Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

α,α’-Dichloro-p-xylene was shown to be mutagenic under the test conditions used in the study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 December 2017 to 14 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guidelines for testing of Chemicals, Section 4, No. 471 “Bacterial Reverse Mutation Test”, 21 July 1997
Deviations:
no
Qualifier:
according to
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
Deviations:
no
Qualifier:
according to
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
EPA Health Effects Test Guidelines, OPPTS 870.5100 “Escherichia coli WP2 and WP2 uvrA Reverse Mutation Assays”, EPA 712-C-96-247, June 1996 (Public Draft)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
No further details specified in the study report.
Target gene:
histidine (his) and tryptophan (trp)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Not specified
Metabolic activation:
with and without
Metabolic activation system:
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 Citoxlab Hungary Ltd. according to Ames et al. and Maron and Ames. 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 a final volume of 1000 mL.

Induction of Liver Enzymes
Male Wistar rats (373-446 g, animals were 9 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 was 03 July 2017.

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 1-3 mL portions, frozen quickly and stored at -80 ± 10ºC.
The date of preparation of S9 fraction for this study was 06 July 2017 (Citoxlab code: E12660).
The sterility of the preparation was confirmed. The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Citoxlab Hungary Ltd. The mean protein concentration of the S9 fraction used was determined to be 28.6 g/L.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.
Test concentrations with justification for top dose:
Preliminary Concentration Range Finding Test (Informatory Toxicity Test): 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item.
Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test): Examined concentrations in the Initial Mutation and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate (with the exception of Salmonella typhimurium TA1537 with and without metabolic activation, where the concentrations were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate in the Confirmatory Mutation Test).
Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test).
In the Initial Mutation Test and Confirmatory Mutation Test same concentrations were used, however in the Confirmatory Mutation Test in case of Salmonella typhimurium TA1537 with and without metabolic activation different concentrations were used.
Vehicle / solvent:
The appropriate vehicle and the behaviour of the test item formulations with the solution of top agar and phosphate buffer were determined in a preliminary compatibility test. All dilutions in the main tests of test item were made in the testing laboratory using DMSO. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent.

Dimethyl sulfoxide (DMSO):
Supplier: VWR
Batch No.: 16F304002
Expiry date: 31 May 2021

Distilled water:
Manufacturer: Hungaro-Gal Kft.
Batch No.: 8130917
Expiry date: 04 March 2018
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine (NPD); 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
DESCRIPTION OF THE TEST PROCEDURE
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (in case of Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation the Plate Incorporation Method was used; in case of Salmonella typhimurium TA1535, TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation the Pre-Incubation Method was used).

Preliminary Compatibility Test
The solubility of the test item* was examined using Dimethyl sulfoxide (DMSO). The test item** was soluble at 100 mg/mL at 100 mg/mL concentration (after approximately 2 minutes vortex) using DMSO as vehicle. DMSO was selected as vehicle (solvent) for the study. The test item was formulated in the selected vehicle (solvent). 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.
*Note: Distilled water was not a suitable vehicle based on the trial formulation of the 17/241-001P study.
**Note: The test item was powdered.

Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the available information and the solubility and compatibility test, 100 mg/mL stock solution 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, 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 solution was prepared in DMSO, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in the Initial Mutation and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate (with the exception of Salmonella typhimurium TA1537 with and without metabolic activation, where the concentrations were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate in the Confirmatory Mutation Test).

Control Groups Used in the Tests
Strain-specific positive and negative (vehicle/solvent) controls, both with and without metabolic activation were included in each test. In addition, untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.

Procedure for Exposure in the Initial Mutation Test and in the Confirmatory Mutation Test
A standard plate incorporation procedure was performed as an Initial Mutation Test and as a Confirmatory Mutation Test in the case of Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation. Bacteria (cultured in Nutrient Broth No.2) 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. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) 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 solution (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 (solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.

