1,2-Benzenediamine, ar-methyl-, reaction products with propylene oxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

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: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,2-Diaminotoluene, propoxylated

EC Number:

918-139-9

Cas Number:

1228577-90-9

Molecular formula:

(C3H6O)n (C3H6O)n (C3H6O)n (C3H6O)n C7H10N2 sum of n: >1 - <8.5

IUPAC Name:

1,2-Diaminotoluene, propoxylated

Details on test material:

purity not indicated by the sponsor

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Type and identity of media: PAA Ready Mix (2 % FBS)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

initial assay: 300, 600, 900, 1200, 1500, 1800, 2100 µg/ml (+/-S9);
independent repeat assay: 150, 300, 600, 900, 1200, 1500, 1800 µg/ml (-S9) and 300, 600, 900, 1200, 1500, 1800, 2100 µg/ml (+S9)

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulfonate (EMS, 900 µg/ml, -S9); dimethylbenzanthracene (DMBA, 20 µg/ml, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 6-8 days

NUMBER OF REPLICATIONS: at least 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

- Mutant frequencies will only be used for assessment, if at least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10% or greater.
- A trial will be considered positive if a concentration-related and in parallel cultures reproducible increase in mutant frequencies is observed. To be relevant, the increase in mutant frequencies should be at least two to three times that of the highest negative or negative control value observed in the respective trial. If this result can be reproduced in a second trial, the test substance is considered to be mutagenic.
- Despite these criteria, a positive result will only be considered relevant, if no significant change in osmolality compared to the negative control can be observed. Otherwise, unphysiological culture conditions may be the reason for the positive result (Scott et al, 1991).
- A test substance will be judged as equivocal if there is no strictly concentration related increase in mutation frequencies but if one or more concentrations induce a reproducible and biologically relevant increase in mutant frequencies in all trials.
- An assay will be considered negative if no reproducible and relevant increases of mutant frequencies were observed.

Statistics:

All acceptable groups are included in the weighted analysis of variance followed by pairwise comparisons to the negative control on a nominal significance level of a =0.05 using the Dunnett test (Dunnett, 1955). The regression analysis part is performed on the basis of the actual concentrations thereby omitting the positive, untreated and negative controls. If there is a significant concentration related increase of the mutant frequency (a = 0.05) in the main analysis the highest concentration will be dropped and the analysis will be repeated. This procedure will be repeated until p > 0.05.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

test substance induced a significant concentration-related cytotoxicity of up to 100 % with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Tercarol 5903 (NLP #10) was evaluated for point mutagenic effects at the hypoxanthine-guanine phosphoribosyl transferase locus (forward mutation assay) in V79 cell cultures after treatment with concentrations of up to and including 2100 µg/ml, both with and without S9 mix. Without and with S9 mix Tercarol 5903 (NLP #10) induced decreases in survival to treatment and decreases in relative population growth. These results revealed a significant concentration-related cytotoxicity of Tercarol 5903 (NLP #10). Without S9 mix precipitation of Tercarol 5903 (NLP #10) in the culture medium was observed at 2100 µg/ml. With S9 mix no precipitation was observed. Without and with S9 mix there was no biologically relevant increase in mutant frequency above that of the negative controls. Ethylmethanesulfonate and dimethylbenzanthracene induced clear mutagenic effects and demonstrated the sensitivity of the test system and the activity of the S9 mix. Based on these results, Tercarol 5903 (NLP #10) is considered to be non-mutagenic in the V79/HPRT forward mutation assay, both with and without metabolic activation.

Applicant's summary and conclusion

Conclusions:

non-mutagenic

Executive summary:

Tercarol 5903 (NLP #10) did not induce mutagenic effects in the in vitro gene mutation assay (HPRT test) with Chinese hamster V79 cells in the presence and absence of a metabolic activation system.