bis(2-ethylhexyl) (4-hydroxy-3,5-dimethoxybenzyl)malonate

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:

2006-03-27 to 2006-08-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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:

2000

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

471-510-2

EC Name:

-

Cas Number:

872182-46-2

Molecular formula:

C28H46O7

IUPAC Name:

1,3-bis(2-ethylhexyl) 2-[(4-hydroxy-3,5-dimethoxyphenyl)methyl]propanedioate

Test material form:

liquid: viscous

Method

Target gene:

HIS operon (S. thyphimurium)

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)

Additional strain / cell type characteristics:

other: frame-shift

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)

Additional strain / cell type characteristics:

other: base-pair substitution

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa

Additional strain / cell type characteristics:

other: base-pair substitution

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: frame-shift

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

hisG 428, rfa + R-factor (pKM101)

Additional strain / cell type characteristics:

other: base-pair substitution

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:
1. Series: 1.6, 8, 40, 200, 1000, 5000 µg/plate
2. Series: 1.6, 8, 20.48, 51.2, 128, 320, 800, 2000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Test material is soluble in DMSO, which is widely used as solvent in this assay

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9, TA98

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, TA100, TA1535

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9, TA1537

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9, TA102

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9, TA98

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9, TA100, TA1535, TA1537, TA102

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
The plates were incubated at 37°C for approximately 48 hours and the number of revertant colonies counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: revertant colonies and examined for a thinning of the background lawn

Rationale for test conditions:

according to Guideline

Evaluation criteria:

The test article was considered to be mutagenic if:
1. the assay was valid
2. Dunnett's test gave a significant response (p < 0.01) and the data set(s) showed a significant dose correlation
3. the positive responses described above were reproducible.

Statistics:

Dunnett's test

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

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

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test substance was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.
An initial toxicity range-finder experiment was carried out in the absence and in the presence of S-9 in strain TA100 only, using final concentrations of the test substance at 1.6, 8, 40, 200, 1000 and 5000 fig/plate, plus negative (solvent) and positive controls. Following these treatments, no evidence of toxicity was observed, but precipitation of the test article (in the form of oily droplets) was present on all test plates treated at 1000 µg/plate and above. These data were considered to be acceptable for mutation assessment and are presented in this report as the TA100 mutagenicity data for Experiment 1.
Experiment 1 treatments of the remaining test strains were performed in the absence and in the presence of S-9, and retained the same test doses as those employed for the range-finder experiment. Following these treatments there was again no evidence of toxicity observed. Precipitation of the test article (in the form of oily droplets) was present on all test plates treated at 1000 pg/plate and above.
Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9, with the maximum test dose reduced to 2000 µg/plate as an estimate of the lower limit of precipitation. Narrowed dose ranges were employed (20.48-2000 fig/plate for all strain treatments except those of strain TA102 in the absence of S-9, where 1.6-2000 fig/plate was employed), in order to examine more closely those concentrations of the test item approaching the maximum test dose, and therefore those considered most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Additional plate incorporation treatments of strain TA100 in the presence of S-9 were also performed, in order to investigate the reproducibility of small increases in revertant numbers seen in Experiment 1. These were performed using a dose range of 20.48-2000 gg/plate. Following all Experiment 2 treatments there was no evidence of toxicity observed, but test article precipitation (in the form of oily droplets) was present on all test plates treated at 2000 µg/plate.
Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

Any other information on results incl. tables

see attached background material

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 substance by examining its ability to revert five histidine-requiring strains of Salmonella typhimurium 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). The investigations for the mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series using triplicate plates were performed.

Results

The test substance was dissolved in DMSO and tested at concentrations ranging from 1.58 to 5000 µg/plate (1st series) and up to 2000 µg/plate in the subsequent 2nd series. Precipitation of the test material in form of oily droplets on the agar plates occurred at concentrations larger or equal to 1000 µg/plate. Toxicity to the bacteria was not observed.
2-Nitrotluorene, Sodium azide, 9-aminoacridine, and Mitomycin C served as strain specific positive control test materials in the absence of S9 mix and 2-aminoanthracene and benzo[a]pyrene were used as controls in the presence of metabolic activation. 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.

Following test substance treatments of all the tester strains, in the absence and in the presence of S-9, increases in revertant numbers that were statistically significant when the data were analysed at the 1% level using Dunnett's test were observed following Experiment 1 treatments of strain TA100 in the presence of S-9 and strain TA102 in the absence of S-9. Neither of these increases were dose-related, occurring as they did at one or more low or intermediate dose levels, nor were they reproducible in a second independent experiment. These increases were therefore considered to have been chance increases arising from normal biological variation, rather than any compound related effect.

As no other strain treatments resulted in any statistically significant increases in revertant numbers, this study was considered to have provided no evidence of any test substance mutagenic activity in this assay system.

Conclusion

The test substance did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to precipitating dose levels in the absence and in the presence of a rat liver metabolic activation system (S-9).