2-methyl-3-(p-tolyl)propionaldehyde

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:

From June 22, 2021 to September 17, 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

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

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-3-(p-tolyl)propionaldehyde

EC Number:

255-410-4

EC Name:

2-methyl-3-(p-tolyl)propionaldehyde

Cas Number:

41496-43-9

Molecular formula:

C11H14O

IUPAC Name:

2-methyl-3-(4-methylphenyl)propanal

Test material form:

liquid

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

- Induction of Liver Enzymes
Male Wistar rats (410-708 g, animals were 9-25 weeks old) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg bw/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed. Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used for S9 preparation used in this study occurred on 11 January 2021.

- 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 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC. The date of preparation of S9 fractions for this study was 14 January 2021.
The sterility of the preparation was confirmed in each case. The protein concentration of the preparation was determined by a chemical analyzer at 540 nm. The mean protein concentration of the S9 fraction used was determined to be 21.1 g/L.
The biological activity in the Salmonella assay of S9 was characterized in each case using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that require metabolic activation by microsomal enzymes. The batches 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.

- 0.2 M Sodium Phosphate Buffer, pH 7.4
*Solution A:
Na2HPO4 x 12 H2O: 71.63 g
Distilled water: q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
*Solution B:
NaH2PO4 x H2O: 27.6 g
Distilled water: q.s. ad 1000 mL
Sterilization was performed at 121°C 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:

Based on the results of the preliminary test, a 100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 1581 μg test item/plate due to cytotoxicity at the higher concentration.
Examined concentrations in Assay 1 were 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.
Examined concentrations in Assay 2 were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.

Vehicle / solvent:

In the study two vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive control chemicals. The following chemicals were used for vehicle (solvent) control groups:

- Dimethyl sulfoxide (DMSO):
Supplier: VWR
Batch No.: 20H054003
Expiry date: 28 July 2025
Purity: 100%

- Distilled water:
Manufacturer: MAGILAB Kft.
Batch No.: 202104032
Expiry date: 29 October 2021

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and distilled water.

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other:

Remarks:

With S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and distilled water.

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other:

Remarks:

Without S9-mix

Details on test system and experimental conditions:

TEST SYSTEM
- Procedure for Growing Cultures
The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37°C in a gyrotory water bath shaker

- Viability of the Testing Cultures
The viability of each testing culture was determined by plating 0.1 mL of the 105, 106, 107 and 108 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37°C.

- The Typical Composition (g/1000 mL) of Minimal Glucose Agar
Glucose: 20.0 g
Magnesium sulfate: 0.2 g
Citric acid: 2.0 g
di-Potassium hydrogenphosphate: 10.0 g
Sodium ammonium hydrogenphosphate: 3.5 g
Agar agar: 13.0 g
Distilled water: q.s. ad1 1000 mL

- Nutrient Broth No.2
Nutrient Broth No.2: 25.0 g
Distilled water: q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

- Nutrient Agar
Nutrient Agar: 20.0 g
Distilled water: q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

- Top Agar for Salmonella typhimurium Strains
*Agar solution:
Agar Bacteriological: 4.0 g
NaCl: 5.0 g
Distilled water: q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
*Histidine – Biotin solution (0.5 mM):
D-Biotin (F.W. 244.31): 122.2 mg
L-Histidine x·HCl x H2O (F.W. 209.63): 104.8 mg
Distilled water: q.s. ad 1000 mL
Sterilization was performed by filtration using a 0.22 μm membrane filter.
*Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM): 100 mL
Agar solution: 900 mL

- Top Agar for Escherichia coli Strain
*Tryptophan solution (2 mg/mL):
L-Tryptophan (F.W. 204.23): 2000 mg
Distilled water: q.s. ad 1000 mL
Sterilization was performed by filtration using a 0.22 μm membrane filter.
*Complete Top Agar for Escherichia coli strain:
Nutrient Broth: 50 mL
Tryptophan solution (2 mg/mL): 2.5 mL
Agar solution (see section 10.2.4.): 947.5 mL

TEST PROCEDURE
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test, an Assay 1 and Assay 2. In the Preliminary Range Finding Test and Assay 1, the plate incorporation method was used and in the Assay 2 the pre-incubation method was used.

- Concentrations
Concentrations for the main tests were selected on the basis of the Preliminary Compatibility Test and Preliminary Range Finding Test. In the Assay 1 and Assay 2, slightly different concentrations were used.

