Sodium 1,2-diisobutoxycarbonylethanesulphonate

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:

27 April 2020 to 16 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", 21 July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse Mutation Test", EPA 712-C-98-247, August 1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008, B.13/14. "Mutagenicity: Reverse Mutation Test Using Bacteria", 30 May 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 1,2-diisobutoxycarbonylethanesulphonate

EC Number:

204-839-5

EC Name:

Sodium 1,2-diisobutoxycarbonylethanesulphonate

Cas Number:

127-39-9

Molecular formula:

C12H22O7S.Na

IUPAC Name:

sodium 1,4-diisobutoxy-1,4-dioxobutane-2-sulfonate

Test material form:

liquid

Remarks:

colourless

Method

Target gene:

Histidine (Salmonella)
Tryptophan (Eschericha coli)

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Charles River Laboratories Hungary Kft. according to Ames et al. [1] and Maron and Ames [2]. The composition of solution refers to 1000 mL.
Male Wistar rats (444-628 g, animals were 17-20 weeks old and 433-642 g, animals were 13-17 weeks old) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed.
Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used for preparation S9 used in this study were 02 September 2019 and 13 January 2020.
On Day 4, the rats were euthanized and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly and stored at -80 ± 10°C. The dates of preparation of S9 fractions for this study were 05 September 2019 and 16 January 2020 (Charles River Laboratories Hungary code: E13142 (it was used in the Preliminary Range Finding Test) and E13222 (it was used in main tests), Expiry date: 05 September 2021 and 16 January 2022).
- method of preparation of S9 mix:
*The S9 Mix (containing 10 % (v/v) of S9)
Salt solution for S9 Mix:
NADP Na 7.66 g
D-glucose-6 phosphate Na 3.53 g
MgCl2 x 6 H2O 4.07 g
KCl 6.15 g
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration through a 0.22 μm membrane filter.
The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix (see above) 400 mL
Prior to addition to the culture medium the S9 mix was kept in an ice bath.
*0.2 M Sodium Phosphate Buffer, pH 7.4
-Solution A (880 mL)
Na2HPO4 x 12 H2O 71.63 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
-Solution B (120 mL)
NaH2PO4 x H2O 27.6 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
0.2 M Sodium phosphate buffer pH 7.4:
Solution A 880 mL
Solution B 120 mL
- concentration or volume of S9 mix and S9 in the final culture medium:
*Assay 1 followed the standard plate incorporation procedure. Bacteria (cultured in Nutrient Broth No.2) 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. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates were properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar 2000 μL
vehicle or test item formulation (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 (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48(±1) hours.
*Assay 2 followed the standard pre-incubation procedure since no biologically relevant increase in the number of revertant colonies was observed in the Assay 1. Bacteria (cultured in Nutrient Broth No.2.) were exposed to the test item 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, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37°C in a shaking incubator.
After the incubation period, 2 mL of molten top agar was 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) hours.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The sterility of the preparation was confirmed in each case. The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Charles River Laboratories Hungary Kft. The mean protein concentration of the S9 fraction used were determined to be 24.5 g/L and 28.0 g/L.
The biological activity in the Salmonella assay of S9 was characterized in each case using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batches of S9 used in this study functioned appropriately.

Test concentrations with justification for top dose:

Preliminary Range Finding Test: Based on the solubility test, a 100 mg solid fraction of test item /mL stock solution was prepared in Distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and 4 times 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 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg solid fraction of test item/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Range Finding Test the plate incorporation method was used.
Test Item Concentrations in the Mutagenicity Tests (Assay 1 and Assay 2): Based on the results of the preliminary test, a 100 mg solid fraction of test item/mL stock solution was prepared in Distilled water, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg solid fraction of test item/plate.
Examined concentrations in Assay 1 were 5000, 1581, 500, 158.1, 50 and 15.81 μg solid fraction of test item/plate.
Examined concentrations in Assay 2 were 5000, 1581, 500, 158.1, 50, 15.8 and 5 μg solid fraction of test item/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; Distilled water

- Justification for choice of solvent/vehicle: In the study two vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive control chemicals. The solubility of the test item was examined using Distilled water. The test item was soluble at 100 mg test item/mL concentrations in Distilled water. Therefore, distilled water was selected as vehicle for the study. The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension in a Preliminary Compatibility Test.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-,nitro-1,2-phenylene-diamine (NPD); in DMSO; 4 µg/plate

Controls 2

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; in DMSO; 2 µg/plate; in all Salmonella strains with S9 and in DMSO 50 µg/plate in E.coli WP2uvrA with S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two main tests: In Assay 1, the plate incorporation method was used. In Assay 2, the pre-incubation method was used.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation in Assay 1); preincubation in Assay 2

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes at 37°C in a shaking incubator (Assay 2)
- Exposure duration/duration of treatment: 48(±1) hours at 37°C (Assay1) and 48(±1) hours plus 20 minutes at 37°C (Assay 2)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: No inhibitory, cytotoxic effect of the test item was observed in Assay 1. In Assay 2 inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Salmonella typhimurium TA100, TA1537 strains and Escherichia coli strain without metabolic activation at 5000 μg solid fraction of test item/plate concentration.

