3-methyl-6-(p-toluidino)-3H-dibenz[f,ij]isoquinoline-2,7-dione

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:

1997

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:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only one plate per dose

Principles of method if other than guideline:

MACROLEX Rot 5B was screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-6-[(4-methylphenyl)amino]-3H-naphthol[1,2,3-de]quinolone-2,7-dione

Cas Number:

81-39-0

Molecular formula:

C24H18N2O2

IUPAC Name:

3-methyl-6-[(4-methylphenyl)amino]-3H-naphthol[1,2,3-de]quinolone-2,7-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

name of test substance: MACROLEX Rot SB
manufacturer: Bayer AG
batch number: 976-012
content: not indicated by the sponsor
approved: not indicated by the sponsor
appearance: black powder
synonyms: C.I. Solvent Red 52; C.I. 68210
chemical name: 3H-Dibenz[f,i,j]isoquionoline-2,7-dione, 3-methyl-6- [(4-methylphenyl) amino]-
molecular weight: 366
CAS No.: 81-39-0

Method

Target gene:

Histidine-deficient mutants

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

There was no indication of a bacteriotoxic effect of MACROLEX Rot SB at doses of up to and including 5000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. At 1581 µg per plate, the substance started to precipitate.

Vehicle / solvent:

MACROLEX Rot SB was dissolved in DMSO and formed a dark violet solution. The positive controls were dissolved in DMSO.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: The positive controls sodium azide, nitrofurantoin, 4-nitro- 1,2-phenylene diamine and cumene hydroperoxide were used without S9 mix; the positive control 2-aminoanthracene was used with S9 mix.

Details on test system and experimental conditions:

Comparable to OECD TG 471

Rationale for test conditions:

Comparable to OECD TG 471

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 150 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guide­ lines may be overruled by good scientific judgement.

In case of questionable results, investigations should con­ tinue, possibly with modifications, until a final evaluation is possible.

Statistics:

not applied

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Evidence of mutagenic activity of MACROLEX Rot 5B was seen. On Salmonella typhimurium TA 1537, TA 98 and TA 102, a bio­ logically relevant increase was found in the mutant count compared to the corresponding negative control. Except for TA 1537 positive response was found only with S9 mix. The lowest effective dose was 16 µg per plate for Salmonella ty­ phimurium TA 1537 and 50 µg per plate for TA 98 and TA 102. The Salmonella/microsome test thus showed MACROLEX Rot 5B to have a mutagenic effect.

The positive controls sodium azide, nitrofurantoin, 4-nitro- 1,2-phenylene diamine, cumene hydroperoxide and 2-amino­ anthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Applicant's summary and conclusion

Executive summary:

MACROLEX Rot 5B was screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These com­ prised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102.

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred at the dose 1581 µg per plate and above.

Evidence of mutagenic activity of MACROLEX Rot 5B was seen. On Salmonella typhimurium TA 1537, TA 98 and TA 102, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Except for TA 1537 positive response was found only with S9 mix. The lowest effective dose was 16 µg per plate for Salmonella ty­ phimurium TA 1537 and 50 µg per plate for TA 98 and TA 102. The Salmonella/microsome test thus showed MACROLEX Rot 5B to have a mutagenic effect.

The positive controls sodium azide, nitrofurantoin, 4-nitro- 1,2-phenylene diamine, cumene hydroperoxide and 2-amino­ anthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.