Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

(2E)-2-methyl-3-phenylacrylaldehyde showed no evidence of mutagenic potential in an OECD Guideline 471 AMES bacterial reverse mutation asssay involving four strains of Salmonella typhimurium and one Escherichia coli strain, in the absence and presence of metabolic activation (±S9).

2-Methyl-3-phenyl-2-propenal showed no convincing evidence of mutagenic potential in good-quality Ames bacterial tests involving seven strains of Salmonella typhimurium and one Escherichia coli strain, with and without added activation (S9). A SOS chromotest with E. coli was also negative.

(2E)-2-methyl-3-phenylacrylaldehyde did not induce any chromosome damage, or damage to the cell division apparatus, in cultured somatic L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system in an OECD Guideline 487 in vitro cytogenicity / micronucleus study .


Link to relevant study records

Referenceopen allclose all

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
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
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
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.
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.
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

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

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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-09-20 to 2019-11-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
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: At room temperature; Protected from light; Under nitrogen atmosphere
- 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
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: L5178Y TK+/- cells supplied by ATCC (American Type Culture Collection, Manassas, USA), were purchased from Biovalley (Marne-La-Vallée, France).
- Suitability of cells: suitable to reveal chemically induced micronuclei
- Normal cell cycle time (negative control): average cell cycle time is approximately 10-12 hours.

For cell lines:
- Absence of Mycoplasma contamination: each batch of frozen cells was checked for the absence of mycoplasma
- Methods for maintenance in cell culture: The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) in liquid nitrogen
- Cell cycle length: approximately 10-12 hours.
- Modal number of chromosomes: 40
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: not specified

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Cell cultures were grown at 37°C in a humidified atmosphere of 5% CO2/95% air in culture medium. The culture medium was RPMI1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL), 0.05% pluronic acid and sodium pyruvate (200 µg/mL). This medium was supplemented by heat-inactivated horse serum at 10% (v/v).
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 : Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA)
- method of preparation of S9 mix : S9 fraction was obtained from the liver of rats treated intraperitoneally with Aroclor 1254 (500 mg/Kg). The S9 fraction (batch No. 4007; protein concentration: 35.4 mg/mL; preserved in sterile tubes at -80°C), was prepared at +5°C immediately before use and maintained at this temperature until added to culture medium.

The composition of S9 mix was as presented in Table 1. under ‘Any other information on materials and methods’.
- concentration or volume of S9 mix and S9 in the final culture medium : +S9: 2%
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 is tested and validated by Moltox for absence of microbiological contamination and its ability to activate benzo(a)pyrene and 2-anthramine (also known as 2-amino anthracene) to mutagenic intermediates.
Test concentrations with justification for top dose:
Preliminary Cytotoxicity Test:
-S9 (3h treatment + 24h recovery): 0.0164, 0.0410, 0.102, 0.256, 0.64, 1.6, 4, and 10 mM
-S9 (24 h treatment + 0 h recovery): 0.0164, 0.0410, 0.102, 0.256, 0.64, 1.6, 4, and 10 mM
+S9 (3h treatment + 24h recovery): 0.0164, 0.0410, 0.102, 0.256, 0.64, 1.6, 4, and 10 mM

Main Cytogenetic Experiment 1:
-S9 (3h treatment + 24h recovery): 0.0156, 0.0313, 0.0625, 0.125, 0.167, 0.188, 0.25, and 0.375 mM
-S9 (24 h treatment + 0 h recovery): 0.0078, 0.0156, 0.0313, 0.0469, 0.0625, 0.0938, 0.125, and 0.188 mM
+S9 (3h treatment + 24h recovery): 0.0313, 0.0625, 0.125, 0.188, 0.25, 0.281, 0.375, and 0.75 mM