Procedure for Exposure in the Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test in the case of Salmonella typhimurium, TA1535 and TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation, since in the Initial Mutation Test positive effect was observed in the case of Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation, the plate incorporation method was used.
For the pre-incubation method, bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, 50 μL of test item formulations or its vehicle (or positive reference controls or their solvent), 100 μL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test item. These tubes (3 tubes per control and 3 tubes for each 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 were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hour.
Rationale for test conditions:
The experimental methods were conducted according to the methods described Ames et al. and Maron and Ames, Kier et al., Venitt and Parry, OECD Guideline No. 471, 1997, Commission Regulation (EC) No. 440/2008, 2008, EPA Guidelines, OPPTS 870.5100, 1998, 1996 and according to the relevant SOPs of Citoxlab Hungary Ltd.
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.

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than two times higher than the reversion rate of the negative (solvent) control.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
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.
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:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
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:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
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:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY CONCENTRATION RANGE FINDING TES (INFORMATORY TOXICITY TEST)
Preliminary Concentration 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.
Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test.
In the Preliminary Concentration Range Finding Test, a dose–related pattern was observed in the number of revertant colonies in Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation. The calculated mutation factor values were over the biologically relevant threshold values of 2 at several concentrations and dose dependence was also observed.
Slight precipitate was observed in the Preliminary Concentration Range Finding Test in Salmonella typhimurium TA98 and TA100 strains without metabolic activation at 5000 μg/plate.
Reduced/slightly reduced background lawn was observed in the Preliminary Concentration Range Finding Test in Salmonella typhimurium TA98 with metabolic activation at 5000 μg/plate. Furthermore, the same effect was observed in Salmonella typhimurium TA98 strain without metabolic activation and Salmonella typhimurium TA100 strain with and without metabolic activation metabolic activation at 5000, 2500 and 1000 μg/plate.

INITIAL AND CONFIRMATORY MUTATION TEST
In the Initial Mutation Test, the plate incorporation method was used, in the Confirmatory Mutation Test the plate incorporation method or the pre-incubation method were 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.
Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation and Confirmatory Mutation Tests were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate (with the exception of Salmonella typhimurium TA1537 with and without metabolic activation, where the concentrations were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate in the Confirmatory Mutation Test).
In the Initial Mutation Test using the plate incorporation method, a clear positive effect of the test item was obtained in Salmonella typhimurium TA98 and TA100 bacterial strains with and without metabolic activation as the calculated mutation factor values were over the biologically relevant threshold value of 2 at several concentrations and dose dependence was also observed.
The observed positive effect was confirmed in the Confirmatory Mutation Test using the plate incorporation method in Salmonella typhimurium TA98 and TA100 bacterial strains with and without metabolic activation. This result demonstrated the positive effect was reproducible and showed dose dependent pattern.
Slight precipitate was observed in the Initial Mutation Test in Salmonella typhimurium TA1535 with and without metabolic activation at 5000 μg/plate. Precipitate/slight precipitate was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA strains with and without metabolic activation at 5000 μg/plate. Furthermore, in Salmonella typhimurium TA98 without metabolic activation at 5000 μg/plate and with metabolic activation at 5000 and 1581 μg/plate. Slight precipitate was observed in Salmonella typhimurium TA100 strain without metabolic activation at 5000 and 1581 μg/plate. The precipitate did not affect the colony counting.
In the Initial Mutation reduced/slightly reduced/ absent background lawn and/or lower numbers of revertant colonies* was observed Test in Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA strains with and without metabolic activation at 5000 and 1581 μg/plate, in Salmonella typhimurium TA100 and TA1537 strains with and without metabolic activation at 5000, 1581 and 500 μg/plate.
Furthermore, reduced background lawn was observed in Salmonella typhimurium TA98 without metabolic activation at 5000 μg/plate.
In the Confirmatory Mutation Test reduced/slightly reduced background lawn and/or lower numbers of revertant colonies* was observed in Salmonella typhimurium TA100 with and without metabolic activation at 5000, 1581 and 500 μg/plate, and in Salmonella typhimurium TA1535 without metabolic activation at 5000, 1581, 500, 158.1 μg/plate and with metabolic activation at 5000 and 1581 μg/plate. Furthermore, in Salmonella typhimurium TA1537 without metabolic activation at 5000 and 1581 μg/plate. Lower numbers of revertant colonies* was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA98 and Escherichia coli WP2 uvrA strains with and without metabolic activation and in Salmonella typhimurium TA537 with and without metabolic activation at 1581 μg/plate. The reduced counts at higher concentrations did not compromise the study; the clear positive results with a dose response of concentrations with acceptable viability is evidence of a true positive finding.
*Note: The lower numbers of revertant colonies (MF<0.50) are considered to indicate an inhibitory effect.

Taking into account the results at all concentrations under all conditions for each bacterial strain, the results for Salmonella typhimurium TA1535 with and without metabolic activation was negative in the Initial Mutation Test* and the results of this strain with metabolic activation was negative in the Confirmatory Mutation Test.
The results for Salmonella typhimurium TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation was clearly negative in the main studies.
The results for Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation using the plate incorporation method were clearly, reproducibly positive, therefore the overall conclusion of this study is clearly Positive.
*Note: In the Confirmatory Mutation Test cytotoxicity was seen in Salmonella typhimurium TA1535 bacterial strain without metabolic activation, with less than 5 concentrations for evaluation, but since the test item is already classified as positive, there is no requirement for further testing.

Summary Tables of the Results

 

Summary Table of Preliminary Concentration Range Finding Test

Concentrations (µg/plate)

Mean values of revertants /Mutation factor (MF)

Salmonella typhimuriumtester strains

TA 98

TA 100

-S9

+S9

-S9

+S9

Untreated control

Mean

18.3

17.0

83.3

108.7

MF

1.04

0.82

0.98

1.18

DMSO control

Mean

17.7

20.7

85.0

92.3

MF

1.00

1.00

1.00

1.00

Distilled water control

Mean

--

--

92.3

--

MF

--

--

1.09

--

5000

Mean

10.0

40.3

16.3

34.7

 

MF

0.57

1.95

0.19

0.38

2500

Mean

58.3

96.7

46.0

47.7

MF

3.30

4.68

0.54

0.52

1000

Mean

105.3

145.7

111.3

112.0

MF

5.96

7.05

1.31

1.21

316

Mean

101.7

119.7

240.0

275.0

MF

5.75

5.79

2.82

2.97

100

Mean

61.3

53.7

194.7

234.7

MF

3.47

2.60

2.29

2.54

31.6

Mean

37.7

38.7

168.0

170.7

MF

2.13

1.87

1.98

1.85

10

Mean

24.7

26.3

118.36

139.7

MF

1.40

1.257

1.39

1.51

NPD (4µg)

Mean

397.3

--

--

--

MF

22.49

--

--

--

2AA (2µg)

Mean

--

2402.7

--

2436.0

MF

--

116.26

--

26.38

SAZ (2µg)

Mean

--

--

1160.0

--

MF

--

--

12.56

--

 

Summary Table of the Initial Mutation Test

Concentrations (µg/plate)

Mean values of revertants /Mutation factor (MF)

Salmonella typhimuriumtest strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

17.7

23.3

91.7

98.3

14.3

10.7

11.3

11.3

45.7

44.7

MF

0.95

0.97

1.00

1.06

1.10

1.00

0.92

0.92

0.92

0.94

DMSO control

Mean

18.7

24.0

91.3

92.3

13.0

10.7

12.3

12.3

49.7

47.3

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

--

--

91.7

--

12.3

--

--

--

45.7

--

MF

--

--

1.00

--

0.95

--

--

--

0.92

--

5000

Mean

14.7

53.0

3.7

8.0

0.0

0.0

0.0

0.7

2.0

4.3

MF

0.79

2.21

0.04

0.09

0.00

0.00

0.00

0.05

0.04

0.09

1581

Mean

131.0

127.7

10.7

7.0

0.00

0.00

1.3

2.3

20.3

23.0

MF

7.02

5.32

0.12

0.08

0.00

0.00

0.11

0.19

0.41

0.49

500

Mean

155.0

187.7

155.3

200.0

11.3

12.0

8.0

11.0

47.7

69.3

MF

8.30

7.82

1.70

2.17

0.87

1.13

0.65

0.89

0.96

1.46

158.1

Mean

79.3

80.3

372.7

368.0

11.0

12.3

9.3

10.7

63.7

83.0

MF

4.25

3.35

4.08

3.99

0.85

1.16

0.76

0.86

1.28

1.75

50

Mean

44.7

46.3

314.7

205.3

10.7

11.7

10.3

8.3

54.7

78.7

MF

2.39

1.93

3.45

2.22

0.82

1.09

0.84

0.68

1.10

1.66

15.81

Mean

33.7

30.0

276.7

226.3

12.3

10.3

12.0

10.3

55.7

69.0

MF

1.80

1.25

3.03

2.45

0.95

0.97

0.97

0.84

1.12

1.46

5

Mean

36.7

35.0

213.0

209.3

12.3

10.0

9.0

10.7

53.0

54.7

MF

1.96

1.46

2.33

2.27

0.95

0.94

0.73

0.86

1.07

1.15

1.581

Mean

28.3

41.3

155.7

208.7

10.7

11.0

9.7

8.7

49.0

49.0

MF

1.52

1.72

1.70

2.26

0.82

1.03

0.78

0.70

0.99

1.04

NPD (4µg)

Mean

410.0

--

--

--

--

--

--

--

--

--

MF

21.96

--

--

--

--

--

--

--

--

--

2AA (2µg)

Mean

--

2433.3

--

2452.0

--

205.3

--

203.3

--

--

MF

--

101.39

--

26.56

--

19.25

--

16.49

--

--

2AA (50µg)

Mean

--

--

--

--

--

--

--

--

--

256.7

MF

--

--

--

--

--

--

--

--

--

5.42

SAZ (2µg)

Mean

--

--

1110.7

--

1100.0

--

--

--

--

--

MF

--

--

12.12

--

89.19

--

--

--

--

--

9AA (50µg)

Mean

--

--

--

--

--

--

409.3

--

--

--

MF

--

--

--

--

--

--

33.19

--

--

--

MMS (2µL)

Mean

--

--

--

--

--

--

--

--

1138.7

--

MF

--

--

--

--

--

--

--

--

24.93

--

 

Summary Table of the Confirmatory Mutation Test

Concentrations (µg/plate)

Mean values of revertants /Mutation factor (MF)

Salmonella typhimuriumtest strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

