Sodium 1,4-dicyclohexyl sulphonatosuccinate

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:

9 April 2020 to 11 June 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 1,4-dicyclohexyl sulphonatosuccinate

EC Number:

245-629-3

EC Name:

Sodium 1,4-dicyclohexyl sulphonatosuccinate

Cas Number:

23386-52-9

Molecular formula:

C16H26O7S.Na

IUPAC Name:

sodium 1,4-bis(cyclohexyloxy)-1,4-dioxobutane-2-sulfonate

Test material form:

liquid

Method

Target gene:

histidine (Salmonella); tryptophan (E. coli)

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:

- source of S9
Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction.
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 documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
The composition of solution refers to 1000 mL.
1. BRUCE N. AMES, JOYCE MCCANN and EDITH YAMASAKI:
Methods for Detecting Carcinogens and Mutagens with the Salmonella /Mammalian-Microsome Mutagenicity Test. Mutation Research, 31: 347-364, 1975
2. DOROTHY M. MARON and BRUCE N. AMES:
Revised Method for the Salmonella Mutagenicity Test. Mutation Research,
113: 173-215, 1983
*Induction of Liver Enzymes
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.
* Preparation of Rat Liver Homogenate S9 Fraction
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 ± 10ºC. The dates of preparation of S9 fractions for this study were 05 September 2019 and 16 January 2020 (Charles River Laboratories Hungary code: E13142 and E13222, 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.
- concentration or volume of S9 mix and S9 in the final culture medium :
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
(S9 Mix (containing 10 % (v/v) of S9)
- 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:

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.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled Water (vehicle for the test item)
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 following chemicals were used for vehicle (solvent) control groups: Dimethyl sulfoxide (DMSO) and Distilled Water
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, DMSO (Dimethyl sulfoxide) and N,N-Dimethylformamide (DMF). The test item was soluble at 100 mg solid fraction of test item /mL concentration using Distilled water, DMSO and DMF. Due to the better biocompatibility 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.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

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

Controls 2

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO 2µg/plate all Salmonella strains and 50 µg/plate 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: Preliminary Range Finding Test, Assay 1 (plate incorporation), Assay 2 (preincubation)

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

Assay 1: TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No
- Exposure duration/duration of treatment: 48(±1) hours at 37°C
Assay 2: TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Yes (20 minutes at 37°C in a shaking incubator)
- Exposure duration/duration of treatment: 20 minutes + 48(±1) hours at 37°C

FOR GENE MUTATION:

Evaluation criteria:

(1)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.
(2) 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.
(3) 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Assay 2 at 5000 µg solid fraction/plate without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Assay 2 at 1581 µg solid fraction/ plate without S9. Reduced colony number in Assy 2 at 5000 µg/solid fraction/plate without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Assay 2 at 1581 µg solid fraction/plate without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Assay 2 at 1581 µg solid fraction/plate without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

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

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):
In the Preliminary Range Finding Test, the plate incorporation method was used. 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 (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg solid fraction of test item/plate.
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: See Table 2 and 3 under Any other information on results incl. tables

Ames test:
- Signs of toxicity: 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. In Assay 1 no inhibitory, cytotoxic effect of the test item was observed in all Salmonella typhimurium bacterial strains and Escherichia coli WP2 uvrA strain with and without metabolic activation.
In Assay 2 inhibitory, cytotoxic effect of the test item (reduced/slightly reduced background lawn development) was observed in all Salmonella typhimurium strains without metabolic activation at 5000 μg solid fraction of test item/plate concentration and in Salmonella typhimurium TA98, TA100 and TA1537 strains without metabolic activation on the plates at the 1581 μg solid fraction of test item/plate concentration.
Reduced colony number was observed in the Assay 2 in Salmonella typhimurium TA1537 strain without metabolic activation on the plates at the 5000 μg solid fraction of test item/plate concentration.
-Revertants:
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 strain at 15.81 μg solid fraction of test item/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.24). However, there was no dose-response relationship, the observed mutation factor values were above the biologically relevant threshold limit 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 50 and 5 μg solid fraction of test item/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.33). 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.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: See under Any other information on results incl. tables
- Negative (solvent/vehicle) historical control data: See under Any other information on results incl. tables

Any other information on results incl. tables

Table 1. Historical Control Data  (Period of 2015-2020)