Main Cytogenetic Experiment 2:
-S9 (3h treatment + 24h recovery): 0.0125, 0.025, 0.05, 0.10, 0.133, 0.15, 0.20, and 0.30 mM
-S9 (24 h treatment + 0 h recovery): 0.0125, 0.025, 0.05, 0.10, 0.12, 0.133 and 0.20 mM
+S9 (3h treatment + 24h recovery): 0.025, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, and 0.40 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: DMSO selected as vehicle based on data provided by the Sponsor and solubility assays performed prior to the start of the study.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
colchicine
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : Duplicate
- Number of independent experiments: 2 (Main Cytogenicity Experiments 1 and Experiment 2) in addition to the preliminary cytotoxicity test

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 3 x 10^5 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Main Cytogenicity Experiments 1 and Experiment 2
-S9: 3 h treatment + 24 h recovery and 24 h treatment + 0 h recovery
+S9: 3 h treatment + 24 h recovery

- Harvest time after the end of treatment (sampling/recovery times): 0 hours (-S9) or 24 hours (-S9 and +S9) depending on the treatment regimen

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After final cell counting, the cells were washed with culture medium containing 10% inactivated horse serum and 1% pluronic acid. The cells were suspended in 49.5% culture medium containing 10% inactivated horse serum, 50% PBS and 0.5% pluronic acid, before being fixed.

Depending on the observation at the end of the recovery period (presence or absence of precipitate and/or cytotoxicity), at least three dose levels of the test material-treated cultures were selected for spreading on slides. Cells were dropped onto clean glass slides and slides were air-dried before being stained for approximately 15 min in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared using the same procedure.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): For each main experiment (with or without S9 mix), micronuclei were analyzed for three concentrations of the test material, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): '

Analysis was performed according to the following criteria:

• micronuclei should be located within the cytoplasma of the cell,
• micronuclei should be clearly surrounded by a nuclear membrane,
• micronuclei should be round or oval in shape,
• the micronucleus area should be less than one-third of the area of the main nucleus,
• micronuclei should be non-refractile (can be distinguished from artefacts such as staining particles),
• micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
• micronuclei should have similar staining intensity to that of the main nuclei,
• micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary,
• only mononucleated cells with a number of micronuclei ≤ 5 were scored to exclude apoptosis and nuclear fragmentation.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Population doubling (PD): Population Doubling (PD) was calculated and used relative to that of the vehicle control. The population doubling is the log of the ratio of the final count at the time of harvesting (N) to the starting count (N0), divided by the log of 2.

PD = [log (N/N0)]/log 2

Mean PD as % of control = (Mean PD treated / Mean PD vehicle control) x 100

- Any supplementary information relevant to cytotoxicity: Cytotoxicity induced by a treatment was evaluated by the decrease in the PD, when compared to the vehicle control (Mean % PD of the vehicle control set to 100%).

Decrease in PD (%) = 100 - Mean PD as % of control

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
Micronuclei were analyzed for three dose levels of the test material, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). The number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture. Analysis was performed under a microscope (1000 x magnification).

- OTHER:
Acceptance Criteria

Each main experiment was considered valid if the following criteria were met:

• the mean PD of the vehicle control had to be ≥ 1 (indicating that cells have undergone mitosis),
• the mean frequency of micronucleated cells in the vehicle control should be consistent with (but not necessarily within) control historical data of the Laboratory. In any case, this frequency should be ≤ 5‰ (i.e. 5⁄1000 or 0.5%),
• a statistically significant increase in the frequency of micronucleated cells has to be obtained in the positive controls over the background frequency of the vehicle control cultures and this frequency should also be greater than or equal to the minimum value of the historical data.
Rationale for test conditions:
The test material was found to be cytotoxic in the preliminary test. Therefore, the highest dose levels selected for the main experiments were based on the levels of cytotoxicity according to the criteria specified in the international regulations.

In the preliminary test, a slight to severe cytotoxicity was observed at concentrations ≥ 0.256 mM following the 3-hour treatment without S9 mix, ≥ 0.0410 mM following the 24-hour treatment without S9 mix and ≥ 0.256 mM following the 3-hour treatment with S9 mix. Based on these results, the highest dose levels selected for the first main experiment were 0.375 mM for the 3-hour treatment without S9 mix, 0.188 mM for the 24-hour treatment without S9 mix and 0.75 mM for the 3-hour treatment with S9 mix.

