Tetraethyl 2,2'-(1,4-phenylenedimethylidyne)bismalonate

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:

13. Mar - 27. Mar 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP conform guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4d, dated May 19, 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: Kanpoan No 287-Environment Protection Agency; Eisei No. 127-Ministry of Health&Welfare, Heisei 09/10/21 Kikyoku No. 2-Ministry of International Trade&Industry

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetraethyl 2,2'-(1,4-phenylenedimethylidyne)bismalonate

EC Number:

228-726-5

EC Name:

Tetraethyl 2,2'-(1,4-phenylenedimethylidyne)bismalonate

Cas Number:

6337-43-5

Molecular formula:

C22H26O8

IUPAC Name:

1,3-diethyl 2-({4-[3-ethoxy-2-(ethoxycarbonyl)-3-oxoprop-1-en-1-yl]phenyl}methylidene)propanedioate

Test material form:

other: solid

Method

Target gene:

His

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

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

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: iin agar (plate incorporation); preincubation

DURATION
- Preincubation period: 100 µL test solution, 500 µL S9 mix/S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37°C for 60 min.
- Exposure duration: 48 h at 37°C

SELECTION AGENT (mutation assays): His deficient medium

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: background growth

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An icrease exceeding the threshold at only one concentration is judged as biologically relevant if reporduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required.

Results and discussion

Test results

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:

cytotoxicity

Remarks:

minor toxic effects

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

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

During the described mutagenicity test and under the experimental conditions reported the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The assay was performed in two independent experiments both with and without liver microsomal activation using Salmonella thyphimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA.

The test item was tested at the following concentrations:

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

The plates incubated with the test item showed normal background up to 5000 µg/plate with and without S9 mix in both experiments.

Minor toxic effects (below the factor 0.5), evident as reduction in the number of revertants, were observed in strain TA 1535 at 333 and 1000 µg/plate without metabolic activation and at 5000 µg/plate with and without metabolic activation in experiment I. In experiment II, a minor toxic effect was observed at 5000 µg/plate without metabolic activation in strain TA 98.

No substantial increase in revertant colony number of any of the five tester strains was observed following the treatment with the test item 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.

In experiment I, with metabolic activation, the number of colonies did not quite reach the lower limit of the historical control data in strains TA 1535 (negative control) and TA 1537 (negative and solvent control). Sinced these deviationts are rather small, these effects are judged to be based upon statistical fluctuations and have no detrimantal impact on the outcome of the study. The historical range of positive controls was exceeded in strain TA 1535 (Exp. II) without metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.