4,4'-bis(chloromethyl)-1,1'-biphenyl

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:

08 March 2016 to 14 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

4,4'-bis(chloromethyl)-1,1'-biphenyl

EC Number:

216-784-4

EC Name:

4,4'-bis(chloromethyl)-1,1'-biphenyl

Cas Number:

1667-10-3

Molecular formula:

C14H12Cl2

IUPAC Name:

4,4'-bis(chloromethyl)-1,1'-biphenyl

Test material form:

solid: particulate/powder

Details on test material:

- Pysical description: White crystalline powder
- Storage conditions: Controlled Room Temperature (15-25oC), protected from humidity, under inert gas.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Provided by sponsor
- Batch/Lot Number: 150701
- Expiration date of the lot/batch: 14 July 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25 °C), protected from humidity, under inert gas

Method

Target gene:

Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia coli strain.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media: Stock cultures were prepared in nutrient broth No. 2.
- Properly maintained: Yes. The strains were stored at -80 ± 10 ºC in the Culture Collection of the Microbiological Laboratory. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- Periodically 'cleansed' against high spontaneous background: Yes. Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate. Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011 to 2014 were (as guide) as follows: Salmonella typhimurium TA98: 9-46, TA100: 54-210, TA1535: 1-46, TA1537: 1-24, Escherichia coli WP2 uvrA: 11-82.

Additional strain / cell type characteristics:

other: S. typhimurium: All strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA- mutation.

Remarks:

The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB) and ampicillin resistance (amp) are checked regularly.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix with activated rat liver S9 fraction.

Test concentrations with justification for top dose:

Preliminary Toxicity Test
5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate

Initial Mutation Test and Confirmatory Mutation Test
1000, 316, 100, 31.6, 10, 3.16, 1.0 and 0.316 μg/plate

Rationale for Concentration Selection
Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test same concentrations were used.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test material was examined using Distilled water, Dimethyl sulfoxide (DMSO) and Acetone. The test material was insoluble at 100 mg/mL concentration using distilled water as vehicle. However, a clear solution was observed at this concentration (after vortex for approximately 5 minutes) using DMSO and Acetone as vehicles. Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study. The test material was formulated in the selected vehicle (solvent).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Two solvents were used, depending on the solubility of the test item and the solubility of the strain specific positive controls: Distilled water and DMSO (initial mutation test and in the confirmatory mutation test).

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine and 2-aminoanthracene

Details on test system and experimental conditions:

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test). In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used, in the Confirmatory Mutation Test in the case of Salmonella typhimurium TA98, TA100, TA1535 strains with and without metabolic activation the plate incorporation method was used; in the case of Salmonella typhimurium TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation the pre-incubation method was used.

- PRELIMINARY CONCENTRATION RANGE FINDING TEST
METHOD OF APPLICATION: In agar (direct plate incorporation)
Based on the available information and the solubility and compatibility test, 100 mg/mL stock solution was prepared in DMSO, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test material.

INITIAL MUTATION TEST AND CONFIRMATORY MUTATION TEST
Based on the results of the preliminary tests, 20 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in seven steps to obtain at eight dosing formulations for lower doses. The maximum test concentration was 1000 μg test material/plate. Examined concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 1000, 316, 100, 31.6, 10, 3.16, 1.0 and 0.316 μg/plate

INITIAL MUTATION TEST PROCEDURE
METHOD OF APPLICATION: In agar (direct plate incorporation)
A standard plate incorporation procedure was performed as an Initial Mutation Test. Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45 °C. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test material and other components were prepared freshly and added to the overlay (45 °C).

The content of the tubes:
top agar 2000 μL
vehicle (solvent) or test item solution (or reference controls) 50 μL
overnight culture of test strain 100 μL
phosphate buffer (pH 7.4) or S9 mix 500 μL

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48±1 hours.

