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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Bacterial Reverse Mutation Assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-09-25 to 2019-11-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Due to unscheduled delay of reporting the Draft Report was issued later than indicated in the Study Plan. This fact had no impact on the results or integrity of the study.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
Due to unscheduled delay of reporting the Draft Report was issued later than indicated in the Study Plan. This fact had no impact on the results or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(2E)-2-methyl-3-phenylacrylaldehyde
EC Number:
701-219-0
Cas Number:
15174-47-7
Molecular formula:
C10H10O
IUPAC Name:
(2E)-2-methyl-3-phenylacrylaldehyde
Test material form:
liquid
Details on test material:
Test item name: (2E)-2-methyl-3-phenylacrylaldehyde
Other name: (E)-α-Methylcinnamaldehyde; trans-α-Methylcinnamaldehyde; Cyprinal
CAS No.: 15174-47-7 (can also be described by alternate CAS No. 101-39-3)
EC number: 701-219-0
Batch/Lot No.: A190809B
Appearance: Clear yellow liquid
Purity: 99.49%
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Sponsor (Emerald Performance Materials, Vancouver, WA 9868); Lot/Batch# A190809B
- Expiration date of the lot/batch: 2021-08-12
- Purity test date: 2019-08-13

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, ≤70% relative humidity), under inert gas (e.g. N2)
- Stability under storage conditions: not stated
- Stability under test conditions: not stated

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Microbiological Laboratory of Charles River Laboratories Hungary Kft
- method of preparation of S9 mix: Male Wistar rats (331-421 g, 7-9 weeks old and 444-628 g, 17-20 weeks old) 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 h before sacrifice when food was removed. Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels.

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.

- concentration or volume of S9 mix and S9 in the final culture medium:
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 6H2O 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 400 mL

Prior to addition to the culture medium the S9 mix was kept in an ice bath.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The sterility of the preparation was confirmed. Sterilization of the salt solution for S9 mix was performed by filtration through a 0.22 μm membrane filter while Sterilization of the sodium phosphate buffer was performed at 121°C in an autoclave.
Test concentrations with justification for top dose:
Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test).

Preliminary Concentration Range Finding Test: 10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
Mutagenicity Test Assay 1: 5, 15.81, 50, 158.1, 500, 1581, 5000 µg/plate
Mutagenicity Test Assay 3: 0.5, 1.581, 15.81, 50, 158.1, 500, 1581 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO) (Supplier: VWR; Batch No.: 18J094039)

- Justification for choice of solvent/vehicle: The solubility of the test material was examined using distilled water, and dimethyl sulfoxide (DMSO). The test material was insoluble (after 1-2 minutes vortex) at 100 mg/mL concentration using distilled water. The test material was soluble (after 1-2 minutes vortex, clear solution) at 100 mg/mL concentration using DMSO. Thus, DMSO was selected as vehicle for the study.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO and Distilled Water
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine (NPD) (-S9: TA98); 2-aminoanthracene (+S9: all Salmonella strains & WP2 uvrA)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 4 (Preliminary Compatibility Test, Preliminary Concentration Range Finding Test (Informatory Toxicity Test), Assay 1 (Plate Incorporation Method), and Assay 3 (Pre-Incubation Method))

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in in agar (plate incorporation - Assay 1); preincubation - Assay 2 (Invalid) and Assay 3 (Additional Experiment)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes at 37ºC
- Exposure duration/duration of treatment: 48 (±1) hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mean number of revertants per plate, the standard deviation, and the mutation factor (mean number of revertants on the test material plate / mean number of revertants on the vehicle control plate) values were calculated for each concentration level of the test material and for the controls.
Rationale for test conditions:
The bacterial reverse mutation test is a microbial assay which detects point mutations induced by chemicals causing base changes or frameshift mutations in the genome of amino-acid requiring strains of Salmonella typhimurium and Escherichia coli. The auxotrophic S. typhimurium and E. coli strains are unable to grow on minimal medium - containing inorganic salts and glucose as a carbon source - except for spontaneous revertants. However, in the presence of a mutagenic agent, some of them can be converted to prototrophs after a reverse mutation to the wild type. These revertants can grow and form colonies in minimal medium. An increased number of the revertant colonies therefore indicates mutagenic activity of the test material.

The bacterial mutagenicity test is used extensively to evaluate substances for mutagenic activity. Some mutagens (e.g.: nitrosamines) are poorly detected in the standard plate incorporation assay, but they can be detected by the pre-incubation assay. Therefore, both approaches have been considered in this study to assess the mutagenicity of the test material.

