Tetrachloro-p-benzoquinone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed OECD and GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrachloro-p-benzoquinone

EC Number:

204-274-4

EC Name:

Tetrachloro-p-benzoquinone

Cas Number:

118-75-2

Molecular formula:

C6Cl4O2

IUPAC Name:

2,3,5,6-tetrachloro-1,4-benzoquinone

Details on test material:

- Name of test material (as cited in study report): Chloranil

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Pre-Experiment/Experiment IA: 0.1; 0.3;1; 3; 10; 33; 100; 333; and 1000 µg/plate
Experiment II without S9mix: 0.06; 0.21;0.63; 2.08; 6.25; 20.8; 62.3; and 208.3 µg/plate
Experiment II with S9mix: 0.63; 2.1; 6.25; 20.8; 62.5; 208.3; 625; and 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was used because of its solubility properties, its relative non-toxicity to the bacteria and the test substance stability in the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II)


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding 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.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item was observed in the test tubes at 1000 - 5000 µg/plate in experiment I, at 625 and 2500 µg/plate in experiment II. Precipitation of the test item was also observed on the incubated agar plates at 2500 and 5000 µg/plate in experiment I. The undissolved particles had no influence on the data recording..

COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Reduced background growth was observed at the following concentrations (µg/plate):
Strains Experiment I Experiment II
without S9 mix with S9 mix without S9 mix without S9 mix with S9 mix
TA 1535 33 - 5000 333 - 5000 33 - 333 20.8 - 208.3 625, 2500
TA 1537 100 - 5000 1000 - 5000 33 - 333 20.8 - 208.3 625, 2500
TA 98 100 - 5000 333 - 5000 100 - 333 20.8 - 208.3 625, 2500
TA 100 33 - 5000 333 - 5000 100 - 333 62.3, 208.3 625, 2500
WP2 uvrA 333 - 5000 1000 - 5000 n.p. 208.3 2500
n.p. = not performed
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at the following concentrations (µg/plate):
Strains Experiment I Experiment II
without S9 mix with S9 mix without S9 mix without S9 mix with S9 mix
TA 1535 100 - 5000 1000 - 5000 100, 333 20.8 - 208.3 625, 2500
TA 1537 333 - 5000 1000 - 5000 100, 333 20.8 - 208.3 625, 2500
TA 98 333 - 5000 2500, 5000 333 20.8 - 208.3 625, 2500
TA 100 100 - 5000 333 - 5000 333 208.3 2500
WP2 uvrA 333 - 5000 2500 , 5000 n.p. / 2500
n.p. = not performed, / = no toxic effects observed

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

 Summary of Results Pre-Experiment and Experiment I

Study Name: 1250201	Study Code: Harlan - CCR 1250201
Experiment: 1250201 VV Plate	Date Plated: 05/06/2009
Assay Conditions:	Date Counted: 08/06/2009

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			15 ± 3	11 ± 5	30 ± 9	158 ± 4	59 ± 13
	Untreated			16 ± 3	12 ± 2	40 ± 7	174 ± 19	49 ± 8
	Chloranil	3 µg		13 ± 2	11 ± 4	32 ± 5	177 ± 14	44 ± 7
		10 µg		12 ± 3	12 ± 4	26 ± 4	178 ± 16	56 ± 6
		33 µg		10 ± 4^R	6 ± 2	31 ± 3	142 ± 56^R	59 ± 3
		100 µg		2 ± 0^M R	^R N	^N R	34 ± 20^M R	44 ± 7
		333 µg		1 ± 1^M R	1 ± 1^M R	1 ± 1^M R	2 ± 2^M R	5 ± 5^M R
		1000 µg		0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	1 ± 1^M R	^N R
		2500 µg		0 ± 0^M R P	0 ± 0^M R P	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
		5000 µg		0 ± 0^M R P	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
	NaN3	10 µg		2020 ± 42			1979 ± 145
	4-NOPD	10 µg				439 ± 3
	4-NOPD	50 µg			104 ± 4
	MMS	3.0 µL						1165 ± 44
With Activation	DMSO			18 ± 4	11 ± 3	33 ± 7	211 ± 96	69 ± 8
	Untreated			14 ± 0	12 ± 7	39 ± 10	160 ± 17	69 ± 6
	Chloranil	3 µg		15 ± 4	15 ± 2	43 ± 4	159 ± 22	64 ± 6
		10 µg		14 ± 2	12 ± 3	33 ± 8	154 ± 19	62 ± 9
		33 µg		18 ± 2	7 ± 1	29 ± 2	148 ± 17	67 ± 2
		100 µg		20 ± 4	10 ± 4	37 ± 7	184 ± 13	63 ± 9
		333 µg		12 ± 3^R	10 ± 2	27 ± 15^R	47 ± 1^R M	60 ± 11
		1000 µg		4 ± 2^M R	1 ± 1^M R	^N R	7 ± 2^M R	35 ± 6^R
		2500 µg		0 ± 0^M R P	0 ± 0^P M R	1 ± 1^P M R	0 ± 0^P M R	10 ± 3^P M R
		5000 µg		0 ± 0^M R P	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R
	2-AA	2.5 µg		246 ± 5	203 ± 15	1507 ± 24	1641 ± 134
	2-AA	10.0 µg						359 ± 81

