Reaction products of 1,4-Benzenedimethanol and 1-naphthol

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:

09 October 2012 to 18 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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

Constituent 1

Reference substance name:

Reaction products of 1,4-Benzenedimethanol and 1-naphthol

EC Number:

834-894-6

Cas Number:

113601-85-7

Molecular formula:

C10H8O to C82H64O5

IUPAC Name:

Reaction products of 1,4-Benzenedimethanol and 1-naphthol

Test material form:

solid

Details on test material:

Name: CAS 113601-85-7
Chemical name: 1,4-Benzenedimethanol, polymer with 1-naphthalenol
Batch/Lot number: 0950986
Description: Brown, solid
Purity: 100%
Expiry date: 08 February 2020
Storage conditions: Controlled room temperature (15-25°C ≤ 70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to ensure personnel health and safety. Causes skin irritation and serious eye irritation.

Specific details on test material used for the study:

No further details specified in the study report

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain / cell type:

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

Cytokinesis block (if used):

Not specified

Metabolic activation:

with and without

Metabolic activation system:

an exogenous metabolic activation system is added in the form of a mammalian microsomal enzyme activation mixture (liver extract, S9 fraction). The activation system uses nicotinamide-adenine dinucleotide phosphate (NADP+)-cytochrome P450 dependent mixed function oxidase enzymes of the liver. The liver extract was obtained from rats, which were pre-treated with phenobarbital and β-naphthoflavone, two inducers of several drug-metabolizing enzymes.

Test concentrations with justification for top dose:

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

Preliminary Solubility Test
Based on the results of the preliminary solubility test, the test item is insoluble in Distilled water. However, it was soluble in Dimethyl sulfoxide (DMSO) and Acetone at 100 mg/mL concentration. Due to the better biocompatibility to the test system, DMSO was selected for solvent of 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.

Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, 100 mg/mL stock formulation 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 and TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)
Based on the results of the preliminary tests, 100 mg/mL stock formulation was prepared from the test item with DMSO, which was diluted by serial dilutions in seven steps to obtain eight dosing formulations. The maximum test concentration was 5000 μg test item/plate in the main tests.
Examined concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate.

Vehicle / solvent:

DMSO and Distilled water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSOand Distilled water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

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

Details on test system and experimental conditions:

Experimental Method
The experimental methods were conducted according to the methods described Ames et al. [1] and Maron and Ames [2], Kier et al. [3], Venitt and Parry [4], OECD Guideline No. 471, 1997 [5], Commission Regulation (EC) No. 440/2008, 2008 [6], EPA Guidelines, OPPTS 870.5100, 1998, 1996 [7][8], and according to the relevant SOPs of CiToxLAB Hungary Ltd.

Procedure for Exposure in the Initial Mutation Test
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. 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 was 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 (solvent) 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 hours.

Procedure for Exposure in the Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed.
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 or concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. 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 hours.

Rationale for test conditions:

A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed

Evaluation criteria:

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.

Statistics:

Not specified

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
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 (vehicle/solvent) and positive controls. In the test each sample (including the controls) was tested in triplicate.
In the Range Finding Test the concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
The observed numbers of revertant colonies were mostly in the normal range. Minor differences were observed in some sporadic cases, but the numbers of revertant had no biological relevance and they were within the historical control range in all cases.
Precipitate / slight precipitate was observed in the preliminary experiment in both tester strains at 5000, 2500, 1000 and 316 μg/plate concentration with or without metabolic concentration.
The experimental results (revertant colony numbers per plate, mutation factors and standard deviations) are detailed in Table 7 (Appendix 2) and in Appendix 3.

INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test, the plate incorporation method; in the Confirmatory Mutation Test, the pre-incubation method was used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain. The Initial Mutation Test and Confirmatory Mutation Test were performed in the presence and absence of metabolic activation system (±S9 mix). Each test was performed with appropriate untreated, negative (vehicle/solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.*

*Note: In the Confirmatory Mutation Test in Escherichia coli WP2 uvrA strain at 5000 μg/plate concentration without metabolic activation, one of the plates was infected. In this case, the mutation factor value was calculated using the data of the two remaining replicates.

