Propanal, 3-(acetyloxy)-2,2-dimethyl-

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:

2016-01-20 to 2016-03-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use, June 2012

Version / remarks:

2012

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:

2,2-dimethyl-3-oxopropyl acetate

EC Number:

811-188-6

Cas Number:

16184-79-5

Molecular formula:

C7H12O3

IUPAC Name:

2,2-dimethyl-3-oxopropyl acetate

Test material form:

liquid

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

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):

CELLS USED
- Source of cells: Trinova Biochem GmbH (Rathenau Str. 2 (earlier: Kerkrader Str. 10));
D-35394 Giessen, Germany

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH (Rathenau Str. 2 (earlier: Kerkrader Str. 10));
D-35394 Giessen, Germany

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

Six (+S9 Mix) and seven (-S9 Mix) concentrations of the test item were tested in the main study. The test concentrations were:
-S9 Mix: 5000; 4000; 2000; 800; 320; 128 and 51.2 μg/plate;
+S9 Mix: 5000; 2000; 800; 320; 128 and 51.2 μg/plate.

Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test (Informatory Toxicity Test).

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: The suitability of the solvent had been determined in the preliminary Solubility Test.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD)

Remarks:

Strain: Salmonella TA 98; without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strain: Salmonella TA 100 and TA 1537; without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Strain: Salmonella TA 1537; without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Strain: E. coli WP2 uvrA; without metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

Strain: all strains used; with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
A dose level is considered toxic if:
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs
- other: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.

Rationale for test conditions:

According to guidelines

Evaluation criteria:

The colony numbers on the control, positive control and the test plates were determined, the mean values, standard deviations and the mutation rates were calculated.

Mutation Rate = Mean revertants at the test item (or control) treatments / Mean revertrants of vehicle control

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:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay 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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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:

not examined

Positive controls validity:

valid

Test results 3

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:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

HISTORICAL CONTROL DATA
- Positive historical control data: see "Any other information on results" table 1
- Negative historical control data: "Any other information on results" table 1

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Inhibitory effects of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case.

Any other information on results incl. tables

Table 1 Historical control values for revertants/plate (for the period of 2008-2015)

		Bacterial strains
Historical control data of DMSO control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	20.9	101.4	10.3	7.9	24.9
		SD	3.5	26.2	1.4	2.5	4.9
		Minimum	10	65	3	2	11
		Maximum	39	150	23	20	44
	+S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	27.1	114.7	12.0	8.8	34.2
		SD	4.0	19.3	1.5	2.1	5.2
		Minimum	15	71	4	3	16
		Maximum	48	161	24	20	56
Historical control data of positive control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	255.6	958.9	842.1	467.4	712.3
		SD	30.7	149.+	134.0	105.7	57.5
		Minimum	123	522	354	109	320
		Maximum	647	1927	1871	1498	1283
	+S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	1224.8	1431.9	165.4	148.0	264.7
		SD	293.8	339.9	35.1	21.3	74.2
		Minimum	409	581	85	68	141
		Maximum	2587	2923	507	407	487

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay shows that under the experimental conditions reported, the test item (acting as direct mutagen, in absence of exogenous metabolic activation) induced gene mutations by base-pair substitution in the genome of the Salmonella typhimurium TA100 tester strain investigated. Therefore, the test item is considered mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item was dissolved in dimethyl sulfoxide (DMSO). In the Initial and Confirmatory Mutation Tests the following concentrations were examined: -S9 Mix: 5000; 4000; 2000; 800; 320; 128 and 51.2 μg/plate; +S9 Mix: 5000; 2000; 800; 320; 128 and 51.2 μg/plate. An Additional Plate Incorporation Test was performed (in parallel with the pre-incubation test) with S. typhimurium TA100 in the absence of metabolic activation (-S9 Mix). In this additional experiment the corresponding Initial Mutation Test part was repeated with the same concentration levels. Bacteria In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were used. In the Additional Plate Incorporation Test the Salmonella typhimurium TA100 strain was investigated. Five bacterial strains were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In parallel with the Confirmatory Mutation Test an Additional Plate Incorporation Test (partial) was performed with Salmonella typhimurium TA100 in the absence of metabolic activation (-S9 Mix). In the performed experiments all of the validity criteria, regarding the investigated strains, negative and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. In the Initial Mutation Test following treatment with the test item significant, biological relevant revertant colony number increases, positive result was noticed at the concentration of 5000 μg/plate in S. typhimurium TA100 in the absence of exogenous metabolic activation (-S9 Mix). The revertant colony number increases showed clear dose-relationship at the further, lower concentration levels. The dose related tendencies, the unequivocal positive results were repeated in subsequent experiments: in the Additional Plate Incorporation Test, and in the Confirmatory Mutation Test. In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.