2-hydroxy-3-phenoxypropyl methacrylate

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:

The study was conducted between 12 March and 01 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 02-04 June 2015 Date on Certificate: 22 September 2015

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-3-phenoxypropyl methacrylate

EC Number:

240-994-5

EC Name:

2-hydroxy-3-phenoxypropyl methacrylate

Cas Number:

16926-87-7

Molecular formula:

C13H16O4

IUPAC Name:

2-hydroxy-3-phenoxypropyl 2-methylprop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 171512
- Expiration date of the lot/batch: 23 December 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25°C, ≤70 relative humidity (RH) %), protected from humidity (tightly closed container)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
All dilutions in the main tests of test item were made in the testing laboratory using Dimethyl sulfoxide (DMSO). Test solutions were freshly prepared at the begining of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 2 hours after preparation.
No. of concentration concentration of the test item Concentration µg/plate)
1 100 mg/mL 5000
2 31.62 mg/mL 1581
3 10 mg/mL 500
4 3.162 mg/mL 158.1
5 1 mg/mL 50
6 0.3162 mg/mL 15.81
7 0.1 mg/mL 5
8 0.03162 mg/mL 1.581
concentrations 1-6 was examined in the Initial Mutation Test using the plate incorporation method, concnetrations 1-7 was examined in the Confirmatory Mutation Test using the pre-incubation method. Concentrations 2-8 was examined in the Complementary Confirmatory Mutation Test in case of Salmonella typhimurium TA1537 strain without metabolic activation.

Method

Target gene:

Not applicable

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented
post-mitochondrial S9 fraction.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Citoxlab Hungary Ltd. according to Ames et al. [1] and Maron and Ames [2]. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
The supplier, batch number and expiry date of the used chemicals described in sections 5.5.3. and 5.5.4 are summarized in Table 5 (section 5.9. Chemicals Used in the Experiments). The composition of solution refers to a final volume of 1000 mL.

Induction of Liver Enzymes
Male Wistar rats (373-446 g, animals were 9 weeks old and 292-387 g, animals were 8 weeks old at the initiation) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed. Euthanasia was performed by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used for preparation S9 used in this study was 03 July 2017 (Citoxlab code: E12660) and 05 January 2018 (Citoxlab code: E12790).

Preparation of Rat Liver-Homogenate S9 Fraction
On Day 4, the rats were euthanized and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 ± 10ºC.
The date of preparation of S9 fraction for this study was 06 July 2017 (Citoxlab code: E12660, Expiry date: 06 July 2019) and 08 January 2018 (Citoxlab code: E12790, Expiry date: 08 January 2020).
The sterility of the preparation was confirmed in each case. The protein concentration of the preparation was determined by a chemical analyser at 540 nm in the Clinical Chemistry Laboratory of Citoxlab Hungary Ltd. The mean protein concentration of the S9 fraction used was determined to be 28.6 g/L and 30.45 g/L. The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. Each batch of S9 used in this study functioned appropriately.

The S9 Mix (containing 10% (v/v) of S9)
Salt solution for S9 Mix:
NADP Na 7.66 g
D-glucose-6 phosphate Na 3.53 g
MgCl2 x 6 H2O 4.07 g
KCl 6.15 g
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration through a 0.22 μm membrane filter.

The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix (see above) 400 mL
Prior to addition to the culture medium the S9 mix was kept in an ice bath.

0.2M Sodium Phosphate Buffer, pH 7.4
Solution A:
Na2HPO4x 12 H2O 71.63 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121oC in an autoclave.

Solution B:
NaH2PO4 x H2O 27.6 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121oC in an autoclave.

0.2M Sodium phosphate buffer pH 7.4:
Solution A 880 mL
Solution B 120 mL

Test concentrations with justification for top dose:

15.81, 50, 158.1, 500, 1581, 5000 µg/plate

The maximum test concentration was 5000 μg test item/plate in the Initial Mutation Test and Confirmatory Mutation Test. The maximum test concentration was 1581 μg test item/plate in the Complementary Confirmatory Mutation Test.

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene diamine; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test as well as in the Complementary Confirmatory Mutation test, the pre-incubation method was used.

DURATION
- Preincubation period: 20 minutes at 37 ºC
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the relevant historical control range in all tester strains of the main tests;
- at least five analysable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occured and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occured in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

According to the guidelines [5] [6] [7] [8], statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Absent/reduced/slightly reduced background lawn was detected in the Confirmatory Mutation Test in all examined bacterial strains with and without metabolic activation on the plates at the concentration levels of 5000 and 1581 µg/plate.
Reduced background lawn was detected in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1537 strain without metabolic activation on the plates at the concentration level of 1581 µg/plate.

