2-methylundecanal

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was assessed for genotoxicity according to OECD guideline 471 and 490 with and without metabolic activation. The test substance was found not to be mutagenic both in the presence and absence of a metabolising system in both tests.

Aldehyde C12 MNA was also assessed in the OECD 487 with and without metabolic activation and was found not to be clastogenic or aneugenic in human lymphocytes under the experimental conditions described.

Link to relevant study records

Reference

Reference 1

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:

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

Specific details on test material used for the study:

Identification: ALDEHYDE C 12 MNA PURE
Appearance: Colourless to pale yellow liquid
Batch: VE00487208
Test item storage: At room temperature protected from light container flushed with nitrogen
Stable under storage conditions until: 09 March 2018 (expiry date)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg bw) & ß-naphthoflavone (100 mg/kg bw)

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: 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30 and 35 μg/ml exposure medium.

In the absence of S9-mix, the dose levels of 25, 30 and 35 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the presence of S9-mix, no dose level with a cell survival below 27% was reached, therefore this experiment was rejected (see Table 6). In the repeat experiment (1A), the following dose-range was selected:
1, 5, 10, 20, 30, 35, 40, 45, 50, 55 and 60 μg/ml exposure medium.
Initially, the experiment in the presence of S9-mix was rejected twice, since no acceptable responses in the solvent controls were obtained. No data is reported.
In the presence of S9-mix, the dose levels of 40 to 60 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.05, 0.1, 0.5, 1, 5, 10, 15 and 20 μg/ml exposure medium.
With S9-mix: 1, 5, 10, 20, 30 and 35 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: 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20 and 25 μg/ml exposure medium.

Vehicle / solvent:

The vehicle for the test item was ethanol (Merck, Darmstadt, Germany).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With Metabolic Activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

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, EC).
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 kept below 1 x 106 cells/ml.

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10-6).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

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 and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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.
Although the mutation frequency of one of the solvent control cultures in the first experiment in the absence of S9-mix and in the second experiment was just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges.
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 suspension growth over the two-day expression period for cultures treated with ethanol was between 15 and 18 (3 hour treatment) and 80 and 86 (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, ALDEHYDE C 12 MNA PURE is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Reference 2

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)

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: ALDEHYDE C 12 MNA PURE
Appearance: Colourless to pale yellow liquid
Batch: VE00487208
Test item storage: At room temperature protected from light container flushed with nitrogen
Stable under storage conditions until: 09 March 2018 (expiry date)

Species / strain / cell type:

lymphocytes: human

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. ALDEHYDE C 12 MNA PURE was tested in the absence and presence of S9-mix.

Lymphocytes (0.4 ml blood of a healthy 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 ALDEHYDE C 12 MNA PURE for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma) was added to the cells
simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time.
The highest tested concentration was based on toxicity observed in Test Facility Study Number 517125 (50 μg/ml).
After 3 hours exposure to ALDEHYDE C 12 MNA PURE 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 re-suspended 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 ALDEHYDE C 12 MNA PURE 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.

Vehicle / solvent:

The vehicle for the test item was ethanol.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine (without metabolic activiation): used as a direct acting aneugen at a final concentration of 0.1 μg/ml for a 3 hour exposure period and 0.05 μg/ml for a 24 hour exposure period.

Details on test system and experimental conditions:

Preparation of Test Item
No correction was made for the purity/composition of the test item.
A solubility test was performed based on visual assessment. The test item was dissolved in ethanol absolute (Merck, Darmstadt, Germany). Amber glassware or containers wrapped in aluminium-foil were used for test item formulations. Test item concentrations were used within 1 hour after preparation. The final concentration of the solvent in the culture medium was 0.5% (v/v).
Any residual volumes were discarded.

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 volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2016) are presented below:
Dose-range finding study: age 34, AGT = 13.4 h
First cytogenetic assay: age 31, AGT = 13.8 h
Cytogenetic assay 1A: age 35, AGT = 13.9 h
Second cytogenetic assay: age 35, AGT = 13.9 h
Cytogenetic assay 2A: age 29, AGT = 14.2 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 (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (Life Technologies) 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., Dartford, United Kingdom) was added.

