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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1-Methylimidazole did not cause gene mutations in Salmonella typhimurium (Ames test), is not mutagenic in the HPRT locus assay in CHO cells and is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9 mix
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500 and 5000 µg/plate (standard plate and preincubation test)
Vehicle / solvent:
- Vehicle used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with metabolic activation: 2-aminoanthracene, without metabolic activation: N-methyl-N' -nitro-N-nitrosoguanidine, 4-nitro-o-phenyleridiamine, 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: standard plate test (SPT) and preincubation test (PIT)
SPT: incubation at 37°C for 48 hours
PIT: preincubation at 37°C for the duration of 20 minutes; incubation at 37°C for 48 hours

NUMBER OF REPLICATIONS: three plates per dose/control
Evaluation criteria:
In general, a substance to be characterised as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control);
- dose-response relationship;
- reproducibility of the results.
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: No bacteriotoxic effect (reduced his- background growth) was observed.
SOLUBILITY: Complete solubility of test substance in aqua dest.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Not-mutagenic in the Ames test (OECD471) using S. typh. TA98, TA100, TA1535 and TA1537 +/- metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Stocks of the CHO cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/β-naphthoflavone induced rat liver S9-mix
Test concentrations with justification for top dose:
See any other information on material and methods incl. tables, table 1 and 2.
Vehicle / solvent:
Due to the good solubility of the test substance in water, culture medium (Ham's F12) was selected as vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 7.12-Dimethylbenz[a]anthracene
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 - 24 hours attachment period
- Exposure duration: In the first experiment the exposure period was 4 hours in the absence and presence of metabolic activation. The second experiment was performed with an exposure period of 24 hours in the absence and 4 hours in the presence of metabolic activation.
- Expression time (cells in growth medium): The exposure period was completed by rinsing several times with HBSS. Then the flasks were topped up with at least 20 mL Ham's F12 medium incl. 10% (v/v) FCS and left to stand in the incubator for about 3 days (4-hour treatment) or 2 days (24-hour treatment). This was followed by the 1st passage. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (2nd passage).
- Selection/fixation time (if incubation with a selection agent): For selection of the mutants, six 75 cm2 flasks with 3x10^5 cells each from every treatment group, if possible, were seeded in 10 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 - 7 days. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.


SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: Duplicate cultures were used for all experimental groups.

DETERMINATION OF CYTOTOXICITY
- Method:
Cloning efficiency 1 (CE1; survival):
For the determination of the influence of the test substance directly after the exposure period, about 200 cells per dose group were seeded in 25 cm² flasks in duplicate using 5 mL Ham's F12 medium incl. 10% (v/v) FCS. After an attachment period of 20 – 24 hours, the cells were treated with the vehicle, test substance or positive control for 4 hours or 24 hours. After exposure, the cells were rinsed several times with HBSS. Then, the cells were cultured in 5 mL Ham's F12 medium incl. 10% (v/v) FCS.
Cloning efficiency 2 (CE2; viability):
For the determination of the mutation rate after the expression period, two aliquots of about 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two flasks (25 cm²) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS each.

In all cases, after seeding the flasks were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted. The absolute and relative cloning efficiencies (%) were calculated for each test group.

CHECK OR DETERMINATION OF FURTHER PARAMETERS
- pH
Changes in pH were recorded by a change in the indicator color of the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two top doses and for the negative controls with and without S9 mix.
- Osmolarity
Osmolarity was measured in at least the top dose and the negative controls with and without S9 mix.
- Solubility
Test substance precipitation was assessed immediately after dosing the test cultures and at the end of treatment.
- Cell morphology
The test cultures of all test groups were examined microscopically for cell morphology at the end of the exposure period, which allows conclusions to be drawn about the cellular attachment.
Evaluation criteria:
Acceptance criteria:
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0.00 -16.43 mutants per 10^6 clonable cells.
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data).
• At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Assessment criteria:
A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.

Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10^6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
The pH value of the test substance preparation was adjusted by adding small amounts of 32% HCl. Osmolarity and pH values were not influenced by test substance treatment.
In this study, in the absence and the presence of S9 mix no precipitation was observed in culture medium up to the highest applied test substance concentration.
There were no adverse observations on cell morphology (cell attachment).