Untreated control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E.coli	TA98	TA100	TA1535	TA1537	E.coli
Mean	20.9	98.3	13.2	8.8	40.6	25.6	104.6	12.3	9.9	44.1
St.dev.	4.4	12.7	3.5	3.2	9.4	5.8	13.2	3.2	3.6	9.4
Range	11-50	67-152	1-33	2-26	14-77	13-54	67-152	3-39	1-29	16-89
n	1296	1297	1302	1314	1302	1311	1314	1314	1323	1299
DMSO control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	20.5	95.4	13.0	8.4	39.6	24.9	101.0	11.8	9.4	43.0
St.dev.	4.2	12.1	3.5	3.1	9.7	5.4	13.5	3.1	3.5	9.5
Range	7-41	60-145	3-34	1-27	12-75	11-50	53-165	2-33	1-29	9-76
n	1428	1419	1425	1449	1425	1443	1443	1449	1455	1431
Distilled water control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	21.5	97.5	13.2	9.3	41.5	25.9	103.1	12.1	10.4	44.6
St.dev.	4.4	12.5	3.3	3.4	9.1	5.6	13.6	3.1	3.7	9.0
Range	13-37	58-150	2-32	3-20	17-72	15-45	59-164	3-34	3-24	13-76
n	288	1332	1332	303	1341	291	1314	1326	300	1320
DMF control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	19.6	93.5	12.9	9.2	42.0	23.8	96.2	11.9	10.5	43.3
St.dev.	3.9	13.3	3.1	3.4	10.8	5.5	13.2	3.0	3.6	10.9
Range	11-33	57-121	6-24	2-18	16-70	11-37	68-143	3-21	3-19	18-72
n	114	114	114	117	111	114	114	114	114	111
Acetone control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	20.9	97.1	12.9	8.2	40.1	25.5	102.2	11.5	9.1	44.1
St.dev.	4.3	10.0	3.6	2.8	8.5	5.6	11.3	3.0	3.2	8.7
Range	11-35	63-126	6-32	2-17	21-63	16-44	66-132	4-19	1-19	20-70
n	219	222	222	225	219	219	222	225	225	219
Positive reference control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	395.9	1130.9	1138.8	413.3	1020.6	2391.6	2408.0	219.0	214.0	239.5
St.dev.	99.4	82.5	102.0	34.0	114.4	146.2	115.7	32.1	22.3	35.6
Range	182-2336	536-1480	568-2004	208-629	488-2496	312-2736	1116-3104	101-418	147-424	127-384
n	1296	1296	1302	1314	1305	1311	1317	1317	1323	1299

TA98: Salmonella typhimurium TA98, TA100: Salmonella typhimurium TA100, TA1535: Salmonella typhimurium TA1535, TA1537: Salmonella typhimurium TA1537, E. coli: Escherichia coli WP2 uvrA; n: number of cases

98: Salmonella typhimurium TA98, TA100: Salmonella typhimurium TA100, TA1535: Salmonella typhimurium TA1535, TA1537: Salmonella

Table 2: 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	21.3	22.7	86.3	111.7	16.7	17.0	12.0	13.3	40.0	43.0
	MF	0.90	0.94	0.95	1.06	1.09	1.02	1.09	1.03	1.00	1.00
DMSO control	Mean	22.0	23.0	--	116.7	--	14.7	130.	12.0	--	42.3
	MF	0.93	0.96	--	1.10	--	0.88	1.18	0.92	--	0.98
Distilled water control	Mean	23.7	24.0	90.7	105.7	15.3	16.7	11.0	13.0	40.0	43.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	16.7	23.7	100.0	111.3	14.0	14.0	10.3	11.0	38.7	41.7
	MF	0.70	0.99	1.10	1.05	0.91	0.84	0.94	0.85	0.97	0.97
1581	Mean	17.7	23.0	98.7	111.3	14.0	12.0	9.7	13.0	38.0	40.3
	MF	0.75	0.96	1.09	1.05	0.91	0.72	0.88	1.00	0.95	0.94
500	Mean	19.0	21.7	108.3	110.3	14.7	14.7	11.0	11.7	39.7	42.7
	MF	0.80	0.90	1.19	1.04	0.96	0.88	1.00	0.90	0.99	0.99
158.1	Mean	17.7	22.0	100.0	109.0	13.0	15.7	10.3	12.3	41.3	42.7
	MF	0.75	0.92	1.10	1.03	0.85	0.94	0.94	0.95	1.03	0.99
50	Mean	17.0	24.0	99.0	120.7	17.7	12.0	10.7	11.7	37.0	42.3
	MF	0.72	1.00	1.09	1.14	1.15	0.72	0.97	0.90	0.93	0.98
15.81	Mean	17.0	20.3	95.7	116.7	16.3	14.0	13.7	9.3	40.0	42.7
	MF	0.72	0.85	1.06	1.10	1.07	0.84	1.24	0.72	1.00	0.99
NDP (4µg)	Mean	409.3	--	--	--	--	--	--	--	--	--
	MF	18.61	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2469.3	--	2472.0	--	212.7	--	210.7	--	--
	MF	--	107.36	--	21.19	--	14.50	--	17.56	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	246.7
	MF	--	--	--	--	--	--	--	--	--	5.83
SAZ (2µg)	Mean	--	--	1128.0	--	1081.3	--	--	--	--	--
	MF	--	--	12.44	--	70.52	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	405.3	--	--	--
	MF	--	--	--	--	--	--	31.18	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1098.7	--
	MF	--	--	--	--	--	--	--	--	24.47	--