Concentrations for the main assays were selected on the basis of the Preliminary Compatibility Test and the Preliminary Concentration Range Finding Test (Informatory Toxicity Test). In Assay 1 and Assay 3, different concentrations were used based on the observed cytotoxicity.

Note: Assay 2 was conducted in this study using the pre-incubation method. However, excessive cytotoxicity was observed at the concentration range of 5000-500 µg/plate in all examined bacterial strains with or without metabolic activation. The number of analyzable doses did not meet the recommendations of the test guidelines and therefore, an additional experiment (Assay 3) was performed in all bacterial strains to complete the study. The experimental conditions were the same as in the Assay 2. Results of the invalid experiment (Assay 2) were not reported.
Evaluation criteria:
Criteria for Validity: The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls were 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 were presented in all strains of the main tests.

Criteria for a Positive Response: The test material was considered mutagenic if:
- a concentration-related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred 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.

Criteria for a Negative Response: The test material was considered non-mutagenic if it produced 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:
According to the guidelines, a 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.

The colony numbers on the untreated / negative (solvent) / positive control and test material treated plates were determined by manual counting. The mean number of revertants per plate, the standard deviation and the mutation factor (mean number of revertants on the test material plate / mean number of revertants on the vehicle control plate) values were calculated for each concentration level of the test material and for the controls using Microsoft ExcelTM software.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA
Remarks:
Assay 1 (Plate Incorporation Assay)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No biologically relevant increase in number of revertants compared to the solvent controls
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity observed at 5000 μg/plate (±S9) for all strains of S. typhimurium and E.coli and at 1581 μg/plate for S. typhimurium (-S9).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA
Remarks:
Assay 3 (Pre-incubation Assay)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No biologically relevant increase in number of revertants compared to the solvent controls
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity observed at 1581-500 μg/plate (±S9) for all strains of S. typhimurium and E.coli.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No precipitate was detected on the plates in the Preliminary Range Finding Test in Salmonella typhimurium strains TA98 and TA100, with or without metabolic activation. No precipitate was detected on the plates in the main tests (Assay 1 and Assay 3) in any examined bacterial strains with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES (if applicable): Preliminary Concentration Range-finding Test
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 detected on the plates in the preliminary experiment.

Inhibitory or toxic effects of the test material (absent background lawn / reduced /slightly reduced background lawn development) was observed in the preliminary experiment in both examined bacterial strains with and without metabolic activation at concentrations of 5000 and 2500 μg/plate.

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

STUDY RESULTS
- Concurrent vehicle negative and positive control data
Mutagenicity Assay 1 (Plate Incorporation Assay) and Mutagenicity Assay 3 (Pre-incubation Assay): 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 in line 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 or without metabolic activation. Data is presented in Tables 3 and 4.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 at concentrations of 158.1 and 5 μg/plate without metabolic activation (the observed mutation factor (MF) value was: 1.35). 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.

In Assay 3 (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 at a 5 μg/plate concentration with metabolic activation (the observed MF was: 1.20). 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.

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 to reflect the biological variability of the test.

Ames test:
- Signs of toxicity : In Assay 1, an inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development) was observed at 5000 μg/plate concentration in Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA, with or without metabolic activation, and at 1581 μg/plate concentration in Salmonella typhimurium TA100 without metabolic activation.

In Assay 3, an inhibitory, cytotoxic effect of the test item (absent / reduced / slightly reduced background lawn development and) was observed at a concentration range of 1581 – 500 μg/plate in all Salmonella typhimurium strains and Escherichia coli WP2 uvrA, with or without metabolic activation.

- Individual plate counts : Provided in Appendix 3 - 5 of the final study report.

- Mean number of revertant colonies per plate and standard deviation : Please see Tables 3 and 4 in the section 'Any other information on results incl. tables'

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please see Table 5 in the section 'Any other information on results incl. tables'
- Negative (solvent/vehicle) historical control data: Please see Table 5 in the section 'Any other information on results incl. tables'
Remarks on result:
other: no mutagenic activity in bacterial strains Salmonella typhimurium TA98, TA100, TA1535 and TA1537 or Escherichia coli WP2 uvrA either in the presence or absence of metabolic activation

