Chemical name of test material (as cited in study report): Benzene, 1,1´-(1,2-ethanediyl)bis-, brominated
- Physical state: solid
- Analytical purity: > 99 %
- Batch No.: 20081010
- Expiration date of the lot/batch: January 08, 2011.
- Storage condition of test material: at room temperature

Method

Target gene:

Genes involved in Histidine synthesis

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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment = Experiment I (plate incorporation test):
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II (pre-incubation test):
33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 4-nitro-o-phenylene-diamine, methyl methane sulfonate, 2-aminoanthracene,

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation);
Experiment II: preincubation;

Experimental Performance:

The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar

In the pre-incubation assay 100 µL test solution (solvent or reference mutagen solution (positive control)), 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After preincubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Statistics:

no statistics performed, as this is not mandatory according to OECD guideline 471.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Remarks:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was observed in the test tubes from 1000 - 5000 µg/plate in experiment I, and at 2500 and 5000 µg/plate in experiment II. Precipitation of the test item was also observed on the incubated agar plates at 2500 and 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I.
Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades.

COMPARISON WITH HISTORICAL CONTROL DATA: the negative controls were within the ranges of the historical control values.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Ames Test Results of the plate incorporation Experiment I (= Pre-Experiment):

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
-	Neg. control	21	8	28	144	55
-	0	15	11	29	142	55
-	3	20	12	26	136	49
-	10	15	10	25	128	54
-	33	18	8	28	138	52
-	100	19	10	23	147	49
-	333	20	8	24	143	58
-	1000	18	10	29	134	59
-	2500	19P	11P	28P	128P	49P
-	5000	16P	8P	19P	121P	50P
Positive controls - S9	Name	NaN3	4-NOPD	4-NOPD	NaN3	MMS
	Concentration (per plate)	10 μg	50 μg	10 μg	10 μg	3.0 μL
	Number of colonies/plate	1966	92	439	2019	1390
+	Neg. control	15	12	32	155	63
+	0	16	11	28	161	62
+	3	17	13	32	163	62
+	10	17	9	30	157	61
+	33	19	11	28	153	61
+	100	18	14	26	154	63
+	333	19	11	30	167	57
+	1000	21	13	31	155	58
+	2500	20P	13P	27P	140P	58P
+	5000	17P	7P	22P	128P	56P
Positive controls + S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentration (per plate)	2.5 μg	2.5 μg	2.5 μg	2.5 μg	10 μg
	Number of colonies/plate	379	265	1859	2699	237

 

 

Table 2: Ames Test Results of the preincubation Experiment II:

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
-	Neg. control	18	10	26	142	51
-	0	19	13	25	126	52
-	33	19	11	26	122	49
-	100	15	12	26	120	48
-	333	18	8	24	117	48
-	1000	12	10	22	121	49
-	2500	15P	12P	27P	121P	56P
-	5000	11P	9P	22P	111P	46P
Positive controls - S9	Name	NaN3	4-NOPD	4-NOPD	NaN3	MMS
	Concentration per plate	10 μg	50 μg	10 μg	10 μg	3.0 μL
	Number of colonies/plate	2008	91	415	2037	353
+	Neg. control	18	13	30	170	49
+	0	19	10	37	152	56
+	33	16	12	37	144	53
+	100	17	9	36	157	56
+	333	16	12	33	151	58
+	1000	15	10	38	145	57
+	2500	18P	11P	33P	133P	55P
+	5000	14P	12P	26P	151P	48P
Positive controls + S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentration per plate	2.5 μg	2.5 μg	2.5 μg	2.5 μg	10 μg
	Number of colonies/plate	304	204	1691	2358	203

 

NaN3 = Sodium azide

MMS = Methyl methane sulfonate

4-NOPD = 4-nitro-o-phenylene-diamine

2-AA = 2-aminoanthracene

P = Precipitate

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, the test substance SH-1 was shown to be non-mutagenic in bacteria.

Executive summary:

This study was performed to investigate the potential of SH-1 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the

Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with SH-1 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, SH-1 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.