 

Table 3: 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	17.3	21.0	82.3	98.3	17.0	17.7	9.3	11.0	42.3	44.0
	MF	0.93	0.84	0.98	0.95	1.02	1.00	1.17	0.89	1.03	1.01
DMSO control	Mean	18.3	23.3	--	94.0	--	17.0	11.0	11.7	--	42.7
	MF	0.98	0.93	--	0.91	--	0.96	1.38	0.95	--	0.98
Distilled water control	Mean	18.7	25.0	84.0	103.3	16.7	17.7	8.0	12.3	41.0	43.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	11.3	21.3	51.7	93.3	8.3	17.0	0.0	6.3	23.0	43.7
	MF	0.61	0.85	0.62	0.90	0.50	0.96	0.00	0.51	0.56	1.00
1581	Mean	16.7	22.0	56.0	102.7	12.0	17.7	8.0	12.0	37.3	47.0
	MF	0.89	0.88	0.67	0.99	0.72	1.00	1.00	0.97	0.91	1.08
500	Mean	16.7	22.3	92.0	103.7	16.7	16.0	10.3	11.3	42.3	47.0
	MF	0.89	0.89	1.10	1.00	1.00	0.91	1.29	0.92	1.03	1.08
158.1	Mean	17.3	23.0	92.7	104.7	17.7	15.3	9.0	11.7	43.0	46.0
	MF	0.93	0.92	1.10	1.01	1.06	0.87	1.13	0.95	1.05	1.05
50	Mean	16.7	20.7	91.3	96.7	15.7	16.7	10.7	9.7	43.7	43.7
	MF	0.89	0.83	1.09	0.94	0.94	0.94	1.33	0.78	1.07	1.00
15.81	Mean	17.0	26.7	94.7	108.0	14.7	17.3	10.0	11.7	41.7	46.7
	MF	0.91	1.07	1.13	1.05	0.88	0.98	1.25	0.95	1.02	1.07
5	Mean	18.0	25.3	82.3	95.0	15.7	16.7	10.7	12.0	40.7	46.0
	MF	0.96	1.01	0.98	0.92	0.94	0.94	1.33	0.97	0.99	1.05
NDP (4µg)	Mean	421.3	--	--	--	--	--	--	--	--	--
	MF	22.98	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2468.0	--	2461.3	--	219.0	--	207.7	--	--
	MF	--	105.77	--	26.18	--	12.88	--	17.80	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	246.7
	MF	--	--	--	--	--	--	--	--	--	5.78
SAZ (2µg)	Mean	--	--	1082.7	--	1217.3	--	--	--	--	--
	MF	--	--	12.89	--	73.04	--	--	---	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	414.7	--	--	--
	MF	--	--	--	--	--	--	37.70	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1056.0	--
	MF	--	--	--	--	--	--	--	--	25.76	--

 

um TA1537, E. coli: Escherichia coli WP2 uvrA; n: number of cases

Applicant's summary and conclusion

Conclusions:

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 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 the Preliminary Concentration Range Finding Test.

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

In Assay 2 inhibitory, cytotoxic effect of the test item (reduced/slightly reduced background lawn development) was observed in all Salmonella typhimurium strains without metabolic activation at 5000 μg solid fraction of test item/plate concentration and in Salmonella typhimurium TA98, TA100 and TA1537 strains without metabolic activation on the plates at the 1581 μg solid fraction of test item/plate concentration.

Reduced colony number was observed in the Assay 2 in Salmonella typhimurium TA1537 strain without metabolic activation on the plates at the 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 substance had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.