METHODS FOR MEASUREMENTS OF GENOTOXICIY:
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 Excel software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Evaluation criteria:

*Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests.
*Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
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.
*Criteria for a Negative Response:
A test article is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

Statistics:

The colony numbers on the untreated / negative (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.

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:

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

Remarks:

in Assay 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

in Assay 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg solid fraction test item/plate concentration in Assay 2 (no cytotoxicity in Assay 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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

Remarks:

in Assay 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

in Assay 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg solid fraction test material/plate concentration in Assay 2 (no cytotoxicity in Assay 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

in Assay 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg solid fraction test item/plate concentration in Assay 2 (no cytotoxicity in Assay 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No precipitate was detected on the plates in the main tests in the all examined bacterial strains with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES (if applicable):
- Preliminary Compatibility Test:
The solubility of the test item was examined using Distilled water. The test item was soluble at 100 mg test item/mL concentrations in distilled water (clear solution). Therefore, distilled water was selected as vehicle for the study. The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension. The test item was soluble in the top solution (clear solution).
-Preliminary Range Finding Test:
Based on the solubility test, a 100 mg solid fraction of test item /mL stock solution was prepared in Distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and 4 times 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 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg solid fraction of test item/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Range Finding Test the plate incorporation method was used. Each sample (including the untreated, negative (solvent) and positive controls) was tested in triplicate. No precipitate was detected on the plates in the preliminary experiment in both examined bacterial strains with and without metabolic activation.
No inhibitory or toxic effects of the test item was observed in the preliminary experiment in both examined bacterial strains with and without metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA100 strain at 15.81 μg solid fraction of test item/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.21). However, there was no dose-response relationship, , the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
In Assay 2 (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 500 μg solid fraction of test item/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.44). However, there was no dose-response relationship, , the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Ames test:
- Signs of toxicity: In the Assay 1 no inhibitory, cytotoxic effect of the test item was observed in all bacterial strains with and without metabolic activation.
In Assay 2 inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Salmonella typhimurium TA100, TA1537 strains and Escherichia coli strain without metabolic activation at 5000 μg solid fraction of test item/plate concentration.

Any other information on results incl. tables

Table 1: Summary Table of the Assay 1

Concentrations (µg solid fraction of test item/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	19.7	21.0	82.3	93.3	14.7	14.7	14.7	11.7	44.3	45.7
	MF	1.00	0.90	1.02	1.03	1.00	0.98	1.26	0.97	1.02	0.99
DMSO control	Mean	19.3	22.3	--	92.0	--	13.3	11.3	11.0	--	45.3
	MF	0.98	0.96	--	1.01	--	0.89	0.97	0.92	--	0.99
Distilled water control	Mean	19.7	23.3	80.7	90.7	14.7	15.0	11.7	12.0	43.3	46.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	17.0	19.0	78.7	92.7	15.7	14.0	11.0	12.3	42.7	44.7
	MF	0.86	0.81	0.98	1.02	1.07	0.93	0.94	1.03	0.98	0.97
1581	Mean	16.7	20.0	74.7	91.7	14.3	14.7	10.7	11.0	43.0	45.0
	MF	0.85	0.86	0.93	1.01	0.98	0.98	0.91	0.92	0.99	0.98
500	Mean	16.0	17.0	74.3	97.7	13.7	15.7	9.0	12.0	43.0	43.3
	MF	0.81	0.73	0.92	1.08	0.93	1.04	0.77	1.00	0.99	0.94
158.1	Mean	16.3	20.3	84.0	97.7	15.0	15.3	9.0	11.7	42.7	46.0
	MF	0.83	0.87	1.04	1.08	1.02	1.02	0.77	0.97	0.98	1.00
50	Mean	16.0	18.0	86.7	99.0	13.3	14.3	9.7	11.7	43.0	45.3
	MF	0.81	0.77	1.07	1.09	0.91	0.96	0.83	0.97	0.99	0.99
15.81	Mean	16.3	18.0	97.3	102.7	13.0	13.7	8.3	10.0	43.7	45.0
	MF	0.83	0.77	1.21	1.13	0.89	0.91	0.71	0.83	1.01	0.98
NPD (4µg)	Mean	428.0	--	--	--	--	--	--	--	--	--
	MF	22.14	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2456.0	--	2473.3	--	215.3	--	208.0	--	--
	MF	--	109.97	--	26.88	--	16.15	--	18.91	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	254.0
	MF	--	--	--	--	--	--	--	--	--	5.60
SAZ (2µg)	Mean	--	--	1057.3	--	1217.3	--	--	--	--	--
	MF	--	--	13.11	--	83.00	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	429.3	--	--	--
	MF	--	--	--	--	--	--	37.88	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1093.3	--
	MF	--	--	--	--	--	--	--	--	25.23	--