Environmental conditions
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 44 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.8 - 37.1°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 physiological saline: 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 Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life Technologies).
The above solution was filter (0.22 m)-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).

First Cytogenetic Assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of ALDEHYDE C 12 MNA PURE 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 re-suspended 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. An additional experiment was performed to obtain appropriate cytotoxicity. This experiment was rejected since it did not meet the acceptability criteria described in the study plan.
Consequently this experiment was repeated.

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 ALDEHYDE C 12 MNA PURE 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. An additional experiment was performed to obtain more data concerning the cytotoxicity of the test item.

Preparation of Slides
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). 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 Charles River Den Bosch 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 Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded
and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

Cytotoxicity Assessment
A minimum of 500 cells (with a maximum deviation of 5%) 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 item or control treatment culture
c = vehicle control culture

Three analyzable 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 Charles River Den Bosch 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. Since the lowest concentration of MMC-C and CP resulted in a positive response the highest concentration was not examined 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:

An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

In case the Chi-square test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The ability of ALDEHYDE C 12 MNA PURE to induce micronuclei in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analyzed was selected based on toxicity, cytokinesis-block proliferation index of 55 ± 5%.

The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database.

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. In addition, the number of mono- and binucleated cells with micronuclei 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.

ALDEHYDE C 12 MNA PURE did not induce a statistically significant and 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 experiments.

Conclusions:

In conclusion, this test is valid and that ALDEHYDE C 12 MNA PURE is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Reference 3

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:

1999-08-12 to 1999-09-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Names of test material (as cited in study report): Aldehyde C12 MNA; undecanal, 2-methyl
- Substance type: Colourless liquid
- Physical state: Liquid
- Analytical purity: No data
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: No data
- Batch No: 8090059
- Expiration date of the batch: No data
- Stability under test conditions: No data
- Storage condition of test material: Cool and dry
- Other: Metabolism by S9: Not known

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable.

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver homogenate

Test concentrations with justification for top dose:

Dose range-finding test: 5 to 5000 µg per plate.
Main study: 15, 50, 150, 500, 1500, 5000 µg per plate.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle:

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 100 µL/plate

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Concentration - 0.7 µg/plate. Vehicle - distilled water. Without S-9 mix for strains TA102, TA1535

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Concentration - 2.5 µg/plate. Vehicle - DMSO. Without S-9 mix for strain TA98.

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Concentration - 50 µg/plate. Vehicle - distilled water. Without S-9 mix for strains TA100 and TA1535, with S9 mix for strains TA98 and TA100

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Concentration - 0.15 µg/plate. Vehicle - distilled water. Without S-9 mix for strain TA102.

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Concentration - 50 µg/plate. Vehicle - DMSO. Without S9 mix for strain TA1537, with S9 mix for strains TA98 and TA100.

Details on test system and experimental conditions:

STRAINS
Strains were obtained from Bruce Ames, University of California, Berkeley, California, USA. For details on the strains used, see the "any other information materials and methods incl. tables" section below.

STORAGE OF TESTER STRAINS
Frozen permanents of the tester strains were stored in liquid nitrogen (at -196 °C). They were prepared from fresh overnight cultures to which 8 % DMSO was added for cryoprotection. From these mater plates were prepared on Vogel-Bonner minimal medium plates enriched with histidine (260 µM) and biotin (3 µM). Ampicillin (25 µg per mL medium), was added to the plates used for strains with the R-factor.

PREPARATION OF CULTURES
Cultures were grown in Oxoid nutrient broth No. 2 (2.5 %) on a shaker at 37 °C for 14-15 hrs to a density of 0.5-3 ×10⁹ cells per mL. They were kept at 4 °C. Only fresh cultures were used for mutagenicity assays.

