Prop-2-yn-1-yl 1H-imidazole-1-carboxylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 26. Jun. 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 30. May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-yn-1-yl 1H-imidazole-1-carboxylate

EC Number:

695-595-2

Cas Number:

83395-38-4

Molecular formula:

C7H6N2O2

IUPAC Name:

prop-2-yn-1-yl 1H-imidazole-1-carboxylate

Test material form:

solid

Specific details on test material used for the study:

Name: 2-propyn-1-yl 1H-imidazole-1-carboxylate
Batch no.: 0802-2#
CAS no.: 83395-38-4
Composition: 2-propyn-1-yl 1H-imidazole-1-carboxylate
Storage: fridge (2 - 8 °C) under inert gas
Expiry date: 11. Jul. 2021
Stability : stable under storage conditions
Appearance: white solid
Purity: 99.1 %
Homogeneity: homogeneous
Production date: 13. Oct. 2020
EC no.: 695-595-2
Molecular formula: C7H6N2O2
Molecular weight: 150.13 g/mol

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Salmonella typhimurium LT2

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation by microsomal enzymes (e.g., benzo(a)pyrene)
S9-mix

Test concentrations with justification for top dose:

5000 µg/plate

Vehicle / solvent:

Dimethylsulfoxide (DMSO)
Demineralized water
Sodium chloride (0.9 % NaCl)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine, C6H7N3O2; CAS-No.: 99-56-9 and 2-Amino-anthracene, C14H11N; CAS-No.: 613-13-8

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE: Two mutation assays were carried out; one with direct plate incorporation and the second with preincubation.

DURATION:
-Plate incorporation method:
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
•100 µL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control). For the positive control MMC 2.5 µL of the stock solu-tion were applied to achieve a final concentration of 0.5 µL/plate.
•500 µL S9-mix or phos-phate buffer (for test without metabolic activation).
•100 µL bacteria suspension.
•2000 µL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ± 1 °C.

-Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:
•100 µL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control). For the positive control MMC 2.5 µL of the stock solu-tion were applied to achieve a final concentration of 0.5 µL/plate.
•500 µL S9-mix or phos-phate buffer (for test without metabolic activation).
•100 µL bacteria suspension.
After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently slewed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C.

NUMBER OF REPLICATIONS: All concentrations were performed with and without S9 mix in triplicate for both tests.

Rationale for test conditions:

All negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the meta-bolic activation system functioned properly.

Evaluation criteria:

-Determination of Titre: The determination of titre should give a number of at least 109 cells/mL, correlating to 100 colonies/plate after dilution.
-Sterility Control: No colony per plate may grow.

All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (mean ± 3 standard deviations). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic ac-tivation and all were within the historical control data ranges.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that 2-propyn-1-yl 1H-imidazole-1-carboxylate is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

Executive summary:

The study procedures described in this report were based on the most recent Guideline OECD 471 (2020) and EU Method B.13/14 (2008).

The test item2-propyn-1-yl 1H-imidazole-1-carboxylatewas tested in theBacterialreverse mutation assay with five strains of Salmonella typhimurium(TA98, TA100, TA102, TA1535 and TA1537).

The test was performed in four experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation and -S9 standing for absence of metabolic activation.

The initial experiment (exp. 1) had to be repeated (exp. 1b) due to an insufficient number of analyzable non-toxic concentrations and as a contamination of the bacterial culture was suspected for TA1535.

Exp. 2 was invalid for the strain TA1537 as a contamination of the bacterial culture was suspected, the experiment was repeated in exp. 2b for TA1537 only.

The invalid experimental part is not reported in this report, but the raw data are kept in the test facility in the GLP- archive.

 

Experiment 1:

In the first experiment,the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

Signs of toxicity were observed towards the strains TA100 (+S9) and TA1537 (+S9).

The results of this experiment showed that none of the concentrations induced a relevant increase in the number of revertants in all evaluated strains, in the presence and the absence of metabolic activation.

Exp. 1 was invalid for the strain TA1535 and not evaluated, the experiment was repeated in exp. 1b.

 

Experiment 1b:

Based on the toxicity results of experiment 1, the experiment was repeated under the same conditions with adapted concentrations for TA100 (+S9), TA1535 (+/-S9) and TA1537 (+S9).

The test item showed no precipitates on the plates at any of the concentrations.

Signs of toxicity were observed towards the strains TA100 (+S9), TA1535 (+/-S9) and TA1537 (+S9). However, at least 5 non-toxic concentrations could be evaluated for mutagenicity.

The results of this experiment showed, that none of the tested concentrations induced an increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2:

Based on the results of the experiments 1 and 1b,the test item was tested up to concentrations of 5000 µg/plate in the presence and absence of S9 mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the test item concentrations.

Signs of toxicity were observed towards the strains TA98 (+S9), TA100 (+S9), TA102 (+S9), TA1535 (+/-S9), for details see8.3.2, page29. However, at least 5 non-toxic concentrations could be evaluated for mutagenicity.

The results of this experiments showed that the test item caused no relevant or dose-related increase in the number of revertants in all evaluated bacteria strains compared to the solvent control, in both the presence and absence of metabolic activation.

Exp. 2 was invalid for the strain TA1537 and not evaluated, the experiment was repeated in exp. 2b for TA1537 only.

 

Experiment 2b:

Based on the results of experiment 1the test item was tested up to a concentration of 5000 µg/plate in the presence and absence of S9 mix in TA1537 using the pre-incubation method.

The test item showed no precipitates on the plates at any of the test item concentrations.

Signs of toxicity were observed towards the strain TA1537 with metabolic activation. However, at least 5 non-toxic concentrations could be evaluated for mutagenicity.

The results of this experiments showed that the test item caused no relevant or dose-related increase in the number of revertants in the bacteria strain TA1537 compared to the solvent control, in both the presence and absence of metabolic activation.

 

Conclusion:

Based on the results of this study it is concluded that2-propyn-1-yl 1H-imidazole-1-carboxylateis not mutagenic in theSalmonella typhimuriumstrains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.