6,6-dimethoxy-2,5,5-trimethylhex-2-ene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Qualifier:

according to guideline

Guideline:

other: European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.49

Version / remarks:

“InVitro Mammalian Cell Micronucleus Test". Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

Principles of method if other than guideline:

List of protocol deviations
1. In the first cytogenetic assay the positive control cultures were exposed to one concentration of CP (15 μg/ml).
Evaluation: The positive control was not examined for micronuclei since this part of the study was repeated in cytogenetic assays 1A, 1B and 1C. This deviation has no influence on the study results.
The study integrity was not adversely affected by the deviation.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Identification: Methyl Pamplemousse
Chemical name (IUPAC): 6,6-dimethoxy-2,2,5-trimethylhex-2-ene
Molecular weight: 186.30
CAS Number: 67674-46-8
EC Number: 266-885-2
Description: Clear colourless liquid (determined at WIL Research Europe B.V.)
Batch: PE00084535
Purity/Composition: See Certificate of Analysis
Test substance storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 15 March 2015 (expiry date)

Target gene:

None

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose range finding test. The highest tested concentration was determined by the solubility of Methyl Pamplemousse in the culture medium.
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

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: 5, 15, 50, 70, 90, 110, 130 and 150 μg/ml culture medium
(3 hours exposure time, 27 hours harvest time).

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C, Colchicine

Remarks:

Without metabolic activation (-S9-mix)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (+S9-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 the international OECD guideline.
Blood was collected from healthy adult, non-smoking, male volunteers (aged < 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2013) are presented below:
Dose range finding study: age 26, AGT = 13.1 h
First cytogenetic assay: age 25, AGT = 12.9 h
Cytogenetic assay 1A: age 22, AGT = 12.8 h
Cytogenetic assay 1B: age 31, AGT = 12.9 h
Cytogenetic assay 1C: age 22, AGT = 12.8 h
Second cytogenetic assay: age 31, AGT = 13.5 h

Cell culture
Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
Culture medium
Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with
20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine
(2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (Invitrogen Corporation) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).
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 (Remel, Europe Ltd., United Kingdom) was added.
Environmental conditions
All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 50 - 91%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 34.5 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per ml: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox); 4 μmol HEPES (Invitrogen Corporation). The above solution was filter (0.22 μm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v)
S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Dose range finding test
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose range finding test. Methyl Pamplemousse was tested in the absence and presence of S9-mix.
Lymphocytes (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of Methyl Pamplemousse for3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B was added to the cells simultaneously with the test substance at the 24 hours exposure time. A vehicle control was included at each exposure time.
The highest tested concentration was determined by the solubility of Methyl Pamplemousse in the culture medium.
After 3 hours exposure to Methyl Pamplemousse in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium with Cytochalasine B (5 μg/ml) and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.
Cytotoxicity of Methyl Pamplemousse in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

First cytogenetic assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of Methyl Pamplemousse for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium with Cytochalasin B (5 μg/ml) and incubated for another 24 hours. Appropriate vehicle and positive controls were included in the first cytogenetic assay.

Second cytogenetic assay
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of Methyl Pamplemousse with cytochalasin B (5 μg/ml) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.

Preparation of slides
To harvest the cells from the cytogenetic assays, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were resuspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic
0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of
96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the WIL Research Europe study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensenbuffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

Cytotoxicity assessment
A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}

CBPI = (No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells) / Total number of cells

t = test substance or control treatment culture
c = vehicle control culture
Three or four analysable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. The highest dose level examined for micronuclei were the cultures that produced 55 ± 5% cytotoxicity. The lowest dose level had little or no cytotoxicity (approximately the same as solvent control). Also cultures treated with an intermediate dose level were examined.

Cytogenetic assessment/scoring of micronuclei
To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with WIL Research Europe study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately.
In case no statistically significant increase was observed in the cultures dosed with the lowest dose of MMC-C, both doses were scored for the presence of micronuclei. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase is observed, this has no effect on the study integrity.
The following criteria for scoring of binucleated cells were used (1 – 2, 6):
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.

The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

Evaluation criteria:

A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided,
p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The ability of Methyl Pamplemousse to induce micronuclei in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analysed was selected based on cytokinesis-block proliferation index of 55 ± 5%.

The number of mono- and binucleated cells with micronuclei found in the solvent control was within the historical control data range, except for one of the duplicate cultures in the second cytogenetic assay in which the number of mononucleated cells with micronuclei was just above the historical control data range.

The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei in the first cytogenetic assay. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Methyl Pamplemousse did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Conclusions:

Finally, it is concluded that this test is valid and that Methyl Pamplemousse is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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 (incl. QA statement)

Type of assay:

other: Mouse Lymphoma Assay

Specific details on test material used for the study:

Test item: 205601/B
Identification: Methyl Pamplemousse
Appearance: Colourless to pale yellow liquid
Batch: AS00239830
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light
Stable under storage conditions until: 11 March 2017 (expiry date)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale: Recommended test system in international guidelines (e.g. OECD).
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).

Metabolic activation:

with and without

Test concentrations with justification for top dose:

In the first experiment, the test item was tested up to concentrations of 95 and 100 μg/ml in the absence and presence of S9-mix, respectively. In the additional second experiment, the test item was tested up to concentrations of 110 μg/ml in the presence of S9-mix. In the third experiment, the test item was tested up to concentrations of 90 μg/ml in the absence of S9-mix.
The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

yes

Remarks:

The solvent for the test item, i.e. dimethyl sulfoxide (DMSO, Merck Darmstadt, Germany).