For the determination of the cell titre, 0.1 mL aliquots of the 1×10⁻⁶ dilution were spread over the surface of complete medium plates (agar - 1.5 %; Difco bacto nutrient broth - 0.8 %; NaCl - 0.5 %). The titre plates were incubated overnight at 37 °C.

METABOLIC ACTIVATION SYSTEM
9000 g supernatant of liver homogenates (S9) was prepared from Sprague-Dawley male rats (8-10 weeks old) induced with Aroclor 1254 (500 mg/kg body weight).

Each S9 lot was tested for protein concentration (Lowry method), sterility, and the ability of enzymatic stimulation of benzo(a)pyrene and 2-aminoanthreacene induced mutagenesis with tester strains TA98, Ta100 and Ta102.

10% S9 mix was freshly prepared for each mutagenicity assay. The co-factors were; distilled water - 0.335 mL; phosphate buffer (0.2 M, pH 7.4) - 0.5 mL; NADP (0.1 M) - 0.04 mL; glucose-6-phosphate (1M) - 0.005 mL; MgCl₂/KCl (0.4 M/1.65 M) - 0.02 mL; S9 fraction 0.1 mL. The S9 mix was kept on ice.

TEST PROCEDURE
The study was performed by the plate incorporation method with and without S9 mix.

Each culture tube initially contained 2 mL of top agar - 0.6 % agar, 0.5 % NaCl, 10 % of 0.5 mM stock solution of L-histidine and D-biotin. The molten agar was kept at 45 °C in sterile culture tubes. To each culture tube, 0.1 mL of bacteria was added followed by the test solution and 0.5 mL of S9 mix or phosphate buffer in the assays without metabolic activation. The test components were mixed in a vortex and immediately poured onto coded minimal agar plates and carefully spread to achieve a uniform distribution of the top agar on the surface of the plate. The minimal agar plates contained 20 to 25 mL of 1.5 % agar in Vogel-Bonner medium E with 2 % glucose. Three parallel plates were prepared for each experimental point. Plates were kept for 48 to 72 hrs at 37 °C in the dark and then examined for the number of revertant colonies (his* revertants). The plates were also examined for the existence of a normal background lawn and/or precipitates and microscopically for microcolony growth.

Where there was any question regarding the nature of the colonies scored as revertants and when positive mutagenic results were obtained, the genotype of revertant colonies would be spot-checked by picking and streaking on histidine-free plates.

To confirm the reversion properties and specificity of the bacterial strains as well as the activity of the liver homogenates used, positive mutagenesis controls were run simultaneously. The genotypes of the tester strains (histidine requirement, spontaneous reversion frequency, sensitivity to UV light, crystal violet and ampicillin) were checked in each experiment. The Vogel-Bonner minimal plates, the test compound, and the S9 mix were checked for sterility.

The experiment was repeated in full after an interval of at least 3 days (first experiment finished 1999-08-26; second started 1999-08-30).

Evaluation criteria:

No data

Statistics:

Standard deviation

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Bacteriotoxic towards strains TA100, TA102, TA1535, TA537 at 500 µg per plate and to strain TA98 at 5000 µg per plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test compound failed to induce a significant increase in the mutation frequency of any of the tester strains in the absence and presence of a metabolic activation system. In one of the experiments without S9 a slight increase of the mutation frequency of strain TA102 was observed - however the effect was not significant and not reproducible.

The estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a χ² test, did not reveal a significant effect at any of the test points in the absence and presence of a metabolising system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The results of the two independent main studies, AM083991 and AM083992, are shown in the following tables (see attached background material):

• Table 1. Results of reverse mutation test AM083991 without metabolic activation.
• Table 2. Results of reverse mutation test AM083991 with metabolic activation.
• Table 3. Results of reverse mutation test AM083992 without metabolic activation.
• Table 4. Results of reverse mutation test AM083991 with metabolic activation.

Conclusions:

The test substance was assessed for genotoxicity according to OECD guidline 471 with and without metabolic activation. The test substance was found not to be mutagenic both in the presence and absence of a metabolising system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The results of the following OECD 471, 487 and 490 tests performed on the substance did not show any evidence of mutagenicity.