In the first main experiment, cytotoxicity was observed at concentrations ≥ 0.125 mM following the 3-hour treatment without S9 mix, ≥ 0.125 mM following the 24-hour treatment without S9 mix and ≥ 0.188 mM following the 3-hour treatment with S9 mix. Based on these results, the highest dose levels selected for the second main experiment were 0.30 mM for the 3-hour treatment without S9 mix, 0.20 mM for the 24-hour treatment without S9 mix and 0.40 mM for the 3-hour treatment with S9 mix.
Evaluation criteria:
Evaluation of a positive response: The test material was considered to have clastogenic and/or aneugenic potential if all the following criteria were met:
• a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
• for at least one dose level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
• a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose levels.

Evaluation of a negative response: The test material was considered clearly negative if none of the criteria for a positive response were met.

The biological relevance of the results was always taken into account when evaluating results.
Statistics:
For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures. This comparison was performed using the χ2 test, unless treated culture data were lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.

To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Preliminary Cytotoxicity Test: At the highest concentration of 10 mM, the pH in the culture medium was approximately 7.1 (as for the vehicle control)
- Data on osmolality: Preliminary Cytotoxicity Test: At the highest concentration of 10 mM, the osmolality in the culture medium was was 351 mOsm/kg H2O (362 mOsm/kg H2O for the vehicle control)
- Precipitation and time of the determination: No precipitate (or emulsion) was observed in the culture medium at the end of the treatment periods.
- Definition of acceptable cells for analysis: Please see 'Criteria for scoring micronucleated cells' under 'Details on test systems and experimental conditions'

RANGE-FINDING/SCREENING STUDIES (Table 3): The concentrations selected for treatment in the preliminary cytotoxicity test were: 0.0164, 0.0410, 0.102, 0.256, 0.64, 1.6, 4 and 10 mM.

An emulsion was observed in the culture medium, at dose levels ≥4 mM following the 3-hour treatment period and at 10 mM at the end of the 24-hour treatment period.

Following the 3-hour treatment without S9 mix, a severe cytotoxicity was observed at concentrations ≥0.256 mM, as shown by a 100% decrease in the PD.

Following the 24-hour treatment without S9 mix, a slight to severe cytotoxicity was observed at concentrations ≥0.0410 mM, as shown by a 30 to 100% decrease in the PD.

Following the 3-hour treatment with S9 mix, a moderate to severe cytotoxicity was observed at concentrations ≥0.256 mM, as shown by a 47 to 100% decrease in the PD

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Mean population doubling and mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Additionally, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells (Table 4 to Table 7).

For all test methods and criteria for data analysis and interpretation:
Short treatment (3h) without S9 mix
- Main Cytogenicity Experiment 1: Relative to the vehicle control, a slight increase in the frequency of micronucleated cells was observed at the concentration of 0.125 mM (p<0.05) together
with a dose response trend (p<0.01)

- Main Cytogenicity Experiment 2: no statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed concentrations relative to the vehicle control (p>0.05). No dose response relationship was demonstrated by the linear regression (p>0.05) and none of the analyzed concentrations showed frequencies of each replicate culture above the vehicle control historical range.

Continuous treatment (24h) without S9 mix
- Main Cytogenicity Experiment 1: No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed concentrations relative to the vehicle control (p>0.05). No dose response relationship was demonstrated by the linear regression (p>0.05) and none of the analyzed concentrations showed frequencies of each replicate culture above the
vehicle control historical range.

- Main Cytogenicity Experiment 2: No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed concentrations relative to the vehicle control (p>0.05). No dose response relationship was demonstrated by the linear regression (p>0.05) and none of the analyzed concentrations showed frequencies of each replicate culture above the
vehicle control historical range.

