N,N'-(2-chloro-5-methyl-1,4-phenylene)bis[3-oxobutyramide]

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:

1996-11-22 to 1997-01-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study documented and performed according to GLP standards and in compliance with OECD Guideline 471.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-(2-chloro-5-methyl-1,4-phenylene)bis[3-oxobutyramide]

EC Number:

255-235-3

EC Name:

N,N'-(2-chloro-5-methyl-1,4-phenylene)bis[3-oxobutyramide]

Cas Number:

41131-65-1

Molecular formula:

C15H17ClN2O4

IUPAC Name:

N-[5-chloro-2-methyl-4-(3-oxobutanamido)phenyl]-3-oxobutanamide

Details on test material:

Identity: P0045
Batch No.: 1130
Storage: RT
Purity: 99.4%
Expiry date: May 18, 1998
Appearance: Brown powder

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test (Toxicity test): 5000, 1580, 500, 158 and 50 microg/plate.
Mutation tests: 5000, 2500, 1250, 625, 313 microg/plate.

Vehicle / solvent:

Dimethylsulphoxide

Controls

Untreated negative controls:

yes

Remarks:

dimethylsulphoxide

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide, 2-Aminoanthracene, 9-Aminoacridine, 2-Nitrofluorene, Cumene hydroperoxide, Sterile distilled water

Details on test system and experimental conditions:

For use in tests sub-cultures were grown in Nutrient Broth No. 2 (Oxoid) at 37°C for 10 hours. This culture provided approximately 2 x 10x9
organisms per ml which was assessed photometrically.

Preparation of liver homogenate S—9 fraction:
Species: Rat.
Strain: Sprague—Dawley derived.
Source: Bantin and Kingman Ltd., The Field
Station, Grimston, Aldbrough, Hull,
North Humberside, HU11 4QE.
Age range: 7 — 8 weeks.
Weight range: <300 g.
Diet: Labsure Rodent Diet LAD 1.

Stimulation of rat liver enzymes:
Mixed—function oxidase systems in the rat liver were stimulated following a single i/p injection of Aroclor 1254 (diluted in Arachis oil to 200 mg/ml) at a dosage of 500 mg/kg. On the fifth day of induction, following an overnight starvation, the rats were killed and their livers aseptically removed.

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity
of a test material is assessed by applying different criteria.
Revertant colonies were counted using a Biotran Automatic Colony Counter. Any toxic effects of the test substance can be detected by a substantial
reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

Statistics:

Not applicable.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity test P0005 was not toxic towards the tester strains.
Therefore, 5000 microg/plate was chosen as the top dose level in the mutation tests.
The mean number of revertant colonies, together with the individual plate counts for P0005 obtained in the first and second mutation test with the
tester strains are shown in the Table below.
No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with P0005 at any dose level,
either in the presence or absence of S-9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Strain	Dose level (microg/plate)	Metabolic activation -/+	Mean revertant colony counts
TA 1535	5000 2500 1250 625 313 0 Untreated	- / + - / + - / + - / + - / + - / + - / +	20 / 17 22 / 15 20 / 20 21 / 18 21 / 18 20 / 19 20 / 17
TA 1537	5000 2500 1250 625 313 0 Untreated	- / + - / + - / + - / + - / + - / + - / +	18 / 23 20 / 26 20 / 24 20 / 24 20 / 24 19 / 26 19 / 23
TA 102	5000 2500 1250 625 313 0 Untreated	- / + - / + - / + - / + - / + - / + - / +	298 / 404 314 / 441 318 / 448 311 / 449 316 / 448 312 / 454 310 / 444
TA 98	5000 2500 1250 625 313 0 Untreated	- / + - / + - / + - / + - / + - / + - / +	27 / 35 28 / 37 28 / 34 29 / 36 28 / 36 28 / 37 27 / 36
TA 100	5000 2500 1250 625 313 0 Untreated	- / + - / + - / + - / + - / + - / + - / +	148 / 160 149 / 158 151 / 164 150 / 159 149 / 161 147 / 164 153 / 168

- Absence

+ Presence

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

lt is concluded that P0045 does not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.

Executive summary:

The genetic toxicity study in vivo (Ames) was performed in year 1996/1997 according to GLP standards and in compliance with OECD Guideline 471.

In the toxicity test, the test substance was assayed at a maximum dose-level of 5000 microgram/plate and four lower dose-levels spaced at approximately half-long intervals. No toxicity was observed in any tester strain even at the highest dose-level tested. The same maximum dose-level was selected for the principal assay.

The test substance did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism.

Therefore, it is concluded that P0045 is not mutagenic in this bacterial system.