CONFIRMATORY MUTATION TEST PROCEDURE
METHOD OF APPLICATION: Preincubation
A pre-incubation procedure was performed as a Confirmatory Mutation Test in the case of Salmonella typhimurium TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation, since in the Initial Mutation Test positive effect was observed in the case of Salmonella typhimurium TA98, TA100, TA1535 strains with and without metabolic activation the plate incorporation method was used.
For the pre-incubation method, bacteria were exposed to the test material both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45 °C.
Before the overlaying, 50 μL of test material formulations or its vehicle (or positive reference controls or their solvent), 100 μL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test material. These tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37 ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for 48±1 hour.

Evaluation criteria:

EVALUATION OF EXPERIMENTAL DATA
The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): Mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

CRITERIA FOR VALIDITY
The study was considered valid if:
- The number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- At least five analysable concentrations were presented in all strains of the main tests.

CRITERIA FOR A POSITIVE RESPONSE
A test material was considered mutagenic if:
- A dose–related increase in the number of revertants occurred and/or;
- A reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- In all strains: The number of reversion was more than twice higher than the reversion rate of the negative (solvent) control.

CRITERIA FOR A NEGATIVE RESPONSE
The test material was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: Salmonella typhimurium TA98, TA100, TA1535 bacterial strains

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No inhibitory, cytotoxic effect of the test item was observed in the main tests.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not valid

Test results 2

Key result

Species / strain:

other: Salmonella typhimurium TA98, TA100, TA1535 and TA1537 bacterial strains

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No inhibitory, cytotoxic effect of the test item was observed in the main tests.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not valid

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material was insoluble at 100 mg/mL concentration using distilled water as vehicle. However, a clear solution was observed at this concentration (after vortex for approximately 5 minutes) using DMSO as the vehicle. Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study.
- Precipitation: Precipitate/slight precipitate was observed in the Preliminary Concentration Range Finding Test in both bacterial strains with and without metabolic activation at 5000, 2500, 1000 and 316 μg/plate concentrations. In the Preliminary Concentration Range Finding Test strong precipitate was observed on the plates in all examined bacterial strains at 5000 and 2500 μg/plate concentrations with and without metabolic activation. This precipitate inhibited the assessment of the background lawn and/or the counting of revertants at these concentrations.
Precipitate/slight precipitate was also observed in the Initial Mutation Test in all examined strains with and without metabolic activation at 1000 and 316 μg/plate concentrations, and in Salmonella typhimurium TA1535 strain without metabolic activation at 100 μg/plate concentration.
Precipitate/slight precipitate was observed in the Confirmatory Mutation Test in all examined strains with and without metabolic activation at 1000 μg/plate concentration and with and/or without metabolic activation at 316 μg/plate concentration, and in Salmonella typhimurium TA98, TA1535, TA1537 strains without metabolic activation at 100 μg/plate concentration.


RANGE-FINDING/SCREENING STUDIES: PRELIMINARY CONCENTRATION RANGE FINDING TES (INFORMATORY TOXICITY TEST)
The plate incorporation method was used for the Preliminary Concentration Range Finding Test. The preliminary test was performed using Salmonella Typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the test each sample (including the controls) was tested in triplicate.
Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test.

In the Preliminary Concentration Range Finding Test, a dose–related increase was observed in the number of revertant colonies in all Salmonella typhimurium strains with and without metabolic activation. The calculated mutation factor values were over the biologically relevant threshold values 2.

Mutation factor values were 18.50, 17.00, 16.31, 18.75, 16.25, 17.97 and 12.63 (in Salmonella typhimurium TA98 strain at all examined concentrations without metabolic activation; respectively); mutation factor values were 27.66, 25.14, 29.60, 32.11, 37.37, 20.17 and 3.66 (in Salmonella typhimurium TA98 strain at all examined concentrations with metabolic activation; respectively).
Mutation factor values were 4.20, 4.85, 5.08, 6.28, 5.88, 5.47 and 6.24 (in Salmonella typhimurium TA100 strain at all examined concentrations without metabolic activation; respectively); mutation factor values were 6.08, 6.35, 9.23, 9.09, 8.50, 4.70 and 1.69 (in Salmonella typhimurium TA100 strain at all examined concentrations with metabolic activation; respectively).

