1,2-Benzenediol, 3,5-dialkyl-, 1,2-dicarboxylate

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

Study period:

12 Oct - 25 Nov 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - 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

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

his operon (S. thyphimurium)

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA97a, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix) (Trinova Biochem, Gießen, Germany), prepared from the livers of male Sprague Dawley rats intraperitoneally treated with 500 mg/kg bw Aroclor 1254

Test concentrations with justification for top dose:

First experiment: 50, 150, 500, 1500 and 5001 µg/plate with and without metabolic activation
Second experiment: 313, 626, 1251, 2501 and 5001 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: DMSO (for the test substance and the positive control substances 4-nitro-1,2-phenylene diamine, 2-amino-anthracene and benzo-a-pyrene) or water (for the positive control substance sodium azide); 0.1 mL for the plate-incorporation and the preincubation treatment
- Justification for choice of solvent/vehicle: DMSO was chosen as vehicle, since the test substance was completely soluble, and this solvent did not have any effect on the viability of bacteria or the number of spontaneous revertants.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine (20 µg/plate, -S9, TA 97a, TA98 and TA 102); sodium azide (1 µg/plate, -S9, TA100 and TA 1535); 2-amino-anthracene (1 µg/plate, +S9, TA 97a, TA100, TA 102 and TA 1535); benzo-a-pyrene (20 µg/plate, +S9, TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION 1: in agar (plate incorporation) for first experiment

DURATION
- Exposure duration: 48 h

METHOD OF APPLICATION 2: preincubation method for the second experiment

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: quadruplicate each in both experiments

DETERMINATION OF CYTOTOXICITY
- Method: determination of titre (cytotoxicity test) and observation of background lawn (plate incorporation and preincubation experiment)

Evaluation criteria:

The test substance was considered to have mutagenic potential, if a significant, reproducible increase in revertant colonies per plate (increase factor ≥ 2) in at least one of the test strains was observed. A concentration-dependent increase over a range of test concentrations was considered to be a sign of mutagenic activity.

Statistics:

Mean values and standard deviations of revertant colonies from each test group were calculated.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA97a, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean numbers of spontaneous revertants in the solvent/vehicle controls of the strains used were all within the normal historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In a preliminary study using the plate incorporation method, each strain was incubated with 1500 and 5001 µg/plate of the test substance. Two replicates for each concentration were performed both in the presence or absence of metabolic activation. For the determination of toxicity, the titre (colonies/plate) was determined in control and treated cultures. In control culture, the titre should give a number of at least 1E+9 cells/mL, correlating to 100 colonies/plate after dilution. A substance is considered non-toxic, if the quotient control titre/treatment titre is below 2. This criterion was fulfilled after treatment with 1500 and 5001 µg/plate of the test substance. In the main study, no signs of toxicity were observed in any strain and at any concentration tested in both experiments.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation)

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(Average of 4 plates ± standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA102	TA1535	TA 97a	TA 98
–	water	105 ± 5	151 ± 24	22 ± 4	97 ± 3	11 ± 3
–	DMSO	88 ± 5	171 ± 9	16 ± 1	108 ± 3	10 ± 2
–	50	79 ± 10	146 ± 9	15 ± 2	108 ± 3	13 ± 1
–	150	81 ± 9	147 ± 17	13 ± 3	110 ± 8	11 ± 1
–	500	96 ± 2	142 ± 9	14 ± 1	132 ± 9	17 ± 3
	1500	90 ± 8	148 ± 9	14 ± 3	104 ± 14	10 ± 2
–	5001	90 ± 10	145 ± 6	14 ± 2	106 ± 9	14 ± 4
Positive controls, -S9	Name	NaN3	4NOPD	NaN3	4NOPD	4NOPD
	Concentrations (μg/plate)	1	20	1	20	20
	Mean No. of colonies/plate (average of 4 ± SD)	495 ± 60	604 ± 99	201 ± 18	456 ± 18	240 ± 19
+	water	128 ± 19	176 ± 17	15 ± 2	108 ± 8	13 ± 6
+	DMSO	112 ± 9	171 ± 4	14 ± 2	115 ± 2	14 ± 3
+	50	86 ± 11	154 ± 5	14 ± 3	102 ± 8	12 ± 2
+	150	80 ± 16	150 ± 8	14 ± 3	101 ± 3	13 ± 2
+	500	91 ± 12	146 ± 8	20 ± 2	105 ± 6	10 ± 3
+	1500	91 ± 15	154 ± 34	16 ± 3	108 ± 5	10 ± 2
+	5001	92 ± 5	149 ± 9	16 ± 4	92 ± 4	14 ± 1
Positive controls, +S9	Name	AAN	AAN	AAN	AAN	BP
	Concentrations (μg/plate)	1	1	1	1	20
	Mean No. of colonies/plate (average of 4 ± SD)	446 ± 63	594 ± 88	201 ± 11	499 ± 22	202 ± 13

Table 2. Test results of experiment 2 (preincubation)

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(Average of 4 plates ± standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA102	TA1535	TA 97a	TA 98
–	water	114 ± 23	162 ± 7	23 ± 6	115 ± 5	17 ± 4
–	DMSO	124 ± 4	153 ± 12	21 ± 3	113 ± 4	10 ± 3
–	313	115 ± 21	154 ± 4	22 ± 2	115 ± 1	11 ± 1
–	626	126 ± 16	148 ± 3	18 ± 3	113 ± 10	10 ± 3
–	1251	123 ± 11	141 ± 15	20 ± 3	109 ± 6	11 ± 3
–	2501	117 ± 8	160 ± 13	19 ± 4	114 ± 1	13 ± 2
–	5001	130 ± 21	150 ± 7	20 ± 1	118 ± 6	10 ± 3
Positive controls, –S9	Name	NaN3	4NOPD	NaN3	4NOPD	4NOPD
	Concentrations (μg/plate)	1	20	1	20	20
	Mean No. of colonies/plate (average of 4 ± SD)	487 ± 35	657 ± 40	122 ± 8	488 ± 56	192 ± 30
+	water	143 ± 11	141 ± 17	17 ± 4	117 ± 5	17 ± 3
+	DMSO	111 ± 15	165 ± 6	22 ± 2	115 ± 12	15 ± 3
+	313	127 ± 23	148 ± 9	19 ± 4	116 ± 4	14 ± 1
+	626	147 ± 8	144 ± 4	14 ± 3	106 ± 4	10 ± 2
+	1251	126 ± 9	143 ± 2	17 ± 2	116 ± 5	11 ± 1
+	2501	134 ± 25	154 ± 13	17 ± 3	118 ± 6	11 ± 3
+	5001	130 ± 7	152 ± 2	15 ± 2	116 ± 6	12 ± 2
Positive controls, +S9	Name	AAN	AAN	AAN	AAN	BP
	Concentrations (μg/plate)	1	1	1	1	20
	Mean No. of colonies/plate (average of 4 ± SD)	618 ± 164	516 ± 60	131 ± 15	460 ± 24	236 ± 26

NaN3 = sodium azide

4NOPD = 4-nitro-1,2-phenylene diamine

AAN = 2-aminoanthracene

BP = benzo-a-pyrene

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative