α,2-dichlorotoluene

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:

September 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1983

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no follow-up experiments were done for confirmation of negative results

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

α,2-dichlorotoluene

EC Number:

210-258-8

EC Name:

α,2-dichlorotoluene

Cas Number:

611-19-8

Molecular formula:

C7H6Cl2

IUPAC Name:

1-chloro-2-(chloromethyl)benzene

Details on test material:

- Source: HOECHST AG

Method

Target gene:

In the Salmonella typhimurium strains (TA 1535, TA 100, TA 1537, TA 1538, TA 98) the amino acid histidine locus is the target gene.
In the Escherichia coli strain (WP2 uvrA) the amino acid tryptophan locus is the target gene.

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538, Escherichia coli WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S-9 mix from Arochlor 1254 induced rats

Test concentrations with justification for top dose:

0.8, 4, 20, 100, 500, 1,500 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: solubility of test item

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

2-aminoanthracene (-S9: 0.5 µg/plate; TA98, TA100, TA1538)

Positive controls:

yes

Remarks:

TA98, TA100, TA1538

Positive control substance:

other: -S9: methylhydrazone derivative (5 µg/plate), +S9: 2-aminoanthracene (0.5 µg/plate)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

2-aminoanthracene (-S9: 1 µg/plate (TA1535, TA 1537), 10 µg/plate (WP2 uvr A))

Positive controls:

yes

Remarks:

TA 1557, TA 1535, WP2 uvr A

Positive control substance:

other: -S9: streptocotocine (5 µg/plate (TA 1535)), 9-aminoacridine (100 µg/plate (TA 1537)), N-ethyl-N-nitro-N-nitrosoguanidine (2 µg/plate, WP2 uvr A); +S9: 2-amioanthracene (1 µg/plate (TA 1535, TA1537), 10 µg/plate (WP2 uvr A))

Details on test system and experimental conditions:

METHOD OF APPLICATION: Overlay (soft) agar (plate incorporation method)

DURATION
- Preincubation period: No preincubation was performed. After mixing of all components, soft agar was distributed directly on the plates
- Exposure duration/ incubation time : Plates with microorganisms / test item situated in the Topagar and Controls were incubated upside down for 48 hours at 37 °C.

SELECTION AGENT (mutation assays): The selection is done by using a minimal agar that contains no histidine for the Salmonella typhimurium strains and a minimal agar that contains no tryptophane for the Escherichia coli strains.

NUMBER OF REPLICATIONS: 4

Evaluation criteria:

The test substance is considered positive in this assay if the following criteria is met:
- Doubling of the revertant colonies compared to controls

Results and discussion

Test results

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Without metabolic activation: >= 500 µg/plate, with metabolic activation: >= 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

see below

Remarks on result:

other: other: Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538, Escherichia coli WP2 uvrA

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The number of revertant colonies did not increase compare
with the solvent control in S. typhimurium , and E. coli WP2 uvrA,

both with and without metabolic activation.

Table 1
========================================================
With        Test         Mean number of
or          substance    revertant colonies/plate
without     dose         --------------------------------
S9mix       (ug/plate)   Base-pair        Frameshift 
                         substitution     type
                         type
                         --------------------------------
                         TA   TA    WP2   TA   TA   TA
                         100  1535  urvA  98   1537 1538
---------------------------------------------------------
            0            154  12    30    25   12   10
            0.8          169  15    26    22   12   12
without     4            159  13    26    22   14   11
(-)         20           152  12    29    19   13   8
            100          186  13    28    14   0.5  9
            500          *    *     13    *    *    *
            1,500        **   **    *     **   **   **
---------------------------------------------------------
            0            163  16    49    30   15   19
            0.8          158  11    52    29   15   16
with        4            151  15    44    32   15   19
(+)         20           175  14    44    32   14   16
            100          188  11    44    23   13   15
            500          123   6    27    18    6   12
          1,500          *    **    19     7    *    *
---------------------------------------------------------
[Positive control without S9mix]
Chemical                 MD   SC   ENNG   MD   9AA  MD
Dose(ug/plate)           5    5     2      5   100   5
Mean number of
Colonies/plate          3405 >5000  633  3170  >5000 3035
---------------------------------------------------------
[Positive control with S9mix]
Chemical                 2AA  2AA   2AA   2AA  2AA  2AA
Dose(ug/plate)           0.5  1     10    0.5   1   0.5
Mean number of
Colonies/plate           715  156   2330  750  136  740
========================================================
MD: Methylhydrazone Derivative, SC: Streptocotocine, 
ENNG: N-Ethyl-N-nitro-N-nitrosoguanidine, 
9AA: 9-Aminoacridine, 2AA: 2-Aminoanthracene
*: no colony growth, **: no bacterial growth

These results have led to the conclusion that OCBC was not
mutagenic under the conditions of this study.

Applicant's summary and conclusion

Executive summary:

In a reverse gene mutation assay in bacteria Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and Escherichia coli strain WP2 uvrA were exposed to o-Chlorobenzylchloride at concentrations of 0, 0.8, 4, 20, 100, 500, 1,500 µg/plate in the presence and absence of mammalian metabolic activation.No preincubation was performed. There was no evidence of induced mutant colonies over background up to a concentration of 1.5 mg o-Chlorobenzylchloride/plate. The positive controls induced the appropriate responses in the corresponding strains. In conclusion, o-Chlorobenzylchloride did not show any mutagenic activity in any tester strains regardless of metabolic activation.