Precipitate/slight precipitate was observed in the Preliminary Concentration Range Finding Test in both bacterial strains with and without metabolic activation at 5000, 2500, 1000 and 316 μg/plate concentrations.
No inhibitory, cytotoxic effect of the test material was observed in the Preliminary Concentration Range Finding Test.


INITIAL AND CONFIRMATORY MUTATION TEST
In the Initial Mutation Test using the plate incorporation method, a clear positive effect of the test item was obtained in Salmonella typhimurium TA98, TA100, TA1535 bacterial strains with and without metabolic activation as the calculated mutation factor values were over the biologically relevant threshold value of 2 and dose dependence was also observed.
In the Initial Mutation Test the calculated mutation factor values were as follows: 28.71; 20.07, 19.00, 13.92, 2.86 in Salmonella typhimurium TA98 strain at 1000, 316, 100, 31.6 and 10 μg/plate concentrations with metabolic activation, respectively, 17.98, 18.08, 19.15, 18.10, 15.35, 7.83 and 2.92 in Salmonella typhimurium TA98 strain at 1000, 316, 100, 31.6, 10, 3.16 and 1.0 μg/plate concentrations without metabolic activation, respectively.

The calculated mutation factor values were as follows 7.84, 8.09, 6.70, 6.12 and 2.01 in Salmonella typhimurium TA100 strain at 1000, 316, 100, 31.6 and 10 μg/plate concentrations with metabolic activation, respectively; 5.67, 6.49, 6.55, 5.76, 6.57 and 2.56 in Salmonella typhimurium TA100 strain at 1000, 316, 100, 31.6, 10 and 3.16 μg/plate concentrations without metabolic activation, respectively.

The calculated mutation factor values were as follows 6.93, 7.10, 6.31 and 3.38 in Salmonella typhimurium TA1535 strain at 1000, 316, 100 and 31.6 μg/plate concentrations with metabolic activation, respectively; 2.71, 2.53, 2.53, 2.12 and 2.09 in Salmonella typhimurium TA1535 strain at 1000, 316, 100, 31.6 and 10 μg/plate concentrations without metabolic activation, respectively.

The observed positive effect was confirmed in the Confirmatory Mutation Test using the plate incorporation method in Salmonella typhimurium TA98, TA100, TA1535 bacterial strains with and without metabolic activation. This result demonstrated the positive effect was reproducible and dose dependent.

In the Confirmatory Mutation Test calculated mutation factor values were as follows: 22.25; 35.73, 33.32, 13.64 and 3.10 in Salmonella typhimurium TA98 strain at 1000, 316, 100, 31.6 and 10 μg/plate concentrations with metabolic activation, respectively, 11.86, 10.35, 11.77, 13.22 and 10.99 in Salmonella typhimurium TA98 strain at 1000, 316, 100, 31.6 and 10 μg/plate concentrations without metabolic activation, respectively and the mutation factor value was 2.33 at 1.0 μg/plate concentrations.
The calculated mutation factor values were as follows 9.11, 10.47, 11.38 and 4.13 in Salmonella typhimurium TA100 strain at 1000, 316, 100 and 31.6 μg/plate concentrations with metabolic activation, respectively; 4.36, 5.29, 4.90, 6.54 and 5.36 in Salmonella typhimurium TA100 strain at 1000, 316, 100, 31.6 and 10 μg/plate concentrations without metabolic activation, respectively.

The calculated mutation factor values were as follows 11.08, 10.36, 10.00 and 3.28 in Salmonella typhimurium TA1535 strain at 1000, 316, 100 and 31.6 μg/plate concentrations with metabolic activation, respectively; 2.93, 2.89, 1.98 and 2.23 in Salmonella typhimurium TA1535 strain at 1000, 316, 100 and 31.6 μg/plate concentrations without metabolic activation, respectively.