 

Table 2:Summary Table of the Assay 2

Concentrations (µg solid fraction of test item/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	18.7	22.7	94.0	99.0	16.3	12.7	12.7	12.3	43.3	46.7
	MF	1.00	1.01	1.06	0.94	1.23	0.97	1.41	1.00	0.98	1.01
DMSO control	Mean	17.3	20.3	--	105.0	--	13.3	11.7	13.3	--	45.3
	MF	0.93	0.91	--	1.00	--	1.03	1.30	1.08	--	0.98
Distilled water control	Mean	18.7	22.3	89.0	105.0	13.3	13.0	9.0	12.3	44.0	46.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	17.7	16.7	63.0	77.7	12.0	10.7	7.3	11.7	19.3	42.0
	MF	0.95	0.75	0.71	0.74	0.90	0.82	0.81	0.95	0.44	0.91
1581	Mean	17.7	21.0	92.7	96.3	12.7	13.7	12.7	12.0	44.7	49.3
	MF	0.95	0.94	1.04	0.92	0.95	1.05	1.41	0.97	1.02	1.06
500	Mean	18.3	20.7	95.0	95.0	12.0	13.7	13.0	12.0	46.0	49.0
	MF	0.98	0.93	1.07	0.90	0.90	1.05	1.44	0.97	1.05	1.06
158.1	Mean	19.0	23.0	95.0	99.3	12.3	12.7	11.7	12.0	42.7	49.0
	MF	1.02	1.03	1.07	0.95	0.93	0.97	1.30	0.97	0.97	1.06
50	Mean	17.3	22.0	84.3	107.3	12.3	12.7	11.3	13.0	41.0	49.0
	MF	0.93	0.99	0.95	1.02	0.93	0.97	1.26	1.05	0.93	1.06
15.81	Mean	19.0	20.7	100.0	102.7	11.0	13.7	10.3	11.7	44.0	47.7
	MF	1.02	0.93	1.12	0.98	0.83	1.05	1.15	0.95	1.00	1.03
5	Mean	17.7	20.7	87.0	105.3	14.0	13.3	10.7	11.0	42.7	49.0
	MF	0.95	0.93	0.98	1.00	1.05	1.03	1.19	0.89	0.97	1.06
NPD (4µg)	Mean	410.7	--	--	--	--	--	--	--	--	--
	MF	23.69	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2453.3	--	2449.3	--	218.3	--	234.0	--	--
	MF	--	120.66	--	23.33	--	16.38	--	17.55	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	246.0
	MF	--	--	--	--	--	--	--	--	--	5.43
SAZ (2µg)	Mean	--	--	1138.7	--	1189.3	--	--	--	--	--
	MF	--	--	12.79	--	89.20	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	412.0	--	--	--
	MF	--	--	--	--	--	--	35.31	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1060.0	--
	MF	--	--	--	--	--	--	--	--	24.09	--

 

Applicant's summary and conclusion

Conclusions:

The registered substance had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The test item 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, a Preliminary Range Finding Test, an Assay 1 (Plate Incorporation Method) and an Assay 2 (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test item was dissolved in Distilled water. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg solid fraction of test item /plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains in the absence and presence of metabolic activation. Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1 were 5000, 1581, 500, 158.1, 50 and 15.81 μg solid fraction of test item/plate and in the Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg solid fraction of test item /plate.

In the assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

No precipitate was detected on the plates in the main tests in all examined Bacterial strains with and without metabolic activation.

No inhibitory, cytotoxic effect of the test item was observed in Assay 1.

In Assay 2 inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Salmonella typhimurium TA100, TA1537 strains and Escherichia coli strain without metabolic activation at 5000 μg solid fraction of test item/plate concentration.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the registered substancehad no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.