Calcium bis[4-[[1-[[(2-chlorophenyl)amino]carbonyl]-2-oxopropyl]azo]-3-nitrobenzenesulphonate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Calcium bis[4-[[1-[[(2-chlorophenyl)amino]carbonyl]-2-oxopropyl]azo]-3-nitrobenzenesulphonate]

EC Number:

276-057-2

EC Name:

Calcium bis[4-[[1-[[(2-chlorophenyl)amino]carbonyl]-2-oxopropyl]azo]-3-nitrobenzenesulphonate]

Cas Number:

71832-85-4

Molecular formula:

C16H13ClN4O7S.1/2Ca

IUPAC Name:

calcium bis[4-({1-[(2-chlorophenyl)carbamoyl]-2-oxopropyl}diazenyl)-3-nitrobenzenesulfonate]

Test material form:

solid: particulate/powder

Method

Target gene:

his and trp

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from aroclor induced rats and syrien hamster

Test concentrations with justification for top dose:

Range finder and first experiment 2.4, 12, 60, 300, 1500 micro g per plate, with and without S9
second experiment 93.75, 187.5, 375, 750, 1500 micro g per plate, with and without S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

other: 2-aminoanthracene: one strain, for standard S9 mix, with S9, 5 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation), second experiment with pre-incubation 30 min at 30°C

DURATION
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: in triplicate

OTHER: The S-9 used for the 'standard' plate-incorporation treatments was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 used for the 'Prival' treatments was prepared from uninduced male Golden Syrian hamsters.

range finder: tested in TA 100, in triplicate +/- S9 mix, with positive and solvent control, incorporation method

counting: Seescan Colony Counter (Seescan pic), Manual counts were performed for all treatments performed at 1500 /micro g/plate, as precipitation of the test agent on these plates prevented an accurate automated count being obtained.

Evaluation criteria:

The test article was considered to be mutagenic if:
1) the assay was valid
2) Dunnett's test gave a significant response (p < 0.01) and the data set showed a significant dose correlation
3) the positive responses described in 2) were reproducible.

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 1500 micrgram/plate

RANGE-FINDING/SCREENING STUDIES: yes, see above under study details

COMPARISON WITH HISTORICAL CONTROL DATA:

the second experiment was done twice due to an error in the first trial (induced instead of uninduced hamster liver)

treatments of strain TA100 in Experiment 1 produced a small increase in revertant numbers (1.3 fold over the negative solvent control counts) in the presence of metabolic activation, that was however statistically significant at the 1% levelwhen the data were analysed using Dunnett's test. However, this increase was not reproduced in the second experiment (either with 'standard' or 'Prival' S-9 mix) and was therefore not attributed to mutagenic activity, but was considered to have been a chance occurrence. This study was therefore considered to have provided no clear evidence of mutagenic activity.

Applicant's summary and conclusion

Conclusions:

It was concluded that the test item did not induce mutation under the conditions employed for this study, both in the absence and in the presence of metabolic activation systems (both 'standard' and 'reductive' S-9 mixes).

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations using the S. typhimurium strains TA1535, TA 1537, TA 1538, TA 98, and TA 100, and the E. coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation and with and without preval modification. Each concentration, including the controls, was tested in triplicate. The test item was tested at five concentrations in the range of 2.4 and 1500 µg/plate (experiment I) and 93.75 to 1500 µg/plate (experiment II).

No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix) or by preval modification.