Bis(2-ethylhexyl) 2-(4-hydroxy-3,5-dimethoxybenzylidene)malonate

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:

2004-09-10 to 2005-03-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

08 Jume 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Method

Target gene:

HIS operon (S. thyphimurium), TRP operon (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor (pKM101)

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor (pKM101)

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

his G 428, rfa +R-factor

Species / strain 6

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

trp-, uvrB

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors

Test concentrations with justification for top dose:

The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg per plate
2nd series: 15.8, 50.0, 158, 500, 1580 µg per plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

cumene hydroperoxide

other: Daunomycin

Remarks:

without S9.mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with S9-mix

Details on test system and experimental conditions:

Bacterial strains were tested in accordance with the plate incorporation method. 3 parallel plates were used for each concentration step of the test material. The incubation of plates was performed at 36-38°C for 2 days. Liver S9 mix from rats pre-treated with beta-naphthoflavone/phenobarbital was used as the metabolic activation system. Two experimental series were performed, containing 10% S9 ind the 1st and 30% S9 in the 2nd series.

Rationale for test conditions:

according to Guidelines.

Evaluation criteria:

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the in-crease in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

Mean Number of Colonies Maximal Mean Number of Colonies over the Actual
(Solvent Control) Solvent Control (Test Material)
<=10 <=9 >=30
<=30 <=19 >= 40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment: "No Increase" "Clear Increase"

All further results, ranging between "no" and "clear", are assessed as "weak in-creases".
Interpretations:
A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the first and second series of the main ex¬periment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if
• a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;

• "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test substance was not mutagenic under the experimental conditions described.

Executive summary:

Purpose

The objective of this study was to evaluate the mutagenic activity of the test item by examining its ability to revert five histidine-requiring strains of Salmonella typhimurium and in a tryptophane-requiring E.coli strain in the absence and in the presence of a rat liver metabolising system (S-9).

Study Design

The procedures used in this study were in accordance with OECD Guideline 471 (adopted 1997), EEC Annex V Test B 13B 14 (2000), UKEMS Guidelines (1990) and ICH Harmonised Tripartite Guideline (1997).

Results

The test material was dissolved in dimethyl sulfoxide (DMSO) and tested at concentrations ranging from 5.00 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate. Toxicity to the bacteria was not observed.
Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Conclusion

The test item is not mutagenic under the test conditons employed.