Experiments with S9 mix
- Main Cytogenicity Experiment 1: The concentrations selected for micronucleus analysis were 0.0625, 0.125 and 0.188 mM. No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed concentrations relative to the vehicle control (p>0.05). No dose response relationship was demonstrated by the linear regression and none of the analysed concentrations produced frequencies of micronucleated cells in either replicate culture above the vehicle control historical range.

- Main Cytogenicity Experiment 2: The concentrations selected for micronucleus analysis were 0.05, 0.10 and 0.15 mM. No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed concentrations relative to the vehicle control (p>0.05). No dose response relationship was demonstrated by the linear regression and none of the analysed concentrations produced frequencies of micronucleated cells in either replicate culture above the vehicle control historical range.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
Without S9 mix (-S9): Following the 3-hour treatment, a moderate to severe cytotoxicity was induced at concentrations ≥0.10 mM, as shown by a 42 to 100% decrease in the Population Doubling (PD). Following the 24-hour treatment, a moderate to severe cytotoxicity was induced at concentrations ≥ 0.10 mM, as shown by a 46 to 91% decrease in the PD.

With S9 mix (+S9): Moderate to severe cytotoxicity was induced at concentrations ≥0.15 mM, as shown by a 45 to 100% decrease in the PD.

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 Tables 8 and 9 under 'Any other information on results incl. tables'.
- Negative (solvent/vehicle) historical control data: Please see Tables 6 and 7 under 'Any other information on results incl. tables'.

Table 3. Preliminary Cytotoxicity Test Results

Treatment

Condition

Treatment

Cell Concentration used for Treatment

(x 104 cells/mL)

Post-treatment cell count

(x 104 cells/mL)

PD

PD as % of control

Decrease in PD (%)

without S9 mix:

3h treatment + 24h recovery

Vehicle Control

30

58.5

1.0

100

 

Test material (mM)

 

0.0164

30

88.5

1.6

162

None

0.0410

30

61.5

1.0

107

None

0.102

30

51.5

0.8

81

19

0.256

30

12.9

#

#

#

0.64

30

1.3

#

#

#

1.6

30

0.1

#

#

#

4 E

30

0.0

#

#

#

10 E

30

0.0

#

#

#

 

without S9 mix:

24h treatment + 0h recovery

Vehicle Control

30

56.5

0.9

100

 

Test material (mM)

 

0.0164

30

52.5

0.8

88

12

0.0410

30

46.7

0.6

70

30

0.102

30

23.6

#

#

#

0.256

30

10.0

#

#

#

0.64

30

2.6

#

#

#

1.6

30

0.4

#

#

#

4

30

1.1

#

#

#

10 E

30

0.1

#

#

#

 

with S9 mix:

3h treatment + 24h recovery

Vehicle Control

30

54.0

0.8

100

 

Test material (mM)

 

0.0164

30

71.5

1.3

148

None

0.0410

30

81.5

1.4

170

None

0.102

30

68.5

1.2

140

None

0.256

30

41.0

0.5

53

47

0.64

30

11.3

#

#

#

1.6

30

3.6

#

#

#

4 E

30

0.3

#

#

#

10 E

30

0.0

#

#

#

PD: population doubling

Vehicle control: DMSO

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

E: emulsion was noted in the culture medium at the end of treatment     

Table 4. Main Cytogenicity Experiment 1: Cytotoxicity Results

Treatment

Cell Concentration used for Treatment

(x 104cells/mL)

Culture

Post-treatment cell count

(x 104cells/mL)

Mean PD

PD as % of control

Decrease in PD (%)

Without S9 mix (-S9): 3-h Treatment + 24-h Recovery

Vehicle Control

30

C1

166.0

2.4

100

 

C2

150.0

Test Material (mM)

 

0.0156

30

C1

143.0

2.2

93

7

C2

138.0

0.0313

30

C1

141.0

2.2

93

7

C2

142.0

0.0625

30

C1

143.0

2.2

92

8

C2

134.0

0.125

30

C1

76.0

1.3

55

45

C2

74.5

0.167

30

C1

24.8

0.0

1

99 (too cytotoxic)