Any other information on results incl. tables

Table 2. Preliminary Concentration Range Finding Test: Results

Concentrations

(µg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimurium

tester strains

TA98

TA100

-S9

+S9

-S9

+S9

Untreated

control

Mean

17.0

18.7

90.0

96.7

MF

0.96

1.00

1.05

1.01

DMSO control

Mean

17.7

18.7

85.3

95.7

MF

1.00

1.00

1.00

1.00

Distilled water

control

Mean

--

--

94.0

--

MF

--

--

1.10

--

5000

Mean

7.7

0.0

0.0

0.0

MF

0.43

0.00

0.00

0.00

2500

Mean

6.0

2.7

0.0

2.0

MF

0.34

0.14

0.00

0.02

1000

Mean

18.3

21.3

65.0

58.3

MF

1.04

1.14

0.76

0.61

316

Mean

18.0

19.0

64.0

83.3

MF

1.02

1.02

0.75

0.87

100

Mean

16.0

21.7

80.7

88.7

MF

0.91

1.16

0.95

0.93

31.6

Mean

20.3

22.0

77.7

86.7

MF

1.15

1.18

0.91

0.91

10

Mean

18.7

22.0

76.3

82.3

MF

1.06

1.18

0.89

0.86

4-nitro-1,2-phenylenediamine (4µg)

Mean

408.0

--

--

--

MF

23.09

--

--

--

2-aminoanthracene

(2µg)

Mean

--

2434.7

--

2464.0

MF

--

130.43

--

25.76

Sodium azide

(2µg)

Mean

--

--

1224.0

--

MF

--

--

13.02

--

Table 3. Results of Plate Incorporation Assay: Assay 1

Concentrations

(µg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimurium tester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated

control

Mean

18.0

19.0

94.7

94.3

14.0

11.3

8.0

9.3

35.7

40.7

MF

1.06

1.00

1.01

0.94

1.20

0.97

0.92

1.04

0.95

0.96

DMSO control

Mean

17.0

19.0

93.3

100.0

11.7

11.7

8.7

9.0

37.7

42.3

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

Distilled water

control

Mean

--

--

84.3

--

13.3

--

--

--

40.0

--

MF

--

--

1.01

--

1.14

--

--

--

1.06

--

5000

Mean

0.0

0.0

1.0

0.0

0.0

0.0

2.7

0.0

9.3

6.0

MF

0.00

0.00

0.01

0.00

0.00

0.00

0.31

0.00

0.25

0.14

1581

Mean

17.3

17.3

39.7

61.0

14.7

14.0

10.3

6.3

23.0

21.7

MF

1.02

0.91

0.43

0.61

1.26

1.20

1.19

0.70

0.61

0.51

500

Mean

19.0

18.7

86.7

98.7

15.0

14.0

10.0

11.3

28.3

43.3

MF

1.12

0.98

0.93

0.99

1.29

1.20

1.15

1.26

0.75

1.02

158.1

Mean

17.0

17.7

90.7

102.3

15.7

14.7

11.7

9.7

32.3

43.0

MF

1.00

0.93

0.97

1.02

1.34

1.26

1.35

1.07

0.86

1.02

50

Mean

18.3

18.3

97.7

100.7

15.0

13.0

11.3

10.0

35.0

44.0

MF

1.08

0.96

1.05

1.01

1.29

1.11

1.31

1.11

0.93

1.04

15.81

Mean

20.0

19.0

83.7

109.0

15.3

14.3

10.7

11.3

34.7

41.7

MF

1.18

1.00

0.90

1.09

1.31

1.23

1.23

1.26

0.92

0.98

5

Mean

18.3

18.3

88.3

106.0

15.0

14.3

11.7

11.0

34.3

43.7

MF

1.08

0.96

0.95

1.06

1.29

1.23

1.35

1.22

0.91

1.03

4-nitro-1,2-phenylenediamine (4 µg)

Mean

413.3

--

--

--

--

--

--

--

--

--

MF

24.31

--

--

--

--

--

--

--

--

--

2-aminoanthracene (2 µg)

Mean

--

2445.3

--

2474.7

--

220.7

--

213.7

--

--

MF

--

128.70

--

24.75

--

18.91

--

23.74

--

--

2-aminoanthracene (50 µg)

Mean

--

--

--

--

--

--

--

--

--

261.0

MF

--

--

--

--

--

--

--

--

--

6.17

Sodium Azide (2 µg)

Mean

--

--

1033.3

--

1037.3

--

--

--

--

--

MF

--

--

10.95

--

77.80

--

--

--

--

--

9-aminoacridine (50 µg)

Mean

--

--

--

--

--

--

414.7

--

--

--

MF

--

--

--

--

--

--

47.85

--

--

--

Methyl-methanesulfonate

(2 µL)

