2-phenylpropionaldehyde-dimethyl acetal

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:

02 January 2019 to 22 January 2019

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labor and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries, 24 November 2000

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-phenylpropionaldehyde-dimethyl acetal

EC Number:

202-022-8

EC Name:

2-phenylpropionaldehyde-dimethyl acetal

Cas Number:

90-87-9

Molecular formula:

C11H16O2

IUPAC Name:

(1,1-dimethoxypropan-2-yl)benzene

Test material form:

liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphtha flavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Experiment 2: 50, 150, 500, 1000, 1500, 3000 and 5000 μg/plate
Experiment 3: 15, 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1 and 2: in agar (plate incorporation)
- Experiment 3: preincubation

DURATION
- Preincubation period: 20 minutes (Experiment 3)
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and 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:

valid

Positive controls validity:

valid

Additional information on results:

Controls:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
- Results for the negative controls (spontaneous mutation rates) are considered to be acceptable.
- The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1:
- There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
- No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
- There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Small, statistically significant increases in TA100 revertant colony frequency were observed at 500, 1500 and 5000 μg/plate in the absence of S9-mix. These increases were within the in-house historical vehicle/untreated control range for the strain and were, therefore considered of no biological relevance. Statistically significant increases in TA1535 revertant colony frequency were also observed at 5000 μg/plate in the absence of S9-mix. Although these counts exceeded the in-house historical untreated/vehicle control range, the increases were still considered to be of no biological relevance because there was no evidence of a dose-response relationship and could not be reproduced in a confirmatory experiment.

Experiment 2:
- There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
- No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
- No biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (repeat of plate incorporation method). Statistically significant increases in TA100 revertant colony frequency were observed in the second mutation test at 5000 μg/plate in the absence of S9-mix, however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.

Experiment 3:
Weakened bacterial background lawns were noted in the third experiment utilizing the pre-incubation modification with a toxic response observed at 5000 μg/plate to all of the tester strains in both the absence and presence of S9-mix.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Any other information on results incl. tables

Table 1: Mean number of revertants without metabolic activation

	TA100			TA1535			WP2			TA98			TA1537
(µg)	Exp 1	Exp 2	Exp 3	Exp 1	Exp 2	Exp 3	Exp 1	Exp 2	Exp 3	Exp 1	Exp 2	Exp 3	Exp 1	Exp 2	Exp 3
Solvent	106	119	130	20	20	27	24	21	16	19	27	16	11	14	12
1.5	123	-	-	26	-	-	18	-	-	18	-	-	9	-	-
5	100	-	-	32	-	-	19	-	-	21	-	-	13	-	-
15	96	-	121	24	-	27	23	-	16	21	-	17	11	-	16
50	109	123	112	15	19	25	23	26	21	15	19	16	10	14	16
150	106	127	123	39	18	26	22	19	15	17	28	22	13	12	18
500	137	115	113	39	18	19	24	21	14	16	27	15	14	14	12
1000	-	132	-	-	18	-	-	20	-	-	21	-	-	13	-
1500	131	132	117	26	15	11	21	21	16	19	20	12	9	15	8
3000	-	137	-	-	13	-	-	17	-	-	17	-	-	9	-
5000	145	140	108	68	17	13	21	17	0	16	19	12	14	7	8

 

Table 2: Mean number of revertants with metabolic activation

	TA100			TA1535			WP2			TA98			TA1537
(µg)	Exp 1	Exp 2	Exp 3	Exp 1	Exp 2	Exp 3	Exp 1	Exp 2	Exp 3	Exp 1	Exp 2	Exp 3	Exp 1	Exp 2	Exp 3
Solvent	111	130	121	11	18	26	25	28	24	25	26	31	15	11	15
1.5	101	-	-	14	-	-	30	-	-	25	-	-	14	-	-
5	109	-	-	11	-	-	26	-	-	24	-	-	9	-	-
15	110	-	104	14	-	22	26	-	25	19	-	25	10	-	13
50	114	138	120	16	12	21	23	22	31	19	35	25	10	14	9
150	128	129	128	13	18	21	28	27	23	22	25	25	14	11	9
500	123	123	129	20	15	22	33	36	23	22	33	23	12	9	10
1000	-	117	-	-	15	-	-	23	-	-	25	-	-	12	-
1500	114	134	116	8	16	12	30	19	21	26	32	25	12	13	7
3000	-	132	-	-	10	-	-	17	-	-	32	-	-	11	-
5000	97	134	85	9	9	8	25	17	14	19	25	11	8	6	6

Applicant's summary and conclusion

Conclusions:

The test material was considered non-mutagenic under the conditions of this test.

Executive summary:

The test substance was evaluated in a bacterial reverse mutation assay performed according to OECD 471 and following GLP using Salmonella typhimurium strains TA98, TA100, TA 1535 and TA1537 and Escherichia coli strain WP2 uvrA both in the presence and absence of an exogenous metabolic activation system (S9 -mix). In the first experiment, test substance concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate were assessed using the plate incorporation assay in triplicate. A small, statistical response was noted for TA1535 in Experiment 1, therefore the experiment was repeated using test concentrations of 50, 150, 500, 1000, 1500, 3000 and 5000 μg/plate. There were no statistically significant increases noted to TA1535 in Experiment 2, therefore, a third, confirmatory experiment was performed using the preincubation method at test concentrations of 15, 50, 150, 500, 1500 and 5000 μg/plate. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the first and second mutation test, the test item induced no visible reduction in the bacterial background lawns of any tester strains. In experiment 3, weakened bacterial background lawns were noted at 5000 μg/plate to all of the tester strains in both the absence and presence of S9-mix. No test item precipitate was observed on any of the plates. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Small, statistically significant increases in TA100 revertant colony frequency were observed in the first mutation test at 500, 1500 and 5000 μg/plate in the absence of S9-mix. These increases were within the in-house historical vehicle/untreated control range for the strain and were, therefore considered of no biological relevance. Statistically significant increases in TA1535 revertant colony frequency were also observed in the first mutation test at 5000 μg/plate in the absence of S9-mix. Although these counts exceeded the in-house historical untreated/vehicle control range, the increases were still considered to be of no biological relevance because there was no evidence of a dose-response relationship and could not be reproduced in further experiments. In the second experiment, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Statistically significant increases in TA100 revertant colony frequency were observed in the second mutation test at 5000 μg/plate in the absence of S9-mix, however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance. In experiment 3, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Although the increases noted for TA1535 in Experiment 1 were quite large (3.5 fold the concurrent vehicle control), they were considered to be of no biological relevance because they could not be reproduced in two further experiments (one of which contained intermediate test item concentration levels in an attempt to qualify the response). There were many different sized colonies noted at the statistically significant dose level (5000 μg/plate) and there may have been an issue with underlying contamination but it was considered that the results should be confirmed in further tests. Therefore, the test material was considered to be non-mutagenic under the conditions of this test.