Examined concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate.
In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.
Using the plate incorporation method (Initial Mutation Test), the highest revertant rates were observed in Salmonella typhimurium TA1535 bacterial strain with metabolic activation at the concentration of 50 and 1.581 μg/plate. The mutation factor values were 1.43. However, there was no dose response, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
Using the pre-incubation method (Confirmatory Mutation Test), the highest revertant rate was observed in Escherichia coli WP2 uvrA bacterial strain without metabolic activation at 1.581 μg/plate concentration. The mutation factor value was 1.30. However, there was no dose response, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
Lower revertant counts compared to the vehicle control were observed in the Initial Mutation Test and Confirmatory Mutation Test at some non-cytotoxic concentrations. However, they had no biological significance and were considered as reflecting the variability of the test system.

Note: Lower or slightly reduced numbers of revertant colonies compared to the vehicle control plates were observed in the Initial Mutation Test in Salmonella typhimurium TA100 strain with metabolic activation in the whole tested concentration range. However, no effect of the background lawn was observed, and lower numbers of revertants compared to the DMSO solvent control were observed for untreated control and Distilled water control also in this strain. Therefore, this fact was not considered as sign of cytotoxicity and had no adverse effect on the results or integrity of the study.
Precipitate / slight precipitate was observed in the Initial Mutation in all examined bacterial strains at 5000, 1581 and 500 μg/plate concentrations with and without metabolic activation, in the Confirmatory Mutation Test in all tester strains at 5000, 1581 and 500 μg/plate concentrations without metabolic activation and at 5000 μg/plate concentration with metabolic activation.
Inhibitory, cytotoxic effect of the test item (reduced numbers of revertant colonies) was observed in the Initial Mutation Test in Salmonella typhimurium TA1535 strain at 5000 and 1581 μg/plate concentrations without metabolic activation and in Salmonella typhimurium TA1537 strain at 5000 μg/plate concentration without metabolic activation.
Stronger inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development and / or reduced numbers of revertant colonies; in some cases pinpoint colonies were also detected) was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5000, 1581 and 500 μg/plate concentrations without metabolic activation, in Salmonella typhimurium TA1535 strain at 5000 and 1581 μg/plate concentrations without metabolic activation, in Escherichia coli WP2 uvrA bacterial strain at 5000 μg/plate concentration without metabolic activation and in Salmonella typhimurium TA1537 strain at 5000 μg/plate concentration with metabolic activation.

Note: Slightly reduced numbers of revertant colonies compared to the vehicle control plates were observed in some other cases in the Confirmatory Mutation Test, although no effect on the background lawn was observed. However, these facts were not considered as sign of cytotoxicity in these cases and they had no adverse effect on the results or integrity of the study.

Any other information on results incl. tables

Genotypes of the Strains Used for Mutagenicity Testing

Strain	Genotype		Mutations		Main DNA target	Plasmid
	trp/his mutation	Type of mutation	Cell wall	DNA-repair
S.typhimuriumTA98	hisD3052	Frameshift	rfa	uvrB	GC	pKM101
S.typhimuriumTA100	hisG46	Base pair substitution	rfa	uvrB	GC	pKM101
S.typhimuriumTA1535	hisG46	Base pair substitution	rfa	uvrB	GC	No
S.typhimurium. TA1537	hisC3076	Frameshift	rfa	uvrB	GC	No
E.coliWP2uvrA	trpE	Base pair substitution	+	uvrA	AT	No

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	Clear solution	5000

Summary of Controls

Type of control	Activation with S9 Mix	Vehicle/ Solvent	Top Agar	Strain-Specific Positive Chemical Solutions	Phosphate Buffer
Untreated	-	-	+	-	+
Untreated	+	-	+	-	-
Vehicle/Solvent	-	+	+	-	+
Vehicle/Solvent	+	+	+	-	-
Positive Reference Control	-	-	+	+	+
Positive Reference Control	+	-	+	+	-

Note: if the solvent of the positive control substance differed from the vehicle (solvent) of the test item, both solvents were run in the assay.