19.0

24.7

102.7

108.3

10.0

11.3

10.7

12.3

48.0

53.7

MF

0.95

1.16

0.94

0.94

0.86

1.00

1.07

0.93

0.97

0.99

DMSO control

Mean

20.0

21.3

109.7

115.3

11.7

11.3

10.0

13.3

49.7

54.0

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

--

--

102.0

--

13.7

--

--

--

50.3

--

MF

--

--

0.93

--

1.17

--

--

--

1.01

--

5000

Mean

0.0

0.0

0.0

0.0

0.0

0.0

--

--

3.7

9.0

MF

0.00

0.00

0.00

0.00

0.00

0.00

--

--

0.07

0.17

1581

Mean

103.7

68.0

0.0

0.0

3.0

0.7

0.3

0.0

35.3

27.0

MF

5.19

3.19

0.00

0.00

0.26

0.06

0.03

0.00

0.71

0.50

500

Mean

153.7

164.0

347.3

366.0

4.0

13.7

8.3

9.7

93.3

94.7

MF

7.68

7.69

3.17

3.17

0.34

1.21

0.83

0.73

1.88

1.75

158.1

Mean

82.7

70.3

690.7

604.0

9.0

15.3

7.3

6.7

67.7

85.3

MF

4.13

3.30

6.30

5.24

0.77

1.35

0.73

0.50

1.36

1.58

50

Mean

32.3

27.3

278.7

268.0

12.0

15.0

10.0

7.7

64.7

67.0

MF

1.62

1.28

2.54

2.32

1.03

1.32

1.00

0.58

1.30

1.24

15.81

Mean

19.3

22.7

156.7

149.0

6.7

10.7

9.3

7.7

63.0

55.0

MF

0.97

1.06

1.43

1.29

0.57

0.94

0.93

0.58

1.27

10.2

5

Mean

18.0

20.0

132.7

129.7

11.7

7.0

11.0

12.3

61.3

52.3

MF

0.90

0.94

1.21

1.12

1.00

0.62

1.10

0.93

1.23

0.97

1.581

Mean

14.7

19.7

121.0

126.7

7.7

16.3

8.3

9.3

46.7

54.7

MF

0.73

0.92

1.10

1.10

0.66

1.44

0.83

0.70

0.94

1.01

0.5

Mean

--

--

--

--

--

--

11.0

10.0

--

--

MF

--

--

--

--

--

--

1.10

0.75

--

--

NPD (4µg)

Mean

376.0

--

--

--

--

--

--

--

--

--

MF

18.80

--

--

--

--

--

--

--

--

--

2AA (2µg)

Mean

--

2389.3

--

2336.0

--

22.0

--

206.0

--

--

MF

--

112.00

--

20.25

--

19.41

--

15.45

--

--

2AA (50µg)

Mean

--

--

--

--

--

--

--

--

--

257.3

MF

--

--

--

--

--

--

--

--

--

4.77

SAZ (2µg)

Mean

--

--

1120.0

--

1205.3

--

--

--

--

--

MF

--

--

10.98

--

88.20

--

--

--

--

--

9AA (50µg)

Mean

--

--

--

--

--

--

426.7

--

--

--

MF

--

--

--

--

--

--

42.67

--

--

--

MMS (2µL)

Mean

--

--

--

--

--

--

--

--

1061.3

--

MF

--

--

--

--

--

--

--

--

21.09

--

 

Historical Control Data

(Period of 2011 – 2016)

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

22.7

103.6

11.8

7.2

33.4

29.7

111.7

11.5

8.9

39.1

St. dev.

5.8

21.4

5.1

3.3

9.7

6.8

19.6

3.9

3.8

9.9

Range

9-50

54-210

1-46

1-24

11-82

10-56

65-204

1-39

1-29

16-89

n

1371

1357

1365

1371

1374

1377

1365

1373

1380

1371

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

21.7

98.9

12.0

7.1

32.3

28.7

109.5

11.3

8.7

38.1

St. dev.

5.7

20.7

5.0

3.3

9.6

7.0

20.7

3.8

3.7

9.7

Range

6-55

40-217

1-43

1-25

7-81

11-67

53-229

2-33

1-29

9-85

n

1482

1473

1479

1485

1482

1487

1476

1487

1491

1482

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

23.5

103.2

11.9

7.8

34.5

30.8

112.2

11.3

9.3

40.3

St. dev.

6.0

22.4

4.9

3.4

9.8

7.1

21.8

3.7

3.7

10.0

Range

11-45

45-215

2-47

2-24

12-84

10-53

64-222

3-39

1-24

13-91

n

367

1359

1365

270

1392

267

1371

1380

267

1383

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

20.4

89.9

11.2

6.9

34.7

28.1

100.3

11.0

8.0

38.0

St. dev.

5.6

17.8

4.7

3.1

12.3

7.0

19.2

3.6

3.1

10.2

Range

8-38

54-152

1-34

1-19

16-99

13-49

60-156

3-21

1-23

17-76

n

216

216

216

216

207

216

216

216

213

207

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

22.6

98.1

12.1

7.4

35.0

29.1

108.1

11.1

8.6

40.5

St. dev.

5.1

15.4

5.8

2.9

9.3

6.7

14.2

3.4

3.3

9.0

Range

11-39

62-160

4-49

1-17

17-62

15-52

66-177

4-22

1-19

17-69

n

278

279

279

279

276

279

279

282

279

279

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

357.2

1229.3

1169.8

454.1

1034.3

2410.2

2429.6

235.1

221.3

257.4

St. dev.

