1,6-Bis(methoxybenzoyloxy)hexane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

The test item is not clastogenic in human lymphocytes under the experimental conditions of the study.

 

The test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

Link to relevant study records

Reference

Reference 1

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:

30 October 2001 - 30 November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study includes data generated according to generally valid and internationally accepted testing guidelines and performed according to GLP. The study was based on the following guidelines: -Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test". (Adopted July 21, 1997). - European Economic Community (EEC). Adapting to technical progress for the twenty-sixth time Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.13/14: "Muta1~enicity: "Reverse Mutation Assay using bacteria". EEC Publication Commission Directive {Published June 8, 2000).

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Remarks:

GLP Statement date: 10/12/2001 and 21/12/2001

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium bacteria and Escherichia coli bacteria

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coIi WP2uvrA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

DOSE RANGE FINDING TEST:
3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of 59-mix.

MUTATION ASSAY:

EXPERIMENT 1: 3, 10, 33, 100, 333 μg/plate in the absence and presence of 59-mix (Strains: TA 1535, TA 1537 and TA98)

EXPERIMENT 2: 3, 10, 33, 100, 333 μg/plate in the absence and presence of 59-mix (Strains: TA 1535, TA 1537, TA98, TA 100 and WP2uvrA)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO

- Justification for choice of solvent/vehicle:
Not specified but likely to be solubility of test substance

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Strain TA1535

Positive control substance:

sodium azide

Remarks:

Without metabolic activation(-S9-mix)

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Strain TA1537

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation(-S9-mix)

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Strain TA98

Positive control substance:

other: Daunomycine

Remarks:

Without metabolic activation(-S9-mix)

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Strain TA100

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation(-S9-mix)

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Strain WP2uvrA

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation(-S9-mix)

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Strains: TA1537, TA1535, TA98, TA100 and WP2uvrA

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation (+S9-mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Preparation of Bacterial cultures:
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml}. Freshly grown cultures of each strain were used for a test.

Permeabilization of the Escherichia coli strain:
WP2uvrA bacteria were washed twice in 0.25 the original volume of ice-cold 0.12 M Tris-HCL buffer pH 8.0, then gently resuspended in 0.2 vol. 0.12 M TrisHCL, 0.5 mM EDTA pH 8.0, and shaken for 2.5 min at 37°C. MgCl2 was then added to a final concentration of 10 mM. The cells were centrifuged and resuspended in the original volume of nutrient broth.

Agar Plates:
Agar plates (0 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18g purified agar (Oxoid, code L2) in Vogel-Bonner Medium E, 20 g glucose.
N.B. The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin and 15 μg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan.

Top Agar:
Top agar medium, containing 0.6% (w/v) purified agar and 0.5% (w/v) NaCl, was heated to dissolve the agar. Samples of 3 ml top agar were transferredinto 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 1 °C.

Environmental conditions:
All incubations were carried out in the dark at 37 ± 1 °C. The temperature was monitored during the experiment.

Evaluation criteria:

A test substance is consideried negative (not mutagenic) in the test if:

a) The total number of reveirtants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.

b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:

a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.

b) The positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done.

Species / strain:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coIi WP2uvrA

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:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

DOSE RANGE FINDING TEST

SETAFIX X 11342 was testeid in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of 59-mix.

Precipitate:

The test substance precipitated in the top agar at concentrations of 100 μg/plate and upwards. Precipitation of SETAFIX X 11342 on the plates was observed at the start and at the end of the incubation period at concentrations of 333 μg/plate and upwards.

Toxicity:

To determine the toxicity of SETAFIX X 11342, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

MUTATION ASSAY

Based on the results of the close range finding test, SETAFIX X 11342 was tested up to concentrations of 333 μg/plate in the absence and presence of S9-mix in two mutation assays.

The first mutation experiment was performed with the strains TA 1535, TA 1537 and TA98 and the second mutation experiment was performed with the strains TA 1535, TA 1537, TA98, TA 100 and WP2uvrA.

Precipitate:

SETAFIX X 11342 precipitated in the top agar at concentrations of 100 and 333 μg/plate. Precipitation of SETAFIX X 11342 on the plates was observed at the start and at the end of the incubation period in all tester strains at the highest concentration tested.