Summary of Range Finding Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains
		TA98		TA100
		-S9	+S9	-S9	+S9
Untreated control	Mean	17.0	20.3	121.0	125.3
	MF	0.86	1.03	0.96	0.82
DMSO control	Mean	19.7	19.7	126.7	153.0
	MF	1.00	1.00	1.00	1.00
Distilled water control	Mean	-	-	-	-
	MF	-	-	0.91	-
5000	Mean	17.3	26.0	134.3	143.3
	MF	0.88	1.32	1.06	0.94
2500	Mean	17.3	18.7	124.7	158.0
	MF	0.88	0.95	0.98	1.03
1000	Mean	17.3	26.0	121.7	145.3
	MF	0.88	1.32	0.96	0.95
316	Mean	16.7	24.3	111.7	165.3
	MF	0.85	1.24	0.88	1.08
100	Mean	21.7	27.3	109.0	142.3
	MF	1.10	1.39	0.86	0.93
31.6	Mean	19.7	26.0	116.3	128.0
	MF	1.00	1.32	0.92	0.95
10	Mean	22.0	27.0	116.3	128.0
	MF	1.12	1.37	0.92	0.84
NPD (4μg)	Mean	235.3	-	-	-
	MF	11.97	-	-	-
2AA (2μg)	Mean	-	2444.0	-	2794.7
	MF	-	124.27	-	18.27
SAZ (2μg)	Mean	-	-	1273.3	-
	MF	-	-	11.10	-

Summary of Initial Mutation Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	25.7	29.3	111.0	127.0	9.0	10.0	8.0	9.7	25.7	39.0
	MF	1.22	0.81	1.03	0.60	1.08	1.30	1.00	1.16	1.01	0.84
DMSO control	Mean	21.0	36.3	108.0	210.7	8.3	7.7	8.0	8.3	25.3	46.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	-	-	124.0	-	7.3	-	-	-	25.3	-
	MF	-	-	1.15	-	0.88	-	-	-	1.00	-
5000	Mean	16.3	29.3	128.3	96.0	2.3	7.3	4.0	5.3	20.7	33.0
	MF	0.78	0.81	1.19	0.46	0.28	0.96	0.50	0.64	0.82	0.71
1581	Mean	21.0	18.7	109.3	130.7	3.3	9.3	7.7	9.3	26.0	41.7
	MF	1.00	0.51	1.01	0.62	0.40	1.22	0.96	1.12	1.03	0.89
500	Mean	22.7	29.3	102.3	119.3	7.3	10.3	7.0	7.3	27.0	31.3
	MF	1.08	0.81	0.95	0.57	0.88	1.35	0.88	0.88	1.07	0.67
158.1	Mean	20.3	30.0	111.7	138.3	8.3	5.3	5.7	6.7	22.7	35.3
	MF	0.97	0.83	1.03	0.66	1.00	0.70	0.71	0.80	0.89	0.76
50	Mean	21.0	29.3	120.3	140.0	8.7	11.0	6.7	6.7	26.3	38.7
	MF	1.00	0.81	1.11	0.66	1.04	1.43	0.83	0.80	1.04	0.83
15.81	Mean	26.0	34.3	103.0	126.7	7.0	8.0	7.7	7.7	22.7	31.7
	MF	1.24	0.94	0.95	0.60	0.84	1.04	0.96	0.92	0.89	0.68
5	Mean	25.3	30.3	109.3	130.7	7.0	10.3	5.7	9.3	25.3	33.0
	MF	1.21	0.83	1.01	0.62	0.84	1.35	0.71	1.12	1.00	0.71
1.581	Mean	21.3	31.7	114.3	123.7	6.7	11.0	7.0	10.3	30.3	30.3
	MF	1.02	0.87	1.06	0.59	0.80	1.43	0.88	1.24	1.20	0.65
NPD (4μg)	Mean	289.3	-	-	-	-	-	-	-	-	-
	MF	13.78	-	-	-	-	-	-	-	-	-
2AA (2μg)	Mean	-	2400.0	-	2273.3	-	214.0	-	125.3	-	-
	MF	-	66.06	-	10.79	-	27.91	-	15.04	-	-
2AA (50μg)	Mean	-	-	-	-	-	-	-	-	-	253.3
	MF	-	-	-	-	-	-	-	-	-	5.43
SAZ (2μg)	Mean	-	-	1244.0	-	1101.3	-	-	-	-	-
	MF	-	-	10.03	-	150.18	-	-	-	-	-
9AA (50μg)	Mean	-	-	-	-	-	-	509.3	-	-	-
	MF	-	-	-	-	-	-	63.67	-	-	-
MMS (2μL)	Mean	-	-	-	-	-	-	-	-	1037.3	-
	MF	-	-	-	-	-	-	-	-	40.95	-