- Preliminary Compatibility Test
The solubility of the test item was examined using Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF) and acetone. Clear solubility was observed at 100 mg/mL concentration using all solvents. Due to the better biocompatibility, DMSO was selected as vehicle for the study. The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

- Preliminary Range Finding Test
Based on the solubility test, a 100 mg/mL stock solution was prepared in DMSO. Six test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and 4 times approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Range Finding Test the plate incorporation method was used.

- Experimental method - Procedure for Exposure in the Assay 1
Assay 1 followed the standard plate incorporation procedure. Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar: 2000 μL
vehicle or test item formulation (or reference controls): 50 μL
overnight culture of test strain: 100 μL
phosphate buffer (pH 7.4) or S9 mix: 500 μL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 67(±1) hours.

- Experimental method - Procedure for Exposure in the Assay 2
Assay 2 followed the standard pre-incubation procedure since no biologically relevant increase in the number of revertant colonies was observed in the Assay 1.
Bacteria (cultured in Nutrient Broth No.2.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48(±1) hours.

Rationale for test conditions:

Test conditions follow the OECD 471 guideline.

Evaluation criteria:

The study was considered valid if:
• the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
• at least five analysable concentrations are presented in all strains of the main tests.

A test item is considered mutagenic if:
• a dose–related increase in the number of revertants occurs and/or;
• a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
• the number of reversions is 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 is 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, 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.

A test article is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

Statistics:

Not used.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98, TA100 and E.coli WP2 uvrA.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Assay 1: at 1581 μg/plate concentration (+/- S9) Assay 2: at 500 and 1581 μg/plate concentration (+/- S9), at 158.1 μg/plate (-S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In Assay 1, the plate incorporation method was used. In Assay 2 the pre-incubation method was used. The assays were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain. The assays were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.
Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1 were 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate and in the Assay 2 were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.
No precipitate was detected on the plates in the main tests in the examined bacterial strains with and without metabolic activation.
Inhibitory, cytotoxic effect of the test item (absent / reduced / slight reduced background lawn development) was observed in the Assay 1 in the examined bacterial strains with and without metabolic activation at 1581 μg/plate concentration. Same cytotoxic effect of the test item (absent / reduced / slight reduced background lawn development) was observed in the Assay 2 in the examined bacterial strains with and without metabolic activation at 1581 and 500 μg/plate concentrations and reduced / slight reduced background lawn development was observed in Assay 2 in the examined bacterial strains without metabolic activation at 158.1 μg/plate concentration.
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA100 bacterial strain at 1.581 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.18). 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 Assay 2 (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 5 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.17). 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 main assays 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.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
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.

Any other information on results incl. tables

Preliminary range finding test:

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

Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.

No precipitate was detected on the plates in the preliminary experiment in both examined bacterial strains with and without metabolic activation.

Inhibitory, cytotoxic effect of the test item (absent / reduced / slight reduced background lawn development) was observed in the preliminary experiment in both examined bacterial strains with and without metabolic activation at 5000, 2500 and 1000 μg/plate concentrations.

Validity of the test:

Untreated, negative (vehicle/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. At least five analysable concentrations were presented in all strains with and without metabolic activation.

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. The study was considered to be valid.

Applicant's summary and conclusion

Conclusions:

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 metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
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 2-methyl-3-(p-tolyl)propionaldehyde had no mutagenic activity on the growth of the 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/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Plate Incorporation Method), an Assay 1 (Plate Incorporation Method) and an Assay 2 (Pre-Incubation Method).
Based on the results of the Compatibility Test, the test item was dissolved in Dimethyl sulfoxide (DMSO). 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 in the absence and presence of metabolic activation. Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1 were 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate and in the Assay 2 were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.
No precipitate was detected on the plates in the main tests in the examined bacterial strains with and without metabolic activation.
Inhibitory, cytotoxic effect of the test item (absent / reduced / slight reduced background lawn development) was observed in the Assay 1 in the examined bacterial strains with and without metabolic activation at 1581 μg/plate concentration. Same cytotoxic effect of the test item (absent / reduced / slight reduced background lawn development) was observed in the Assay 2 in the examined bacterial strains with and without metabolic activation at 1581 and 500 μg/plate concentrations and reduced / slight reduced background lawn development was observed in Assay 2 in the examined bacterial strains without metabolic activation at 158.1 μg/plate concentration.
In the assays 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.
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 analyzable 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 2-methyl-3-(p-tolyl)propionaldehyde had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.