Toxicity

The bacterial background lawn was not reduced at all concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Number of revertants:

All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

The negative control values were within our laboratory background historical control data ranges. The strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except for TA1537 in the presence of S9 -mix (second experiment). However, since this value was just without the limit of the range, the validity of the test was considered to be not affected.

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that SETAFIX X 11342 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

 

The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain.

 

SETAFIX X 11342 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

 

In the dose range finding test, SETAFIX X 11342 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. SETAFIX X 11342 precipita1ted on the plates at dose levels of 333 μg/plate and upwards. The bacterial background lawn was not reduced at all concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

In the first and in the second mutation assay, SETAFIX X 11342 was tested up to concentrations of 333 μg/plate in the absence and presence of S9-mix. SETAFIX X 11342 precipitated on the plates at this dose level. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

 

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

 

SETAFIX X 11342 did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

Based on the results of this study it is concluded that SETAFIX X 11342 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 January 2004 - 26 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study includes data generated according to generally valid and internationally accepted testing guidelines and performed according to GLP. The study was based on the following guidelines: - OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted 21st July 1997). - European Economic Community (EEC). Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity; B.10: "Mutagenicity: In Vitro Mammalian Chromosome Aberration Test".

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

.The study integrity was not adversely affected by the deviations.

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

.The study integrity was not adversely affected by the deviations.

GLP compliance:

yes

Remarks:

GLP Statement Date: 01-04-2004

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Cultured Human Lymphocytes

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-miix

Test concentrations with justification for top dose:

Dose Range Finding Test: 1, 3, 10, 33 and 100 μg/ml (culture medium with and without S9-mix)

First Cytogenetic Assay: 10, 33 and 100 μg/ml (culture medium with and without S9-mix)

Second Cytogenetic Assay: 10, 33 and 100 μg/ml (culture medium with and without S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl Sulfoxide (DMSO)

- Justification for choice of solvent/vehicle:
Not specified but likely to be solubility

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

not specified

Positive controls:

yes

Remarks:

Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation (-S9-mix)

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

not specified

Positive controls:

yes

Remarks:

Solvent for positive controls: Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (+S91-mix)

Details on test system and experimental conditions:

TEST SYSTEM
Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (e.g. EPA, OECD, EEC).
These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemicals

Blood was collected from healthy adult, non-smoking male volunteers. The Average Generation Time (AGT) of the cells is presented below:
Dose range finding study: age 29, AGT = 15.8 h (Nov. 2003)
First cytogenetic assay: age 33, AGT = 14.2 h (Nov. 2003)
Second cytogenetic assay: age 38, AGT = 16.8 h (Feb. 2004)

CELL CULTURE:

Blood samples:
Blood samples were taken from healthy adult male volunteers by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

Culture medium:
Culture medium consisted of RPMI 1640 medium (lnvitrogen Corporation, Breda, The Netherlands), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (lnvitrogen Corporation), L-glutamine (2 mM) {Merck), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (lnvitrogen Corporation) and 30 U/ml heparin.

Lymphocyte cultures:
Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium {in the absence and presence of S9-mix respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Murex, Dartford, England) was added.

Environmental conditions:
All incubations were carried out in a humid atmosphere (80-100%) containing 5 ± 0.5% C02 in air in the dark at 37 ± 1°C. ThE~ temperature, humidity and C02-percentage were monitored throughout the experiment.

Metabolic activation system:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats (6), which were obtained from Charles River (Sulzfeld, Germany).

Evaluation criteria:

Data evaluation:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) it induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) a statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cellls with chromosome aberrations.

Acceptability of the assay:
A chromosome aberration test was considered acceptable if it met the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group was compared to that of the solvent control using Chi-square statistics.

If P is small (P< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence level.

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Cultured peripheral human lymphocytes

Remarks:

Migrated from field 'Test system'.

DOSE RANGE FINDING TEST

At a concentration of 100 μg/ml Setafix X 11342 precipitated in the culture medium. Therefore, a concentration of 100 μg/ml was used as the highest concentration of Setafix X 11342.

In the dose range finding test lt>lood cultures were treated with 1, 3, 10, 33 and 100 μg Setafix X 11342 /ml culture mE~dium with and without S9-mix.