COMPARISON WITH HISTORICAL CONTROL DATA:
In this study, no relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolizing system. In both experiments, after 4 and 24 hours treatment with the test substance, the values for the corrected mutation frequencies (MFcorr.: 0.00 – 6.13 per 10^6 cells) were close to the respective vehicle control values (MFcorr.: 1.38 – 2.18 per 10^6 cells) and clearly within the range of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 16.43 per 10^6 cells; with S9 mix: MFcorr.: 0.00 – 15.83 per 10^6 cells).

The positive control substances EMS (without S9 mix; 300 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 78.86 – 497.43 per 10^6 cells; with S9 mix: MFcorr.: 148.67 – 346.92 per 10^6 cells) were within our historical positive control data range (without S9 mix: MFcorr.: 47.35 – 1 338.10 per 10^6 cells; with S9 mix: MFcorr.: 131.35 –
1250.00 per 10^6 cells).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In both experiments, in the absence and presence of S9 mix, no cytotoxicity indicated by reduced relative cloning efficiency of about or below 20% relative survival was observed up to the top dose applied.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Under the experimental conditions of the study, the test substance is not mutagenic in the HPRT locus assay (OECD476) under in vitro conditions in CHO cells +/- metabolic activation.
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
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:
other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Stocks of the V79 cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for; mycoplasma contamination, karyotype stability, plating efficiency (=colony forming ability) incl. vital staining.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/β-naphthoflavone induced rat liver S9-mix
Test concentrations with justification for top dose:
1st experiment
13.3, 26.6, 53.1, 106.3, 212.5, 425.0, 850.0 μg/mL (4-hour exposure, with and without S9 mix)

2nd experiment
26.6, 53.1, 106.3, 212.5, 425.0, 850.0 μg/mL (24-hour exposure, without S9 mix)
159.4, 318.8, 637.5, 850.0 µg/mL (4-hour exposure, with S9 mix)

3rd experiment
159.4, 318.8, 637.5, 850.0 µg/mL (4-hour exposure, with S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium (Minimal Essential Medium: MEM)
- Justification for choice of solvent/vehicle: the vehicle was chosen due to the good solubility of the test substance in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; preincubation

DURATION
- Preincubation period: 6 hours
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 20 hours (when 4 hours exposure) or none (when 24 hours exposure)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

STAIN (for cytogenetic assays): Wrights solution (modified May-Grünwald solution) and counterstain with Giemsa/Titrisol solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: relative increase in cell count (RICC)
Evaluation criteria:
As a rule, at least 1000 cells per culture, means at least 2000 cells per dose group, were evaluated and the number of micronucleus-containing cells was recorded.
The analysis of micronuclei was carried out following the criteria of Countryman and Heddle:
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only cells clearly surrounded by a nuclear membrane were scored.
Slides were coded before microscopic analysis. Cultures with few isolated cells were not analysed for micronuclei.

The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells.
- The number of cells containing micronuclei in the negative control was within the range of the historical negative control data.
- The positive control substances both with and without S9 mix induced a significant increase in the number of micronucleated cells.

A test substance is considered "positive" if the following criteria are met:
- A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
- The number of micronucleated cells exceeds both the value of the concurrent negative control and the range of the historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
- The number of micronucleated cells in the dose groups is not significant increased above the concurrent negative control value and is within the range of the historical negative control data.
Statistics:
The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH values were not influenced by test substance treatment.
The pH of the stock solutions was adjusted to physiological values using small amounts of HCl. No precipitation of the test substance in culture medium was observed.

RANGE-FINDING/SCREENING STUDIES:
In the pretest the parameters pH value and osmolarity were not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations measured.
In addition, no test substance precipitation in culture medium occurred up to the highest applied concentration of 85 μg/mL at the end of treatment in the absence and presence of S9 mix.
After 4 hours treatment in the absence and presence of S9 mix and after 24 hours treatment in the absence of S9 mix no cytotoxicity was observed up to the highest applied concentration by reduced RICC of about or below 40 - 50%.