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO, Merck Darmstadt, Germany).

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale: Recommended test system in international guidelines (e.g. OECD).
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 106 cells/ml.

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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, an increase in the mutation frequency above the GEF was only observed at the toxic dose level of 90 μg/ml with a RTG of 27%. This mutagenic response could not be repeated in an additional experiment.
Since the meaningful increase (above the positive threshold, GEF) in the mutation frequency at the TK locus in the presence of S9-mix is only observed at a toxic concentration with a RTG of 27% and could not be repeated in an additional experiment, the biological relevance of this increase is doubtful. Therefore the test results in the presence of S9-mix are considered equivocal under the experimental conditions described in the report. The test item was not mutagenic in the current test in the absence of S9-mix.

Conclusions:

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 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. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The growth rate over the two-day expression period for cultures treated with DMSO was between 12 and 17 (3 hours treatment) and 106 and 121 (24 hours 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 an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, an increase in the mutation frequency above the GEF was only observed at the toxic dose level of 90 μg/ml with a RTG of 27%. This mutagenic response could not be repeated in an additional experiment.

Since the meaningful increase (above the positive threshold, GEF) in the mutation frequency at the TK locus in the presence of S9-mix is only observed at a toxic concentration with a RTG of 27% and could not be repeated in an additional experiment, the biological relevance of this increase is doubtful. Therefore the test results in the presence of S9-mix are considered equivocal under the experimental conditions described in the report. The test item was not mutagenic in the current test in the absence of S9-mix.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): HR 02/197007
- Physical state: colourless to pale yellowish, clear liquid
- Purity test date: 20 February 2002
- Lot/batch No.: 000003
- Expiration date of the lot/batch: 31 August 2002
- Storage condition of test material: 7±2 °C, protected from light and moisture

Target gene:

Histidine gene of S. typhimurium

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from the livers of male Wistar rats treated with phenobarbitone/B-naphtoflavone

Test concentrations with justification for top dose:

Experiment 1: 0.05, 0.16, 0.5, 1.6 and 5 mg/plate (all strains)
Experiment 2: 0.05, 0.16, 0.5, 1.6 and 5 mg/plate (TA 97a, TA 98, TA 102); 0.016, 0.05, 0.16, 0.5 and 1.6 mg/plate (TA 100, TA 1535)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test material was insoluble in water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

See Table 7.6.1/1

Positive control substance:

sodium azide

other: ICR 191 acridine mutagen dihydrochloride, 2-aminoanthracene, 4-nitro-1,2-phenylenediamine, nitrofurantoine, cumene hydroperoxide, danthron

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours (72 hours for TA 102 - S9 in Experiment 2) at 37±1 °C

NUMBER OF REPLICATIONS: triplicate plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: growth assessment of the bacterial background lawn

OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay was considered valid if the following criteria were met:
1. The following genotypes of the tested strains had to be confirmed:
- Histidine auxotrophy
- Ampicillin resistance
- Tetracycline resistance (only TA 102)
- UV-sensitivity (except TA 102)
- Growth inhibition with crystal violet (rfa-mutation)

2. Titer of the overnight culture had to be ≥ 10^8 cells/mL

3. Negative controls had to be within the following ranges:
- TA 97a ± S9: 150-450
- TA 98 ± S9: 15-50
- TA 100 ± S9: 60-200
- TA 102 ± S9: 300-600
- TA 1535 ± S9: 5-30

4. The induction rates of the positive controls had to be ≥ 2

Evaluation criteria:

Induction rate of the mean values was calculated for each concentration (= revertant colonies of test item/ revertant colonies of the corresponding negative control)
The test item was to be interpretated as mutagenic if there was a concentration effect relationship and the induction rate ≥ 2.

Statistics:

None

Species / strain:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See Table 7.6.1/2

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: the test material is not classified as a VOC
- Water solubility: the test material was solubilised in DMSO to improve solubility
- Precipitation: none observed


RANGE-FINDING/SCREENING STUDIES: See table 7.6.1/2.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges of the laboratory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/2: Cytotoxicity results

Strain	S9	Lowest cytotoxic concentration (mg/plate)
TA 97a	-	5
	+	-
TA 98	-	-
	+	-
TA 100	-	5
	+	5
TA 102	-	-
	+	-
TA 1535	-	5
	+	5

Conclusions:

HR 02/197007 is not mutagenic with and without metabolic activation in S. typhimurium strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 were exposed to HR 02/197007 diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. In a preliminary toxicity assay, citotoxicity was assessed between 0.016 and 5 mg/plate, depending on bacterial strain type.

The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation.

HR 02/197007 caused reduction in the growth of the bacterial background lawn at 5 mg/plate for TA 100 and TA 1535 with and without metabolic activation and for TA 97a without metabolic activation. All strains were tested between 0.05 and 5 mg/plate in a first experiment and between 0.016 and 1.6 mg/plate in a second experiment, when citotoxicity was observed at 5 mg/plate. The same experimental conditions were repeated in the second experiment when no citotoxicity was observed.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

 

HR 02/197007 is not mutagenic with and without metabolic activation in S. typhimurium strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)