FIRST CYTOGENETIC ASSAY

Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:

Without and with S9-mix: 10, 33 and 100 μg Setafix X 11342/ml culture medium (3 h 1:ixposure time, 24 h fixation time).

All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix Setafix X 111342 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

SECOND CYTOGENETIC ASSAY

To obtain more information about the possible clastogenicity of Setafix X 11342, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Setafix X 11342 in the absencE of S9 mix for 24 or 48 hours. In the· presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Setafix X 11342. The following dose levels were selected for the seicond cytogenetic assay:

Without S9-mix: 10, 33 and 100 μg Setafix X 11342/ml culture medium (24 hand 48 h exposure time, 24 hand 48 h fixation time)

With S9-mix: 10, 33 and 100 μg Setafix X 11342/ml culture medium (3 h exposure time, 48 h fixation time)

All doses were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix Setafix X 11342 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Conclusions:

Interpretation of results (migrated information):
negative

The test is valid and that Setafix X 11342 is not clastogenic in human lymphocytes under the experimental conditions of this study.

Executive summary:

 

The objective of this study was to evaluate Setafix X 11342 for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

 

The effect of Setafix X 11342 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix) is summarised. The possible clastogenicity of Setafix X 11342 was tested in two independent experiments.

 

The study procedures described in this report were based on the following guidelines:

- OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted 21st July 1997).

- European Economic Community (EEC). Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity; B.10: "Mutagenicity: In Vitro Mammalian Chromosome Aberration Test".

 

Batch PP 03 of Setafix X 11342 was a light yellowish solid with a purity of >95%. The test substance was crushed in a test tube and dissolved in dimethyl sulfoxide at concentrations of 33 mg/ml and lower.

 

In the first cytogenetic assay, Setafix X 11342 was tested up to 100 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. Setafix X 11342 precipitated in the culture medium at this dose level.

 

In the second cytogenetic assay, Setafix X 11342 was tested up to 100 μg/ml for a 24 hand 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of 1.8% (v/v) S9-fraction Setafix X 11342 was also tested up to 100 μg/ml for a 3 h exposure time with a 48 h fixation time.

 

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

Setafix X 11342 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, in two independently repeated experiments.

 

Finally, it is concluded that this test is valid, and that Setafix X 11342 is not clastogenic in human lymphocytes under the experimental conditions of this study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Jul 2017 to 04 Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Identification: Weylchem di-ester
Batch: DECG020911
Purity: 94.9%

Target gene:

testing the ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/--3.7.2C mouse lymphoma cells [American Type Culture Collection, (ATCC, Manassas, USA) (2001)]

Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH and was prepared from rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

Test concentrations with justification for top dose:

First Mutagenicity Test: Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test: Without and with S9-mix: 1.22, 2.44, 4.88, 9.75, 19.5, 39, 78 and 156 μg/ml exposure medium.

Second Mutagenicity Test: Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 1.22, 2.44, 4.88, 9.75, 19.5, 39, 78, 117 and 156 μg/ml exposure medium.

Since the test item was not toxic and difficult to dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium.

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide (DMSO, SeccoSolv, Merck Darmstadt, Germany).

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

historical control data range

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
In the first experiment, cell cultures were exposed for 3 hours to the test item in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test item in exposure medium for 24 hours in the absence of S9-mix.

For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4000000 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency and the mutation frequency.

SELECTION AGENT
For determination of the cloning efficiency the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.

For determination of the mutation frequency a total number of 960000 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (trifluorothymidine-selection), with the exception of the positive control groups (methyl methanesulfonate and cyclophosphamide) where a total number of 960000 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (trifluorothymidine-selection). The microtiter plates for cloning efficiency and mutation frequency were incubated for 11 or 12 days. After the incubation period, the plates for the trifluorothymidine-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma) to each well. The plates for the cloning efficiency and mutation frequency were scored with the naked eye or with the microscope.

DETERMINATION OF THE MUTANT COLONIES:
The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.