113.8

207.5

204.2

169.7

141.7

317.7

291.6

135.9

56.2

113.4

Range

152-2336

536-2120

208-2440

149-2104

488-1708

312-4918

1192-5240

101-2216

117-838

125-2512

n

1371

1359

1365

1371

1377

1378

1365

1377

1380

1371

TA98:Salmonella typhimuriumTA98; TA100:Salmonella typhimuriumTA100; TA1535:Salmonella typhimuriumTA1535; TA1557:Salmonella typhimuriumTA1537; E. coli: Escherichia coli WP2uvrA; n: number of cases

Conclusions:
The reported data of the mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item α,α’-Dichloro-p-xylene (Batch Number: EWP6O) was shown to be mutagenic under the test conditions used in this study.
Executive summary:

The test item α,α’-Dichloro-p-xylene 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 Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (in case of Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation the Plate Incorporation Method was used; in case of Salmonella typhimurium TA1535, TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation the Pre-Incubation Method was used).

 

Based on the results of a solubility test, the test item was formulated in DMSO. Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Preliminary Concentration Range Finding Test, the test item concentrations in the Initial Mutation Test (5 strains) and in the Confirmatory Mutation Test (5 strains) were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate (with the exception of Salmonella typhimurium TA1537 with and without metabolic activation, where the concentrations were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 µg/plate in the Confirmatory Mutation Test).

 

In the Preliminary Concentration Range Finding Test, in the Initial Mutation Test and Confirmatory Mutation Test in case of Salmonella typhimurium TA98 and TA100 bacterial strains with and without metabolic activation, a clear, reproducible positive effect was obtained using the plate incorporation method.

 

Slight precipitate was observed in the Preliminary Concentration Range Finding Test in Salmonella typhimurium TA98 and TA100 strains without metabolic activation at 5000 µg/plate.

Slight precipitate was observed in the Initial Mutation Test in Salmonella typhimurium TA1535 with and without metabolic activation at 5000 µg/plate.

Precipitate/slight precipitate was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA strains with and without metabolic activation at 5000 µg/plate. Furthermore, in Salmonella typhimurium TA98 without metabolic activation at 5000 µg/plate and with metabolic activation at 5000 and 1581 µg/plate. Slight precipitate was observed in Salmonella typhimurium TA100 strain without metabolic activation at 5000 and 1581 µg/plate.

The precipitate did not affect the colony counting.

 

Reduced/slightly reduced background lawn was observed in the Preliminary Concentration Range Finding Test in Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation at higher concentrations.

 

Reduced/slightly reduced/ absent background lawn and/or lower numbers of revertant colonies* was observed in the Initial Mutation Test in Salmonella typhimurium TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation at higher concentrations, furthermore reduced background lawn was observed in Salmonella typhimurium TA98 without metabolic activation at 5000 µg/plate.

Reduced/slightly reduced background lawn and/or lower numbers of revertant colonies* was observed in the Confirmatory Mutation Test in all examined strains with and without metabolic activation at higher concentrations.

 

*Note: The lower numbers of revertant colonies (MF<0.50) are considered to indicate an inhibitory effect.

 

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 analysable** concentrations were presented in all strains of the main tests. The tests were considered to be valid.

 

**Note: In the Confirmatory Mutation Test excessive cytotoxicity was observed in Salmonella typhimurium TA1535 bacterial strain without metabolic activation using the pre-incubation method. However, in the Initial Mutation Test and in the Confirmatory Mutation Test clearly positive results were observed in Salmonella typhimurium TA98 and Salmonella typhimurium TA100 strains with and without metabolic activation.

 

Since the overall conclusion of this study is clearly Positive, no additional Complementary Confirmatory Test is required because the results are fully sufficient to make a regulatory Positive conclusion. Cytotoxicity in Salmonella typhimurium TA1535 bacterial strain was seen without metabolic activation, with less than 5 concentrations for evaluation, but since the test item is already classified as positive, there is no requirement for further testing.

 

Although one of the validity criteria was not fulfilled in case of Salmonella typhimurium TA1535 bacterial strain without metabolic activation (due to cytotoxicity no five analyzable doses were observed), but due to scientific reasons further experiment is not considered to be required because Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation showed clearly positive results. Therefore, this fact had no impact on the study on the results or integrity of the study and this change is not considered to be a deviation.