C2

36.7

0.188

30

C1

80.5

1.4

58

42

C2

75.5

0.25

30

C1

51.5

0.9

38

62 (too cytotoxic)

C2

61.0

0.375

30

C1

19.8

#

#

#

C2

12.5

Positive Controls

 

Mitomycin C

 (0.2 µg/mL)

30

C1

105.0

1.8

76

24

C2

107.0

Colchicine

(0.5 µg/mL)

30

C1

35.0

0.1

3

97

C2

28.5

Without S9 mix (-S9): 24-h Treatment + 0-h Recovery

Vehicle Control

30

C1

87.0

1.3

100

 

C2

62.0

Test Material (mM)

 

0.0078

30

C1

96.5

1.6

123

None

C2

87.5

0.0156

30

C1

88.5

1.7

126

None

C2

100

0.0313

30

C1

79.5

1.6

120

None

C2

99.0

00469

30

C1

87.0

1.7

131

None

C2

110.0

0.0625

30

C1

89.5

1.5

115

None

C2

81.5

0.0938

30

C1

60.0

1.2

90

10

C2

75.5

0.125

30

C1

44.3

0.4

31

69 (too cytotoxic)

C2

35.0

0.188

30

C1

25.8

0.3

26

74 (too cytotoxic)

C2

50.0

Positive Controls

 

Mitomycin C

 (0.05 µg/mL)

30

C1

50.7

0.9

66

34

C2

59.0

Colchicine

(0.5 µg/mL)

30

C1

21.8

#

#

#

C2

22.0

With S9 mix (+S9): 3-h Treatment + 24-h Recovery

Vehicle Control

30

C1

161.0

2.4

100

 

C2

149.0

Test Material (mM)

 

0.0313

30

C1

141.0

2.3

97

3

C2

156.0

0.0625

30

C1

147.0

2.3

96

4

C2

144.0

0.125

30

C1

110.0

1.9

79

21

C2

108.0

0.188

30

C1

58.5

1.3

54

46

C2

87.5

0.25

30

C1

50.3

0.5

22

78 (too cytotoxic)

C2

35.7

0.281

30

C1

79.5

1.3

55

45

C2

69.5

0.375

30

C1

51.0

0.6

27

73 (too cytotoxic)

C2

42.3

0.75

30

C1

17.0

#

#

#

C2

21.2

Positive Controls

 

Cyclophosphamide (3 µg/mL)

30

C1

93.0

1.8

74

26

C2

109.0

PD: population doubling

Vehicle control: DMSO

C1: Culture 1

C2: Culture 2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

                            

Table 5. Main Cytogenicity Experiment 2: Cytotoxicity Results

Treatment

Cell Concentration used for Treatment

(x 104cells/mL)

Culture

Post-treatment cell count

(x 104cells/mL)

Mean PD

PD as % of control

Decrease in PD (%)

Without S9 mix (-S9): 3-h Treatment + 24-h Recovery

Vehicle Control

30

C1

102.0

1.6

100

 

C2

86.0

Test Material (mM)

 

0.0125

30

C1

92.5

1.6

94

6

C2

83.5

0.025

30

C1

78.0

1.4

86

14

C2

82.0

0.05

30

C1

76.5

1.4

85

15

C2

81.0

0.10

30

C1

57.5

0.8

49

51

C2

47.3

0.133

30

C1

55.5

0.6

38

62 (too cytotoxic)

C2

37.3

0.15

30

C1

57.5

0.8

47

53

C2

45.0

0.20

30

C1

36.7

0.0

2

98 (too cytotoxic)

C2

25.0

0.30

30

C1

22.2

#

#

#

C2

11.2

Positive Controls

 

Mitomycin C

 (0.2 µg/mL)

30

C1

72.0

1.2

71

29

C2

63.0

Colchicine

(0.5 µg/mL)

30

C1

21.8

#

#

#

C2

13.5

Without S9 mix (-S9): 24-h Treatment + 0-h Recovery

Vehicle Control

30

C1

86.5

1.5

100

 