COMPARISON WITH HISTORICAL CONTROL DATA:
In this study, no biologically relevant increase in the number of micronucleated cells was observed either without S9 mix or after the addition of a metabolizing system. In all valid experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.4 – 1.7% micronucleated cells) were close to the concurrent negative control values (0.5 – 1.4% micronucleated cells) and within our historical negative control data range (0.1 - 1.8% micronucleated cells).
The positive control substances EMS (without S9 mix; 500 μg/mL) and CPP (with S9 mix; 2.5 μg/mL) induced statistically significant increased micronucleus frequencies in three independently performed experiments. In this study, in the absence and presence of metabolic activation the frequency of micronucleated cells (2.5 – 25.4% micronucleated cells) was clearly above the range of our historical negative control data range (0.1 - 1.8% micronucleated cells) and close to or within our historical positive control data range (2.3 – 23.5% micronucleated cells).
In the 2nd Experiment in the presence of a metabolizing system the positive control group showed not the expected increase in the number of cells containing micronuclei (CPP 2.5 μg/mL: 1.7% micronucleated cells; historical positive control data range: 2.3 – 23.5%). Thus, this part of the 2nd Experiment with metabolic activation did not fulfill the acceptance criteria and the results are excluded from the assessment of the genotoxic potency.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The proliferation index (PI) is based on the scoring of 1000 cells per culture (2000 cells per test group) for the different test groups without and with metabolic activation and includes the measurement of colony size.
In this study, in the absence and the presence of S9 mix no cytotoxicity indicated by reduced PI values was observed.
In addition, in all experiments in the absence and the presence of S9 mix no growth inhibition indicated by reduced cell counts was observed.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Under the experimental conditions described (MNT/OECD487), the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells +/- metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

1-Methylimidazol was tested in the Ames reverse mutation assay (performed according to OECD guideline 471) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 at 20 to 5000 µg/plate (standard plate and preincubation test) with and without metabolic activation (1989). 1-Methylimidazol was not cytotoxic at the tested concentrations. No precipitation of the test substance was observed. Under the conditions tested, 1-methylimidazol was not mutagenic in any of the Salmonella typhimurium strains.

In a GLP-compliant OECD 476 guideline study, the substance 1-Methylimidazol was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (2012). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). In the first experiment the treatment period was 4 hours without S9 mix (0; 106.25; 212.50; 425.00; 850.00μg/mL) and with S9 mix (0; 106.25; 212.50; 425.00; 850.00μg/mL). In the second experiment the treatment period was 24 hours without S9 mix (0; 106.3; 212.5; 425.0; 850.0μg/mL) and 4 hours with S9 mix (0; 125.0; 250.0; 500.0; 850.0μg/mL). Following an expression phase of about 6 – 8 days the cells were maintained in selection medium for an additional week for formation of hprt mutants. Finally, the Giemsa stained colonies were counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance 1-Methylimidazol is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

In a GLP-compliant OECD 487 guideline study 1-Methylimidazol was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) (2013). Three independent experiments were carried out with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the doses tested in the first experiment were 0; 13.3; 26.6; 53.1; 106.3; 212.5; 425.0; 850.0 μg/mL (4 hours exposure, with and without S9 mix). In the second experiment the doses tested were 0; 26.6; 53.1; 106.3; 212.5; 425.0; 850.0 μg/mL (24 hours exposure, without S9 mix) and 0; 159.4; 318.8; 637.5; 850.0 μg/mL (4 hours exposure, with S9 mix). In the third experiment the doses tested were 0; 159.4; 318.8; 637.5; 850.0 μg/mL (4 hours exposure, with S9 mix). A sample of 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group.The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei, except in the 2nd Experiment in the presence of metabolic activation. Therefore, this experimental part was judged invalid and it was excluded from the assessment of the genotoxic potency. In this study, no cytotoxicity indicated by reduced relative increase in cell count (RICC) or proliferation index (PI) was observed up to the highest required test substance concentration. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other.

Thus, under the experimental conditions described, 1-Methylimidazol is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Based on the results of the in vitro genotoxicity studies, 1-methylimidazole does not need to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.