A positive effect of the test item was obtained in the Confirmatory Mutation Test using the pre-incubation method in Salmonella typhimurium TA1537 strain with and without metabolic activation as the calculated mutation factor values were over the biologically relevant threshold value of 2 and dose dependence was also observed.
In the Confirmatory Mutation Test calculated mutation factor values were as follows: 4.73, 4.20 and 3.93 in Salmonella typhimurium TA1537 at 1000, 316 and 100 μg/plate concentrations with metabolic activation, respectively; 2.16 and 2.00 in Salmonella typhimurium TA1537 at 316 and 100 μg/plate concentrations without metabolic activation, respectively.

Taking into account the results at all concentrations under all conditions for each bacterial strain, the results for Escherichia coli WP2 uvrA with and without metabolic activation were negative. The results for Salmonella typhimurium TA98, TA100, TA1535 with and without metabolic activation using the plate incorporation method were reproducibly positive. Positive results were observed using the pre-incubation method in Salmonella typhimurium TA1537 with and without metabolic activation.

HISTORICAL CONTROL DATA
- Positive historical control data: The mean values of revertant colonies of the positive control plates were within the historical control range in all strains.
- Negative (solvent/vehicle) historical control data: The mean values of revertant colonies of the solvent control plates were within the historical control range in all strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
There were no signs of inhibitory, cytotoxic effect of the test item in the Preliminary Experiment and in the main tests in all examined bacterial strains at all concentrations with or without metabolic activation.

Remarks on result:

other: Initial Mutation Test using the plate incorporation method

Any other information on results incl. tables

Preliminary compatibility test

Based on the available information and the results of the solubility testing, DMSO was selected as vehicle (solvent) of the study. The results of the Preliminary Compatibility Test are summarized below.

 

The Solubility of the Test Item in DMSO

Concentration of test item in DMSO (mg/mL)	Solubility in DMSO	Solubility in the top solution (test item formulation 50 μL + phosphate buffer 500 μL + top agar 2 mL)	Test item concentration in the test tube(μg/tube)
100	clear solution	strong precipitate, opalescence	5000
50	clear solution	precipitate, opalescence	2500
20	clear solution	precipitate, opalescence	1000
6.32	clear solution	slight precipitate, slight opalescence	316
2	clear solution	slight opalescence	100
0.632	clear solution	clear solution	31.6

 

Validity of the test

Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analyzable concentrations were presented in all strains with and without metabolic activation.

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Table 1: Summary Table of the Initial Mutation Test

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Salmonella thyphimuriumtester strains			Escherichia coli
		TA98	TA100	TA1535	TA1537	Wp2 uvrA
-	Untreated DMSO Distilled H20 1000 316 100 31.6 10 3.16 1.0 0.316	25.7 17.3 -- 311.7 313.3 332.0 313.7 266.0 135.7 50.7 25.3	109.7 99.0 105.7 561.3 642.7 648.0 570.7 650.7 253.3 143.3 132.3	10.7 11.3 8.3 30.7 28.7 28.7 24.0 23.7 13.7 14.7 13.0	7.0 6.7 -- 10.0 8.7 6.0 9.3 8.7 6.7 8.7 8.7	33.3 30.3 37.0 42.7 40.0 38.0 40.0 31.7 33.0 29.0 33.7
+	Untreated DMSO Distilled H20 1000 316 100 31.6 10 3.16 1.0 0.316	28.3 29.0 -- 832.7 582.0 551.0 403.7 83.0 31.0 30.7 24.3	116.7 115.0 -- 901.3 930.7 770.7 704.0 230.7 154.7 134.3 131.0	11.3 9.7 -- 67.0 68.7 61.0 32.7 17.0 16.3 10.3 8.3	8.3 10.0 -- 15.0 12.7 15.0 12.0 9.0 7.7 6.0 8.3	31.3 35.7 -- 40.0 41.3 43.3 33.7 29.7 31.3 35.7 34.3
Positive Controls
-	Name	NPD	SAZ	SAZ	9AA	MMS
	Concentration (µg/plate)	4	2	2	50	2µL
	Mean no. colonies/plate	412.0	1074.7	1058.7	401.3	958.7
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2	2	2	2	50
	Mean no. colonies/plate	2434.7	2421.3	229.3	214.0	217.3

