Allyl 2-cyanoacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation Reverse Mutation Assay using Bacteria (Salmonella typhimurium and Escherichia coli) with Allyl 2-cyanoacrylate

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Comply “Chemicals Act” of the Federal Republic of Germany, Appendix 1 to § 19a, last amended by Article 4 of the Act of October 23, 2020 (BGBl. I p. 2232); Consensus Document of the National and Länder Working Party on Good Laboratory Practice“) on the archiving and storage of records and materials, 5 May 1998; OECD Principles of Good Laboratory Practice (as revised in 1997) OECD Environmental Health and Safety Publications; Series on Principles of Good Laboratory Practice and Compliance Monitoring - Number 1. Environment Directorate, Organisation for Economic Co-operation and Development, Paris 1998. The OECD Principles of Good Laboratory Practice are accepted by regulatory authorities throughout the European Community, USA and Japan. This study was assessed for compliance with the study plan and the Standard Operating Procedures of Eurofins Munich. The study and/or the test facility are inspected periodically by the Quality Assurance Unit according to the corresponding SOPs. These inspections and audits are carried out by the Quality Assurance Unit, personnel independent of staff involved in the study. A signed quality assurance statement, listing all performed audits, is included in the report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test",
adopted 21 July 1997, corrected 26 June 2020; Commission Regulation (EC) No. 440/2008 B.13/14: ”Mutagenicity – Reverse Mutation Test using Bacteria“, dated May 30, 2008; EPA Health Effects Test Guidelines, OCSPP 870.5100 "Bacterial Reverse Mutation Test" EPA 712-
C-98-247, August 1998

Deviations:

not applicable

Remarks:

The OECD Guideline for Testing of Chemicals, Section 4, No. 471 – Bacterial Reverse Mutation Test - recommends using a combination of S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101).

Principles of method if other than guideline:

none

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Allyl 2-cyanoacrylate

EC Number:

230-796-7

EC Name:

Allyl 2-cyanoacrylate

Cas Number:

7324-02-9

Molecular formula:

C7H7NO2

IUPAC Name:

prop-2-en-1-yl 2-cyanoprop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

Name: Allyl 2-cyanoacrylate
CAS No.: 7324-02-9
Batch No.: L220FD9037
Molecular Weight: 137 g/mol
Physical State: liquid
Colour: clear, yellowish
Active Components: 96.5 %
Expiry Date: 31 March 2021
Storage Conditions: 2 – 8 °C, protected from light

Method

Target gene:

Four strains of S. typhimurium and one strain of E. coli WP2 uvrA (pKM101) with the following
characteristics were used:
TA98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA1537: his C 3076; rfa-; uvrB-: frame shift mutations
E. coli: WP2 uvrA (pKM101): trp-; uvrA-: base-pair substitutions

Species / strain

Species / strain / cell type:

bacteria, other: S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
Experiment II:
0.0050, 0.0158, 0.050, 0.158, 0.50 and 1.58 μL/plate
Toxic effects of the test item were observed in experiment I in tester strain TA100 at concentrations of 0.316 μL/plate and higher (without metabolic activation) and in tester strain TA1535 at a concentration of 5.0 μL/plate (without metabolic activation).

Vehicle / solvent:

yes

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

A. dest., DMSO, were treated in the same way as all dose groups

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Toxic effects of the test item were observed in experiment I in tester strain TA100 at concentrations of 0.316 μL/plate and higher (without metabolic activation) and in tester strain TA1535 at a concentration of 5.0 μL/plate (without metabolic activation)

Remarks:

S. typhimurium: TA100, TA1535; NaN3; sodium azide
S. typhimurium: TA98, TA1537; 4-NOPD; 4-nitro-o-phenylene-diamine
E. coli WP2 uvrA (pKM101);MMS; methylmethanesulfonate
S. typhimurium: TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA
(pKM101); 2-AA; 2-am

Details on test system and experimental conditions:

Pre-Experiment for Toxicity
The toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).
Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
The test item was tested in the pre-experiment at the following concentrations:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate

Exposure Concentrations
The test item concentrations to be applied in the main experiments were chosen according to the
results of the pre-experiment 5.0 μL/plate was selected as the maximum concentration for experiment I and 1.58 μL/plate for experiment II. The concentration ranges covered two logarithmic decades. Two independent experiments were performed at the following concentrations:
Experiment I:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
Experiment II:
0.0050, 0.0158, 0.050, 0.158, 0.50 and 1.58 μL/plate
As the results of the pre-experiment were in accordance with the criteria of validity (10.8), these were reported as a part of the main experiment I.
Experimental Performance
For the plate incorporation method, the following materials were mixed in a test tube and poured over
the surface of a minimal agar plate:
50 μL Test solution at each dose level or solvent control,
100 μL Negative control or reference mutagen solution (positive control),
500 μL S9 mix (for testing with metabolic activation) or S9 mix
substitution buffer (for testing without metabolic activation),
100 μL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of
the strain),
2000 μL Overlay agar.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).

Statistics:

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at
least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Toxic effects of the test item were observed in experiment I in tester strain TA100 at concentrations of 0.316 μL/plate and higher (without metabolic activation) and in tester strain TA1535 at a concentration of 5.0 μL/plate (without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item Allyl 2-cyanoacrylate was investigated for its potential to induce gene mutations
according to the plate incorporation test (experiment I and II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
Experiment I:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
Experiment II:
0.0050, 0.0158, 0.050, 0.158, 0.50 and 1.58 μL/plate
Precipitation of the test item was observed in all tester strains used in experiment I and II. In
experiment I precipitation was noted at concentrations of 0.316 μL/plate and higher (with and without metabolic activation), and in experiment II precipitation was observed at concentrations of 0.5 μL/plate and higher (with and without metabolic activation). The observed precipitation did not interfere with the scoring; thus it did not impact the results.
Toxic effects of the test item were observed in experiment I in tester strain TA100 at concentrations of 0.316 μL/plate and higher (without metabolic activation) and in tester strain TA1535 at a concentration of 5.0 μL/plate (without metabolic activation).
The reduction in the number of revertants down to a mutation factor of 0.5 found in experiment II in tester strain TA1537 at a concentration of 0.050μL (without metabolic activation) was regarded as not biologically relevant due to the lack of dose-dependency and the lack of concomitant clearing of the background lawn.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Allyl 2-cyanoacrylate at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met. The negative and solvent control plates (with and without metabolic activation) were mostly within the historical control data range with the exception of a slightly higher spontaneous reversion frequency in tester strain TA100 in experiment I (without metabolic activation, mean: 153 and 176 for the negative and solvent controls, respectively). Since no technical failure occurred, the data were considered acceptable for addition to the laboratory historical database. The observed slight increase can be attributed to biological variability and was regarded as not biologically relevant and did not influence the validity of the results.

Remarks on result:

other:

Remarks:

Allyl 2-cyanoacrylate is considered to be non-mutagenic in this bacterial reverse mutation assay.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Allyl 2-cyanoacrylate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Allyl 2-cyanoacrylate is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item Allyl 2-cyanoacrylate was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I and experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
Experiment II:
0.0050, 0.0158, 0.050, 0.158, 0.50 and 1.58 μL/plate
Precipitation of the test item was observed in all tester strains used in experiment I and II (with and without metabolic activation).
Toxic effects of the test item were observed in experiment I in tester strain TA100 at concentrations of 0.316 μL/plate and higher (without metabolic activation) and in tester strain TA1535 at a concentration of 5.0 μL/plate (without metabolic activation).
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Allyl 2-cyanoacrylate at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.