 

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

In conclusion, the test item α,α’-Dichloro-p-xylene (Batch Number: EWP6O) was shown to be mutagenic under the test conditions used in this study.

Endpoint:
genetic toxicity in vitro, other
Type of information:
(Q)SAR
Remarks:
Predicted using DEREK Nexus
Adequacy of study:
supporting study
Study period:
25 January 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
QSAR prediction
Principles of method if other than guideline:
Data is QSAR data.
Type of assay:
other: QSAR assessment using DEREK Nexus
Key result
Remarks on result:
mutagenic potential (based on QSAR/QSPR prediction)
Remarks:
Predicted result using DEREK Nexus

Due to an alkylating agent Di-chloro xylene triggered PLAUSIBLE alerts for chromosome damage and mutagenicity in vitro. Alkyl halides are electrophilic species that are capable of directly alkylating DNA. Positive results in the in vitro chromosome aberration test have been reported for several compounds.

However, a single negative result has been reported in the CCRIS database§§. This study was conducted in Salmonella typhimurium strain TA98 in the absence of metabolic activation. Derek Nexus notes that such compounds are positive “notably in Salmonella typhimurium strains TA100 and TA1535”. Given the absence of additional strains and lack of metabolic activation the positive alert triggered cannot be dismissed. Overall, the evidence suggests that Di-chloro xylene has the potential to be an in vitro genotoxin.

Conclusions:
Positive for genotoxicity following in-silico assessment using DEREK Nexus.
Executive summary:

The genotoxicity has been assessed by assessment of the structure for structural alerts compared to known alerts contained in the DEREK Nexus database. On the basis of the structure the substance is considered to be genotoxic.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

The test item α,α’-Dichloro-p-xylene 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 Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (in case of Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation the Plate Incorporation Method was used; in case of Salmonella typhimurium TA1535, TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation the Pre-Incubation Method was used).

 

Based on the results of a solubility test, the test item was formulated in DMSO. Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Preliminary Concentration Range Finding Test, the test item concentrations in the Initial Mutation Test (5 strains) and in the Confirmatory Mutation Test (5 strains) were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate (with the exception of Salmonella typhimurium TA1537 with and without metabolic activation, where the concentrations were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 µg/plate in the Confirmatory Mutation Test).

 

In the Preliminary Concentration Range Finding Test, in the Initial Mutation Test and Confirmatory Mutation Test in case of Salmonella typhimurium TA98 and TA100 bacterial strains with and without metabolic activation, a clear, reproducible positive effect was obtained using the plate incorporation method.

Slight precipitate was observed in the Preliminary Concentration Range Finding Test in Salmonella typhimurium TA98 and TA100 strains without metabolic activation at 5000 µg/plate.

Slight precipitate was observed in the Initial Mutation Test in Salmonella typhimurium TA1535 with and without metabolic activation at 5000 µg/plate.

Precipitate/slight precipitate was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA strains with and without metabolic activation at 5000 µg/plate. Furthermore, in Salmonella typhimurium TA98 without metabolic activation at 5000 µg/plate and with metabolic activation at 5000 and 1581 µg/plate. Slight precipitate was observed in Salmonella typhimurium TA100 strain without metabolic activation at 5000 and 1581 µg/plate.

The precipitate did not affect the colony counting.

 

Reduced/slightly reduced background lawn was observed in the Preliminary Concentration Range Finding Test in Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation at higher concentrations.

Reduced/slightly reduced/ absent background lawn and/or lower numbers of revertant colonies was observed in the Initial Mutation Test in Salmonella typhimurium TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation at higher concentrations, furthermore reduced background lawn was observed in Salmonella typhimurium TA98 without metabolic activation at 5000 µg/plate.

Reduced/slightly reduced background lawn and/or lower numbers of revertant colonies was observed in the Confirmatory Mutation Test in all examined strains with and without metabolic activation at higher concentrations.

 

The reported data of the mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item α,α’-Dichloro-p-xylene (Batch Number: EWP6O) was shown to be mutagenic under the test conditions used in this study.

Justification for classification or non-classification