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	R M P N	Reduced background growth Manual count Precipitate Analysis not possible

   Summary of Results Pre-Experiment and Experiment I A

Study Name: 1250201	Study Code: Harlan - CCR 1250201
Experiment: 1250201 VVa Plate	Date Plated: 15/06/2009
Assay Conditions:	Date Counted: 18/06/2009

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100
Without Activation	DMSO		21 ± 5	13 ± 2	27 ± 1	126 ± 21
	Untreated		14 ± 4	10 ± 5	25 ± 5	163 ± 15
	Chloranil	0.1 µg	18 ± 4	12 ± 4	23 ± 7	134 ± 10
		0.3 µg	15 ± 4	13 ± 3	34 ± 6	136 ± 15
		1 µg	21 ± 5	14 ± 2	25 ± 7	132 ± 11
		3 µg	25 ± 5	10 ± 2	27 ± 4	147 ± 10
		10 µg	15 ± 5	12 ± 4	27 ± 1	157 ± 30
		33 µg	17 ± 8^R	13 ± 2^R	35 ± 2	152 ± 10
		100 µg	2 ± 2^M R	1 ± 0^M R	^N R	95 ± 7^M R
		333 µg	0 ± 0^R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R
	NaN3	10 µg	2160 ± 26			2071 ± 121
	4-NOPD	10 µg			266 ± 17
	4-NOPD	50 µg		78 ± 1
	MMS	3.0 µL

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 4-NOPD MMS	sodium azide 4-nitro-o-phenylene-diamine methyl methane sulfonate	M R N	Manual count Reduced background growth Analysis not possible

    Summary of Results Experiment II

Study Name: 1250201	Study Code: Harlan - CCR 1250201
Experiment: 1250201 HV2 Pre	Date Plated: 02/07/2009 / 14/07/2009*
Assay Conditions:	Date Counted: 08/07/2009 / 20/07/2009*

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		13 ± 2	16 ± 1	34 ± 8	106 ± 5	60 ± 11
	Untreated		14 ± 1	14 ± 3	38 ± 10	142 ± 18	50 ± 2
	Chloranil	0.1 µg	16 ± 4	12 ± 5	32 ± 8	128 ± 3	62 ± 15
		0.3 µg	14 ± 1	11 ± 2	28 ± 1	113 ± 16	58 ± 14
		1 µg	15 ± 2	13 ± 1	33 ± 6	119 ± 10	62 ± 5
		3 µg	16 ± 4	10 ± 2	30 ± 3	120 ± 8	54 ± 5
		10 µg	14 ± 1	11 ± 2	24 ± 4	111 ± 13	63 ± 1
		33 µg	4 ± 3^R	3 ± 2^R	6 ± 2^M R	131 ± 17	58 ± 11
		100 µg	6 ± 1^M R	1 ± 1^M R	1 ± 2^M R	81 ± 6^R	49 ± 7
		333 µg	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R	37 ± 4^M R
	NaN3	10 µg	1770 ± 60			1756 ± 71
	4-NOPD	10 µg			366 ± 24
	4-NOPD	50 µg		94 ± 4
	MMS	3.0 µL					577 ± 12
With Activation	DMSO		18 ± 3	15 ± 2	41 ± 3*	129 ± 9	69 ± 3
	Untreated		20 ± 3	16 ± 1	41 ± 7*	148 ± 8	62 ± 6
	Chloranil	1 µg	18 ± 3	17 ± 3	39 ± 7*	134 ± 24	69 ± 8
		3 µg	20 ± 4	16 ± 5	37 ± 3*	133 ± 19	63 ± 7
		10 µg	20 ± 6	16 ± 1	35 ± 1*	131 ± 9	73 ± 7
		33 µg	12 ± 3	16 ± 3	35 ± 9*	151 ± 6	62 ± 4
		100 µg	13 ± 3	16 ± 3	31 ± 3*	133 ± 8	64 ± 8
		333 µg	14 ± 1	12 ± 2	29 ± 9*	140 ± 6	54 ± 15
		1000 µg	3 ± 2^M R	3 ± 1^M R	5 ± 2^M R*	76 ± 3^R	54 ± 9
		2500 µg	0 ± 0^M R	0 ± 0^M R	0 ± 0^M R*	0 ± 0^M R	17 ± 5^M R
	2-AA	2.5 µg	253 ± 30	161 ± 5	1294 ± 136*	1808 ± 17
	2-AA	10.0 µg					338 ± 8

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	R M	Reduced background growth Manual count

* repeat experiment

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without S9 mix

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Chloranil is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Chloranil to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations (µg/plate):

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000

Pre-Experiment/Experiment IA: 0.1; 0.3; 1; 3; 10; 33; 100; and 333

Experiment II without S9mix: 0.06; 0.21;0.63; 2.08; 6.25; 20.8; 62.5; and 208.3

Experiment II with S9mix: 0.63; 2.1; 6.25; 20.8; 62.5; 208.3; 625; and 2500

Reduced background growth was observed with and without metabolic activation at the higher concentrations in all strains used.

Distinct toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Chloranil at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.