C2

86.5

Test Material (mM)

 

0.0125

30

C1

103.0

1.7

114

None

C2

97.0

0.025

30

C1

111.0

1.8

115

None

C2

91.5

0.05

30

C1

80.5

1.4

94

6

C2

81.0

0.10

30

C1

53.5

0.8

54

46

C2

52.3

0.12

30

C1

36.3

0.4

28

72 (too cytotoxic)

C2

44.3

0.133

30

C1

47.0

0.8

52

48

C2

57.0

0.20

30

C1

40.0

0.1

9

91 (too cytotoxic)

C2

26.0

Positive Controls

 

Mitomycin C

 (0.05 µg/mL)

30

C1

58.5

1.1

72

28

C2

69.5

Colchicine

(0.5 µg/mL)

30

C1

20.2

#

#

#

C2

20.3

With S9 mix (+S9): 3-h Treatment + 24-h Recovery

Vehicle Control

30

C1

104.5

1.9

100

 

C2

127.0

Test Material (mM)

 

0.025

30

C1

126.0

1.9

95

5

C2

90.5

0.05

30

C1

119.0

1.8

91

9

C2

85.0

0.10

30

C1

76.5

1.4

73

27

C2

85.0

0.15

30

C1

55.5

1.0

50

50

C2

62.5

0.20

30

C1

30.8

0.5

25

75 (too cytotoxic)

C2

53.0

0.25

30

C1

57.0

1.0

49

51

C2

59.0

0.30

30

C1

42.0

0.3

18

82 (too cytotoxic)

C2

34.0

0.40

30

C1

40.7

0.0

1

99 (too cytotoxic)

C2

20.2

Positive Controls

 

Cyclophosphamide (3 µg/mL)

30

C1

77.0

1.3

69

31

C2

75.0

PD: population doubling

Vehicle control: DMSO

C1: Culture 1

C2: Culture 2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

Table 6. Main Cytogenicity Experiment 1: Cytogenic Results

Treatment

Mean PD as % of control

Culture

No. of Cells Analyzed

Number of Cells with ‘n’ Micronuclei

Total Micronucleated Cells

Frequency of Micronucleated Cells (‰)

n = 1

n = 2

n = 3

n = 4

n = 5

Per culture

Per dose

Per culture

Per dose

Without S9 mix (-S9): 3-h Treatment + 24-h Recovery

Vehicle Control

100

C1

1000

1

0

0

0

0

1

2

1.0

1.0

C2

1000

1

0

0

0

0

1

1.0

Test Material (mM)

 

0.0156

93

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

0.0313

93

C1

1000

2

0

0

0

0

2

3

2.0

1.5

C2

1000

1

0

0

0

0

1

1.0

0.0625

92

C1

1000

3

0

0

0

0

3

4

3.0

2.0

C2

1000

1

0

0

0

0

1

1.0

0.125

55

C1

1000

7

0

0

0

0

7

11

7.0

5.5

C2

1000

4

0

0

0

0

4

4.0

0.167

1

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

0.188

58

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

0.25

38

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

0.375

#

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

Positive Controls

 

Mitomycin C

(0.2 µg/mL)

76

C1

1000

77

1

0

0

0

78

156

78.0

78.0 ***

C2

1000

76

2

0

0

0

78

78.0

Colchicine

(0.5 µg/mL)

3

C1

1000

18

0

0

0

0

18

37

18.0

18.5 ***

C2

1000

19

0

0

0

0

19

19.0

Without S9 mix (-S9): 24-h Treatment + 0-h Recovery

Vehicle Control

100

C1

1000

0

0

0

0

0

0

2

0.0

1.0

C2

1000

2

0

0

0

0

2

2.0

Test Material (mM)

 

0.0078

123

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

0.0156

126

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

0.0313

120

C1

1000

2

0

0

0

0

2

5

2.0

2.5

C2

1000

3

0

0

0

0

3

3.0

0.0469

131

C1

1000

2

0

0

0

0

2

5

2.0

2.5

C2

1000

3

0

0

0

0

3

3.0

0.0625

115

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

0.0938

90

C1

1000

1

0

0

0

0

1

5

1.0

2.5

C2

1000

4

0

0

0

0

4

4.0

0.125

31

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

0.188

26

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

Positive Controls

 