Mean

--

--

--

--

--

--

--

--

1081.3

--

MF

--

--

--

--

--

--

--

--

27.03

--

Table 4. Results of Pre-incubation Assay: Assay 3

Concentrations

(µg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimurium tester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated

control

Mean

17.7

26.7

92.3

121.3

16.3

16.7

11.7

10.3

39.0

45.3

MF

0.96

1.01

0.99

1.11

0.89

1.22

1.30

0.91

0.99

1.00

DMSO control

Mean

18.3

26.3

93.3

109.0

18.3

13.7

9.0

11.3

39.3

45.3

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

Distilled water

control

Mean

--

--

92.7

--

18.3

--

--

--

39.0

--

MF

--

--

0.99

--

1.00

--

--

--

0.99

--

1581

Mean

9.7

15.7

0.0

44.0

9.3

5.0

0.0

0.0

9.7

15.0

MF

0.53

0.59

0.00

0.40

0.51

0.37

0.00

0.00

0.25

0.33

500

Mean

8.3

19.0

57.7

71.7

10.7

5.7

2.7

2.7

10.7

18.7

MF

0.45

0.72

0.62

0.66

0.58

0.41

0.30

0.24

0.27

0.41

158.1

Mean

14.0

25.3

83.7

102.3

12.7

14.3

7.3

8.3

39.3

43.3

MF

0.76

0.96

0.90

0.94

0.69

1.05

0.81

0.74

1.00

0.96

50

Mean

18.3

26.0

94.3

98.0

17.0

15.3

8.0

9.7

38.3

44.7

MF

1.00

0.99

1.01

0.90

0.93

1.12

0.89

0.85

0.97

0.99

15.81

Mean

20.0

28.3

96.3

96.7

17.3

13.7

9.0

8.3

38.7

43.3

MF

1.09

1.08

1.03

0.89

0.95

1.00

1.00

0.74

0.98

0.96

5

Mean

17.7

26.3

94.0

101.3

15.3

16.3

8.0

9.3

37.0

44.7

MF

0.96

1.00

1.01

0.93

0.84

1.20

0.89

0.82

0.94

0.99

1.581

Mean

18.3

27.0

92.0

101.0

12.7

16.0

8.0

10.7

40.3

44.3

MF

1.00

1.03

0.99

0.93

0.69

1.17

0.89

0.94

1.03

0.98

0.5

Mean

18.7

27.3

92.0

96.0

17.7

15.0

9.0

9.3

38.0

44.0

MF

1.02

1.04

0.99

0.88

0.96

1.10

1.00

0.82

0.97

0.97

4-nitro-1,2-phenylenediamine (4 µg)

Mean

406.7

--

--

--

--

--

--

--

--

--

MF

22.18

--

--

--

--

--

--

--

--

--

2-aminoanthracene (2 µg)

Mean

--

2449.3

--

2406.7

--

212.3

--

209.3

--

--

MF

--

93.01

--

22.08

--

15.54

--

18.47

--

--

2-aminoanthracene (50 µg)

Mean

--

--

--

--

--

--

--

--

--

244.7

MF

--

--

--

--

--

--

--

--

--

5.40

Sodium Azide (2 µg)

Mean

--

--

1205.3

--

1188.0

--

--

--

--

--

MF

--

--

13.01

--

64.80

--

--

--

--

--

9-aminoacridine (50 µg)

Mean

--

--

--

--

--

--

421.3

--

--

--

MF

--

--

--

--

--

--

46.81

--

--

--

Methyl-methanesulfonate

(2 µL)

Mean

--

--

--

--

--

--

--

--

1044.0

--

MF

--

--

--

--

--

--

--

--

26.77

--

Table 5. Historical Control Data (2011-2018)

 

without metabolic activation (-S9)

with metabolic activation (+S9)

TA98

TA100

TA1535

TA1537

E. coli

TA98

TA100

TA1535

TA1537

E. coli

Untreated control data

Mean

22.3

101.5

12.1

7.9

36.3

27.4

108.9

11.6

9.4

41.3

St. dev.

5.4

19.5

4.6

3.5

10.8

6.7

18.4

3.6

3.8

10.3

Range

9-50

54-210

1-46

1-26

11-82

10-56

65-204

1-39

1-29

16-89

n

1860

1846

1857

1866

1875

1878

1869

1877

1881

1875

DMSO control data

Mean

21.4

97.5

12.1

7.7

35.3

27.6

106.4

11.4

9.1

40.4

St. dev.

5.3

18.8

4.5

3.4

10.7

6.7

19.3

3.9

3.7

10.2

Range

6-55

40-217

1-43

1-27

7-81

11-67

53-229

2-33

1-29

9-85

n

2007

1992

2004

2016

2019

2024

2013

2027

2028

2022

Distilled water control data

Mean

23.0

101.0

12.1

8.7

37.4

29.0

108.9

11.4

10.0

42.3

St. dev.

