α-chloro-o-xylene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 May 2018 - 06 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 31, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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:

α-chloro-o-xylene

EC Number:

209-013-8

EC Name:

α-chloro-o-xylene

Cas Number:

552-45-4

Molecular formula:

C8H9Cl

IUPAC Name:

1-(chloromethyl)-2-methylbenzene

Test material form:

liquid

Specific details on test material used for the study:

purity: 98.3 g / 100 g (substance characterization available)
physical state / appearance: colorless to yellowish, clear, liquid
storage conditions: +2 to +8°C

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Tinova Biochem GmbH (Gießen, Germany)
- Suitability of cells: as published by Ames et al.
Regular checking of the properties of the Salmonella typhimurium and Escherichia coli strains regarding the membrane permeability, ampicillin resistance; UV sensitivity, and amino acid requirement as well as normal spontaneous mutation rates is performed in Envigo CRS GmbH according to Ames et al. (1977) and Maron and Ames (1983). Thus, it is ensured that the experimental conditions set down by Ames are fulfilled.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

According to the current OECD Guideline No. 471 the maximum concentration should be 5000 μg/plate, unless limited by toxicity or solubility of the test item.
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment met the acceptance criteria (cf. 4.8.3), thus, it is reported as experiment I. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II:
Strain TA 100: 1, 3, 10; 33; 100; 333; 1000; and 2500 μg/plate
The remaining strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria (Maron et al.; 1981)

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

sterility controls

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-Aminoanthracene (2-AA)

Details on test system and experimental conditions:

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

For each strain and dose level, including the controls, three plates were used.

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

Experiment II (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37°C for 60 minutes:
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension (cf. test system, pre-culture of the strains)

After pre-incubation 2000 μL overlay agar (approx. 45°C) was added to each tube. Then, the mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL of the stock solution, 500 μl S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of two-fold or above (strains TA 98, TA 100, and WP2 uvrA) or of three-fold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2500 µg/plate, except for Experiment II without S9 mix (precipitation was observed)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2500 µg/plate, except for experiment I without S9 mix (precipitation was observed)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: test item precipitated in the overlay agar in the test tubes from 2500 μg/plate up to the highest investigated dose in all strains with S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 μg/plate up to the highest investigated dose in all strains with and without S9 mix. The undissolved particles had no influence on the data recording.

Any other information on results incl. tables

Summary of Experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		11 ± 2	10 ± 5	23 ± 7	208 ± 10	44 ± 5
	Untreated		10 ± 5	11 ± 3	33 ± 8	198 ± 13	42 ± 10
	2-Methylbenzyl chloride	3 μg	11 ± 3	10 ± 3	25 ± 5	201 ± 25	39 ± 10
		10 μg	11 ± 0	11 ± 4	28 ± 4	211 ± 12	30 ± 4
		33 μg	15 ± 2	8 ± 2	27 ± 5	214 ± 6	35 ± 6
		100 μg	15 ± 1	12 ± 3	24 ± 6	216 ± 14	51 ± 7
		333 μg	9 ± 4	9 ± 4	20 ± 1	168 ± 31	40 ± 3
		1000 μg	11 ± 1	11 ± 3	21 ± 5	52 ± 12	24 ± 4
		2500 μg	4 ± 1^PM	5 ± 1^PM	11 ± 3^PM	48 ± 8^PM	23 ± 3^P
		5000 μg	4 ± 1^PM	3 ± 1^PM	4 ± 1^PM	33 ± 7^PM	23 ± 8^P
	NaN3	10 μg	1331 ± 117			1790 ± 101
	4-NOPD	10 μg			539 ± 32
	4-NOPD	50 μg		75 ± 10
	MMS	2.0 μL					842 ± 11
With Activation	DMSO		15 ± 2	8 ± 2	43 ± 3	207 ± 6	46 ± 13
	Untreated		15 ± 4	12 ± 2	44 ± 13	213 ± 5	55 ± 8
	2-Methylbenzyl chloride	3 μg	13 ± 2	11 ± 1	45 ± 9	194 ± 21	55 ± 4
		10 μg	16 ± 6	9 ± 3	41 ± 5	179 ± 18	43 ± 14
		33 μg	18 ± 4	10 ± 4	42 ± 7	202 ± 22	57 ± 6
		100 μg	21 ± 2	13 ± 4	40 ± 11	213 ± 8	60 ± 10
		333 μg	17 ± 5	13 ± 1	38 ± 6	224 ± 2	52 ± 13
		1000 μg	16 ± 5	15 ± 1	33 ± 7	106 ± 16	47 ± 11
		2500 μg	17 ± 4^PM	11 ± 2^PM	17 ± 3^P	61 ± 16^PM	44 ± 8^P
		5000 μg	4 ± 1^PM	0 ± 1^PM	1 ± 1^PM	8 ± 3^PM	16 ± 2^PM
	2-AA	2.5 μg	417 ± 34	195 ± 29	3686 ± 348	4705 ± 78
	2-AA	10.0 μg					465 ± 6

 

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

 

 

 

 

  

Summary of Experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		10 ± 1	9 ± 3	19 ± 4	176 ± 8	36 ± 8
	Untreated		12 ± 2	6 ± 3	23 ± 3	207 ± 5	41 ± 3
	2-Methylbenzyl chloride	1 μg				174 ± 9
		3 μg	8 ± 2	9 ± 3	24 ± 7	175 ± 10	29 ± 3
		10μg	12 ± 4	9 ± 2	26 ± 7	198 ± 15	30 ± 9
		33μg	10 ± 1	8 ± 3	29 ± 5	203 ± 10	36 ± 9
		100μg	13 ± 1	10 ± 1	26 ± 6	213 ± 14	40 ± 11
		333μg	12 ± 2	10 ± 1	19 ± 6	109 ± 14	25 ± 4
		1000μg	6 ± 1	6 ± 1	23 ± 3	77 ± 26	24 ± 8
		2500μg	8 ± 3^P	1 ± 1^P	16 ± 3^P	2 ± 2^P	35 ± 8^P
		5000μg	6 ± 1^P	0 ± 0^P	0 ± 1^P		1 ± 1^P
	NaN3	10 μg	1220 ± 4			1886 ± 47
	4-NOPD	10 μg			470 ± 28
	4-NOPD	50 μg		84 ± 11
	MMS	2.0 μL					346 ± 45
With Activation	DMSO		14 ± 5	14 ± 2	34 ± 5	152 ± 10	39 ± 3
	Untreated		13 ± 4	13 ± 3	41 ± 3	214 ± 5	48 ± 7
	2-Methylbenzyl chloride	1 μg				124 ± 23
		3 μg	10 ± 2	14 ± 2	31 ± 4	150 ± 12	45 ± 15
		10μg	12 ± 2	14 ± 3	38 ± 2	122 ± 10	45 ± 2
		33μg	14 ± 4	14 ± 4	35 ± 9	139 ± 19	51 ± 3
		100μg	15 ± 5	12 ± 3	35 ± 9	173 ± 26	62 ± 11
		333μg	11 ± 4	18 ± 5	39 ± 8	72 ± 20	52 ± 5
		1000μg	7 ± 2	15 ± 5	33 ± 6	55 ± 15	31 ± 4
		2500μg	1 ± 1^P	11 ± 5^P	6 ± 1^P	2 ± 1^P	2 ± 1^P
		5000μg	0 ± 0^P	1 ± 1^P	0 ± 1^P		1 ± 1^P
	2-AA	2.5 μg	341 ± 20	209 ± 13	2915 ± 654	2773 ± 347
	2-AA	10.0 μg					404 ± 13

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P	Precipitate