The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Remarks:

vehicle control served as negative control

Positive controls validity:

valid

Additional information on results:

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate (MMS) and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. Although in the second experiment the response of MMS was just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The suspension growth over the two-day expression period for cultures treated with DMSO was between 20 and 26 (3 hour treatment) and 51 and 53 (24 hour treatment).

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.

In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

Conclusions:

In conclusion, the test item is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of the test item by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

 

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

 

The study procedures described in this report were based on the most recent OECD guideline.

 

Batch DECG020911 of the test item was a slightly yellow solid. The test item was dissolved in dimethyl sulfoxide.

 

In the first experiment, the test item was tested up to concentrations of 156 μg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. The test item precipitated in the culture medium at the dose levels of 78 and 156 μg/ml.

 

In the second experiment, the test item was tested up to concentrations of 130 μg/ml in the absence of S9-mix. The incubation time was 24 hours. No severe toxicity was observed at this dose level in the absence of S9-mix. The test item precipitated in the culture medium at the dose levels of 115 and 130 μg/ml.

 

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

 

Positive control chemicals, methyl methanesulfonate (MMS) and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. Although in second experiment the response of MMS was just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

 

In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

 

In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

AMES TEST

The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain.

 

SETAFIX X 11342 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

 

In the dose range finding test, SETAFIX X 11342 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. SETAFIX X 11342 precipita1ted on the plates at dose levels of 333 μg/plate and upwards. The bacterial background lawn was not reduced at all concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

In the first and in the second mutation assay, SETAFIX X 11342 was tested up to concentrations of 333 μg/plate in the absence and presence of S9-mix. SETAFIX X 11342 precipitated on the plates at this dose level. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

 

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

 

SETAFIX X 11342 did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

Based on the results of this study it is concluded that SETAFIX X 11342 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

CHROMOSOME ABERRATION TEST

The objective of this study was to evaluate Setafix X 11342 for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

 

The effect of Setafix X 11342 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix) is summarised. The possible clastogenicity of Setafix X 11342 was tested in two independent experiments.

 

The study procedures described in this report were based on the following guidelines:

- OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted 21st July 1997).

- European Economic Community (EEC). Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity; B.10: "Mutagenicity: In Vitro Mammalian Chromosome Aberration Test".

 

Batch PP 03 of Setafix X 11342 was a light yellowish solid with a purity of >95%. The test substance was crushed in a test tube and dissolved in dimethyl sulfoxide at concentrations of 33 mg/ml and lower.

 

In the first cytogenetic assay, Setafix X 11342 was tested up to 100 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix. Setafix X 11342 precipitated in the culture medium at this dose level.

 

In the second cytogenetic assay, Setafix X 11342 was tested up to 100 μg/ml for a 24 hand 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of 1.8% (v/v) S9-fraction Setafix X 11342 was also tested up to 100 μg/ml for a 3 h exposure time with a 48 h fixation time.

 

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

Setafix X 11342 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, in two independently repeated experiments.

 

Finally, it is concluded that this test is valid, and that Setafix X 11342 is not clastogenic in human lymphocytes under the experimental conditions of this study.

 

IN VITRO MAMMALIAN CELL GENE MUTATION TESTS USING THE THYMIDINE KINASE GENE

The objective of this study was to evaluate the mutagenic potential of the test item by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

 

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

 

The study procedures described in this report were based on the most recent OECD guideline.

 

Batch DECG020911 of the test item was a slightly yellow solid. The test item was dissolved in dimethyl sulfoxide.

 

In the first experiment, the test item was tested up to concentrations of 156 μg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. The test item precipitated in the culture medium at the dose levels of 78 and 156 μg/ml.

 

In the second experiment, the test item was tested up to concentrations of 130 μg/ml in the absence of S9-mix. The incubation time was 24 hours. No severe toxicity was observed at this dose level in the absence of S9-mix. The test item precipitated in the culture medium at the dose levels of 115 and 130 μg/ml.

 

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

 

Positive control chemicals, methyl methanesulfonate (MMS) and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. Although in second experiment the response of MMS was just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

 

In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

 

In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for classification or non-classification

AMES TEST

The test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

CHROMOSOME ABERRATION TEST

The test item is not clastogenic in human lymphocytes under the experimental conditions of the study.

 

MAMMALIAN CELL GENE MUTATION TEST

The test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.