NPD = 4-nitro-1,2-phenylene-diamine

2AA = 2-aminoanthracene

SAZ = Sodium azide

9AA = 9-aminoacridine

MMS = Methyl-methanesulfonate

Table 2: Summary Table of the Confirmatory Mutation Test

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Salmonella thyphimuriumtester strains			Escherichia coli
		TA98	TA100	TA1535	TA1537	Wp2 uvrA
-	Untreated DMSO Distilled H20 1000 316 100 31.6 10 3.16 1.0 0.316	17.7 23.0 -- 272.7 238.0 270.7 304.0 252.7 24.3 53.7 23.7	108.7 116.0 114.0 505.3 613.3 568.0 758.7 621.3 110.7 161.7 109.3	12.7 14.7 11.3 43.0 42.3 29.0 32.7 22.0 7.0 12.3 12.3	6.3 6.3 -- 11.0 13.7 12.7 9.7 12.0 5.3 4.3 5.7	30.3 33.7 30.3 55.3 38.0 36.7 37.3 35.7 33.0 33.3 24.3
+	Untreated DMSO Distilled H20 1000 316 100 31.6 10 3.16 1.0 0.316	23.7 24.3 -- 541.3 869.3 810.7 332.0 75.3 25.7 23.7 26.0	130.3 129.3 -- 1178.7 1354.0 1472.0 534.7 180.0 138.3 138.7 137.3	11.7 8.3 -- 92.3 86.3 83.3 27.3 15.0 11.3 16.3 11.7	7.7 5.0 -- 23.7 21.0 19.7 7.7 6.0 7.3 8.0 5.3	40.0 39.0 -- 53.3 44.3 45.0 44.7 42.0 35.7 39.3 35.0
Positive Controls
-	Name	NPD	SAZ	SAZ	9AA	MMS
	Concentration (µg/plate)	4	2	2	50	2µL
	Mean no. colonies/plate	380.0	1124.0	1195.3	401.3	1040.0
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2	2	2	2	50
	Mean no. colonies/plate	2417.3	2424.0	202.7	204.0	229.3

NPD = 4-nitro-1,2-phenylene-diamine

2AA = 2-aminoanthracene

SAZ = Sodium azide

9AA = 9-aminoacridine

MMS = Methyl-methanesulfonate

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test material had mutagenic activity in all Salmonella typhimurium bacterial strains with and without metabolic activation while no mutagenic activity was observed in Escherichia coli WP2 uvrA bacterial strain.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The test material was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats. The study included a Preliminary Compatibility Test, an Initial Mutation Test (Plate Incorporation Method and Confirmatory Mutation Test (in the case of Salmonella typhimurium TA98, TA100, TA1535 strains with and without metabolic activation the Plate Incorporation Method was used; in the case of Salmonella typhimurium TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation the Pre-Incubation Method was used). Based on the results of the Preliminary Concentration Range Finding Test, the test material concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 1000, 316, 100, 31.6, 10, 3.16, 1.0 and 0.316 μg/plate. In the Preliminary Concentration Range Finding Test in the case of Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation, in the Initial Mutation Test and Confirmatory Mutation Test in the case of Salmonella typhimurium TA98, TA100, TA1535 bacterial strains, a clear, reproducible positive effect was obtained using the plate incorporation method. In the Confirmatory Mutation Test a positive effect was obtained in Salmonella typhimurium TA537 bacterial strain with and without metabolic activation using the pre-incubation method. There were no signs of inhibitory, cytotoxic effect of the test item in the Preliminary Experiment and in the main tests in all examined bacterial strains at all concentrations with or without metabolic activation. In both of the main test conducted, the test material induced gene mutations by base pair changes or frameshifts in the genome of the strains used. The validity criteria of the test was fulfilled therefore the test was valid.

Under the conditions of the study, the test material had mutagenic activity in all Salmonella typhimurium bacterial strains with and without metabolic activation while no mutagenic activity was observed in Escherichia coli WP2 uvrA bacterial strain.