2,2'-Methylenebis(4-aminophenol) dihydrochloride

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to the OECD Guideline and GLP

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Reference 2

Reference Type:

other: abstract

Title:

Unnamed

Year:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC Directive 92/69, L 383 A, Annexe V, B 14

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

Constituent

Details on test material:

Ro 1525 corresponds to Colipa A 155
Ro 1525
SAT 9803 75
Batch-No.: Ro-Rn 6567-083

Method

Target gene:

TA1537: his C 3076; da-; uvrB- frame shift mutations
TA 98: his D 3052; rfa-; uvrB-;R-factor frame shift mutations
TA1535: his G 46; rfa-; uvrB- base-pair substitutions
TA 100: his G 46; rfa-; uvrB;R-factor base-pair substitutions
TA 102: his G 428; rfa'; uvrB';R-factor base-pair substitutions

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium strain: TA 98, TA 100, TA 102, TA 1535, TA 1537

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitallß-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats.

Test concentrations with justification for top dose:

33, 100, 333, 1000, 2500 and 5000 ug/plate

Vehicle / solvent:

deionised water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: sodium azide (TA1535, TA 100), 4-nitro-o-phenylene-diamine (TA 1537, TA 98), methyl methane sulfonate (TA 102). With metabolic activation: 2-aminoanthracene (all strain)

Details on test system and experimental conditions:

The histidine dependent strains are derived fiom S. typhimurium strain LT2 through a
mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation
they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the
cell wall more easily. A hrther mutation causes an inactivation of the excision repair
system. The latter alteration includes mutational processes in the nitrate reductase and biotin
genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA
98 and TA 100 and TA 102 the R-factor plasmid pKM 10 1 carries the ampicillin resistance
marker. The strain TA 102 does not contain the uvrB--mutation and is excision repair
proficient. Additionally, TA 102 contains the multicopy plasmid pAQ 1 carrying the hisG428
mutation (ochre mutation in the hisG gene ) and a tetracycline resistance gene.

Evaluation criteria:

Regular checking of the properties of the strains regarding the membrane permeability,
ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in
the laboratory of RCC Cytotest Ce11 Research according to Ames et al. (1). In this way it
was ensured that the experimental conditions set down by Arnes were fulfilled.

Results and discussion

Test results

Species / strain:

other: TA 98, TA 100, TA 102, TA 1535, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Salmonella typhimurium

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the
experimental conditions reported, the test article did not induce gene mutations by base pair
changes or fiamechifis in the genome of the strains used.
with S9 mix
/
without S9 mix
1000 - 5000
Therefore, Ro 1525 is considered to be non-mutagenic in this Salmonella typhimurium
reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Ro 1525 to induce gene mutations

according to the plate incorporation test (experiment I) and the pre-incubation test

(experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98,

TA 100. and TA 102.

The assay was performed in two independent experiments both with and without h e r

microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test article was tested at the following concentrations:

33; 100; 333; 1000; 2500; and 5000 pg/plate

The background growth was reduced at concentrations as low as 333 pglplate in the first

and at 100 pg/plate and above in the second experiment without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was

observed following treatment with Ro 1525 at any dose level, neither in the presence nor

absence of metabolic activation (S9 mix). There was also no tendency of higher mutation

rates with increasing concentrations in the range below the generally acknowledged border

of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase

of induced revertant colonies.