5.5

20.3

4.4

3.5

10.7

6.7

20.3

3.4

3.7

10.1

Range

11-45

45-215

2-47

2-24

12-84

10-53

64-222

3-39

1-24

13-91

n

399

1863

1869

405

1905

402

1875

1887

402

1893

DMF control data

Mean

20.4

90.2

11.4

7.7

37.3

27.1

98.9

11.1

8.9

40.0

St. dev.

5.2

16.8

4.3

3.4

12.8

6.8

18.0

3.3

3.5

11.0

Range

8-38

54-152

1-34

1-19

16-99

11-49

60-156

3-21

1-23

17-76

n

276

276

276

276

267

276

276

276

273

267

Acetone control data

Mean

22.4

97.7

12.1

7.5

36.2

28.5

106.8

11.1

8.8

41.3

St. dev.

5.0

14.7

5.6

2.9

9.6

6.6

14.2

3.4

3.3

9.1

Range

11-39

62-160

4-49

1-17

17-63

15-52

66-177

4-22

1-19

17-70

n

314

315

315

318

312

315

315

318

318

315

Positive reference control data

Mean

368.8

1208.5

1165.0

444.3

1034.2

2410.8

2425.1

229.2

219.0

255.4

St. dev.

100.9

185.0

179.4

147.1

140.2

274.5

252.0

117.0

49.0

98.2

Range

152-2336

536-2120

208-2440

149-2104

488-2496

312-4918

1192-5240

101-2216

117-838

125-2512

n

1860

1848

1857

1866

1878

1878

1869

1881

1881

1875

TA98: Salmonella typhimurium TA98

TA100: Salmonella typhimurium TA100

TA1535: Salmonella typhimurium TA1535

TA1537: Salmonella typhimurium TA1537

E. coli: Escherichia coli WP2uvrA

n: number of cases

Applicant's summary and conclusion

Conclusions:
Based on the results observed, the test material ((2E)-2-methyl-3-phenylacrylaldehyde)) did not induce gene mutations by base pair changes or frameshifts in the genome of the strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and strain WP2 uvrA of Escherichia coli. (2E)-2-methyl-3-phenylacrylaldehyde) was therefore determined to exhibit no mutagenic activity in this bacterial reverse mutation assay.
Executive summary:

In a key Guideline OECD 471 bacterial reverse mutation test, the test material ((2E)-2-methyl-3-phenylacrylaldehyde)) in dimethyl sulfoxide (DMSO) (vehicle) was evaluated for potential mutagenic activity using histidine-requiring auxotroph strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and a tryptophan-requiring auxotroph strain of Escherichia coli (WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital / β-naphthoflavone induced rats.

 

A preliminary compatibility test, a preliminary concentration range finding test, and two main mutagenicity assays (Assay 1 - Plate Incorporation Method and Assay 3 - Pre-Incubation Method) were conducted in the study. In the preliminary range finding test, test material concentrations of 10, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate were evaluated using Salmonella typhimurium strains TA98 and TA100 in the absence and presence of metabolic activation. Based on the results of this preliminary test, the test material was tested in the main mutagenicity assays as described below:

 

Assay 1 (plate incorporation method): 5, 15.81, 50, 158.1, 500, 1581, and 5000 µg/plate

 

Assay 3 (pre-incubation method): 0.5, 1.581, 5, 15.81, 50, 158.1, 500, and 1581 µg/plate

 

No precipitate was observed on the plates in strains TA98 and TA100 of Salmonella typhimuirum in the absence or presence of metabolic activation (±S9) in the preliminary range-finding test. Cytotoxic effects such as absent, reduced or slightly reduced background lawn development were observed at higher concentrations in the preliminary test as well as in the main mutagenicity assays in all tested bacterial strains in the presence and absence of metabolic activation.

 

In the main mutagenicity assays (Assay 1 and Assay 3), the number of revertant colonies were not observed to show any biologically relevant increase compared to the solvent controls. No reproducible dose related trends or indication of any treatment-related effect was observed in these assays. The mean number of revertant colonies in the negative (vehicle/solvent) control plates were observed to be consistent with the historical control range and the positive controls showed the expected increase in the number of revertant colonies.

 

Based on the results observed, the test material ((2E)-2-methyl-3-phenylacrylaldehyde)) did not induce gene mutations by base pair changes or frameshifts in the genome of the strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and strain WP2 uvrA of Escherichia coli. (2E)-2-methyl-3-phenylacrylaldehyde) was therefore determined to exhibit no mutagenic activity in this bacterial reverse mutation assay.