Any other information on results incl. tables

PRELIMINARY COMPATIBILITY TEST

Based on the available information and the results of the solubility testing, DMSO was selected as vehicle (solvent) of the study. The results of the Preliminary Compatibility Test are summarized in Table 6.

Table 6:The Solubility of the Test Item inDMSO

Concentration of test item in DMSO (mg/mL)	Solubility in DMSO	Solubility in the top solution (test item formulation 50 µL + phosphate buffer 500 µL + top agar 2 mL)	Test item concentration in the test plate (µg/tube)
100	clear solution	slightly opalescent, small oily drops	5000
50	clear solution	clear solution	2500

 

PRELIMINARY CONCENTRATION RANGE FINDING TEST

In the Preliminary Concentration Range Finding Test (Informatory Toxicity Test), the plate incorporation method was used. This test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 strains in the presence and absence of metabolic activation system (± S9 mix) with appropriate untreated, negative (solvent) and positive controls. In the test, each sample (including the controls) was tested in triplicate.

Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate were examined in the Preliminary Concentration Range Finding Test.

In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).

 

No precipitate was observed in the Preliminary Concentration Range Finding Test in any bacterial strains with and without metabolic activation.

 

Reduced/slightly reduced background lawn was detected in the Preliminary Concentration Range Finding Test in both bacterial strains with and/or without metabolic activation on the plates at the concentration level of 5000µg/plate.

 

The experimental results (revertant colony numbers per plate, mutation factors and standard deviations) are detailed in Table 7 (Appendix 2) and in Appendix 3.

INITIAL, CONFIRMATORY AND COMPLEMENTARY CONFIRMATORY MUTATION TESTS

In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test as well as in the Complementary Confirmatory Mutation Test, the pre-incubation method was used.

The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain in the presence and absence of a metabolic activation system (±S9 mix) with appropriate untreated, negative (solvent) and positive controls. The Complementary Confirmatory Mutation Test was carried out using Salmonella typhimurium TA1537 strain in the absence of a metabolic activation system. In the main tests, each sample (including the controls) was tested in triplicate.

In the Initial Mutation Tests, Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect

In the Initial Mutation Test (using plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 500 μg/plate concentration with metabolic activation(the observed mutation factor value was: MF: 1.28).However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.

In the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test (using the pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 15.81 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.19). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.

No precipitate was observed in the main testsinall examined bacterial strains with and without metabolic activation.

Reduced/slightly reduced background lawn was detected in the Initial MutationTest in all examined Salmonella typhimurium strains without metabolic activation on the plates at the concentration level of 5000µg/plate. Absent/reduced/slightly reduced background lawn was detected in the Confirmatory Mutation Test in all examined bacterial strains with and without metabolic activation on the plates at the concentration levels of 5000 and 1581µg/plate.

Reduced background lawn was detected in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1537 strain without metabolic activation on the plates at the concentration level of 1581µg/plate

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

The experimental results (revertant colony numbers per plate, mutation factors, and standard deviations) are summarized in Tables 8-9 (Appendix 2) and Appendix 4 and 5.

VALIDITY OF THE TESTS

Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains.

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test.

At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate.

The study was considered to be valid.

Appendices containing results tables are attached to this record.

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item PHPM (Batch Number: 171512) had no mutagenic activity on the applied bacterial strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats.

 

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Plate Incorporation Method), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and Complementary Confirmatory Mutation Test (Pre-Incubation Method).

 

Based on the results of the Preliminary Compatibility Test, the test item was dissolved in DMSO at a concentration of 100 mg/mL.Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate, in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate, in the Complementary Confirmatory Mutation Test were 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

 

In the Initial Mutation Test, Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent control. There were no dose-related trends and no indication of any treatment-related effect.

 

No precipitate was observed in the preliminary test and in the main tests in all examined bacterial strains with and without metabolic activation.

Reduced/slightly reduced background lawn was detected in the Initial MutationTest in all examined Salmonella typhimurium strains without metabolic activation on the plates at the concentration level of 5000µg/plate.

Absent/reduced/slightly reduced background lawn was detected in the Confirmatory Mutation Test in all examined bacterial strains with and without metabolic activation on the plates at the concentration levels of 5000 and 1581µg/plate.

Reduced background lawn was detected in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1537 strain without metabolic activation on the plates at the concentration level of 1581µg/plate.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item PHPM (batch number: 171512) had no mutagenic activity on the applied bacterial strains under the test conditions used in this study.