 

Summary of Confirmatory Mutation Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	22.0	26.7	110.0	122.3	7.0	11.0	5.7	12.0	34.0	38.3
	MF	1.02	0.91	1.08	0.93	0.68	0.87	0.71	1.50	1.23	0.91
DMSO control	Mean	21.7	29.3	102.0	132.0	10.3	12.7	8.0	8.0	27.7	42.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	-	-	119.0	-	7.0	-	-	-	44.3	-
	MF	-	-	1.17	-	0.68	-	-	-	1.60	-
5000	Mean	2.0	20.3	18.7	96.0	3.3	8.7	1.0	1.0	11.0	34.0
	MF	0.09	0.69	0.18	0.73	0.32	0.68	0.13	0.13	0.40	0.80
1581	Mean	4.0	25.3	25.0	103.7	1.7	10.0	9.3	6.7	26.0	41.7
	MF	0.18	0.86	0.25	0.79	0.16	0.79	1.17	0.83	0.94	0.98
500	Mean	14.0	27.0	66.0	98.3	7.0	5.7	3.7	6.0	31.0	37.0
	MF	0.65	0.92	0.65	0.74	0.68	0.45	0.46	0.75	1.12	0.87
158.1	Mean	20.7	27.0	97.0	107.7	8.7	8.7	5.3	7.0	27.3	44.0
	MF	0.95	0.92	0.95	0.82	0.84	0.68	0.67	0.88	0.99	1.04
50	Mean	17.0	21.7	93.7	111.7	4.7	12.7	7.0	9.7	31.3	49.3
	MF	0.78	0.74	0.92	0.85	0.45	1.00	0.88	1.21	1.13	1.17
15.81	Mean	16.0	21.0	91.0	119.3	7.7	10.0	4.3	7.7	28.0	38.7
	MF	0.74	0.72	0.89	0.90	0.74	0.79	0.54	0.96	1.01	0.91
5	Mean	18.7	32.7	101.0	132.	7.3	12.7	6.0	7.7	24.3	33.3
	MF	0.86	1.11	0.99	1.00	0.71	1.00	0.75	0.96	0.88	0.79
1.581	Mean	15.0	34.3	100.3	119.3	9.3	11.7	7.7	6.0	36.0	40.7
	MF	0.69	1.17	0.98	0.90	0.90	0.92	0.96	0.75	1.30	0.96
NPD (4μg)	Mean	298.7	-	-	-	-	-	-	-	-	-
	MF	13.78	-	-	-	-	-	-	-	-	-
2AA (2μg)	Mean	-	2374.7	-	2468.0	-	192.3	-	207.3	-	-
	MF	-	80.95	-	18.7	-	15.18	-	25.92	-	-
2AA (50μg)	Mean	-	-	-	-	-	-	-	-	-	235.3
	MF	-	-	-	-	-	-	-	-	-	5.56
SAZ (2μg)	Mean	-	-	1202.7	-	1134.7	-	-	-	-	-
	MF	-	-	10.11	-	162.1	-	-	-	-	-
9AA (50μg)	Mean	-	-	-	-	-	-	364	-	-	-
	MF	-	-	-	-	-	-	45.5	-	-	-
MMS (2μL)	Mean	-	-	-	-	-	-	-	-	1102.7	-
	MF	-	-	-	-	-	-	-	-	24.87	-

Applicant's summary and conclusion

Conclusions:

The test item SN-475N had no mutagenic activity in the bacterium tester strains under the test conditions used in this study

Executive summary:

The test item SN-475N 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 Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Solubility Test and available information, the test item was dissolved in DMSO. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate.

Inhibitory, cytotoxic effect of the test item was observed in the Initial Mutation Test and/or Confirmatory Mutation Test in all tester strains without metabolic activation and in Salmonella typhimurium TA1537 strain with metabolic activation.

Precipitate / slight precipitate was observed in all tester strains in the main tests in the higher concentration range (1-3 concentrations).

The mean values of revertant colonies of the 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 tests were 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 test item SN-475N had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.