Mitomycin C

(0.05 µg/mL)

66

C1

1000

12

0

0

0

0

12

40

12.0

20.0 ***

C2

1000

28

0

0

0

0

28

28.0

Colchicine

(0.5 µg/mL)

#

C1

1000

29

0

0

0

0

29

57

29.0

28.5 ***

C2

1000

26

2

0

0

0

28

28.0

With S9 mix (+S9): 3-h Treatment + 24-h Recovery

Vehicle Control

100

C1

1000

1

0

0

0

0

1

3

1.0

1.5

C2

1000

2

0

0

0

0

2

2.0

Test Material (mM)

 

0.0313

97

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

0.0625

96

C1

1000

1

0

0

0

0

1

2

1.0

1.0

C2

1000

1

0

0

0

0

1

1.0

0.125

79

C1

1000

0

0

0

0

0

0

2

0.0

1.0

C2

1000

2

0

0

0

0

2

2.0

0.188

54

C1

1000

3

0

0

0

0

3

4

3.0

2.0

C2

1000

1

0

0

0

0

1

1.0

0.25

22

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

0.281

55

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

0.375

27

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

0.75

#

C1

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

Positive Controls

 

Cyclophosphamide

(3 µg/mL)

74

C1

1000

37

2

0

0

0

39

71

39.0

35.5 ***

C2

1000

31

1

0

0

0

32

32.0

PD: Population Doubling

Vehicle control: DMSO

C1: Culture 1

C2: Culture 2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

Statistics: 2 x 2 contingency table:

*: p < 0.05

***: p < 0.001

Table 7. Main Cytogenicity Experiment 2: Cytogenic Results

Cod

Treatment

Mean PD as % of control

Culture

No. of Cells Analyzed

Number of Cells with ‘n’ Micronuclei

Total Micronucleated Cells

Frequency of Micronucleated Cells (‰)

n = 1

n = 2

n = 3

n = 4

n = 5

Per culture

Per dose

Per culture

Per dose

Without S9 mix (-S9): 3-h Treatment + 24-h Recovery

80

Vehicle Control

100

C1

1000

1

0

0

0

0

1

3

1.0

1.5

46

C2

1000

2

0

0

0

0

2

2.0

 

Test Material (mM)

 

 

0.0125

94

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

74

0.025

86

C1

1000

1

0

0

0

0

1

3

1.0

1.5

48

C2

1000

2

0

0

0

0

2

2.0

57

0.05

85

C1

1000

2

0

0

0

0

2

4

2.0

2.0

63

C2

1000

2

0

0

0

0

2

2.0

42

0.10

49

C1

1000

0

0

0

0

0

0

0

0.0

0.0

76

C2

1000

0

0

0

0

0

0

0.0

 

0.133

38

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

 

0.15

47

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

 

0.20

2

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

 

0.30

#

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

Positive Controls

 

45

Mitomycin C

(0.2 µg/mL)

71

C1

1000

51

1

0

0

0

52

90

52.0

45.0 ***

65

C2

1000

36

2

0

0

0

38

38.0

60

Colchicine

(0.5 µg/mL)

#

C1

1000

8

0

0

0

0

8

23

8.0

11.5 ***

52

C2

1000

14

1

0

0

0

15

15.0

 

Without S9 mix (-S9): 24-h Treatment + 0-h Recovery

 

Vehicle Control

100

C1

1000

3

0

0

0

0

3

5

3.0

2.5

 

C2

1000

2

0

0

0

0

2

2.0

 

Test Material (mM)

 

 

0.0125

114

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

 

0.025

115

C1

1000

2

0

0

0

0

2

3

2.0

1.5

 

C2

1000

1

0

0

0

0

1

1.0

 

0.05

94

C1

1000

1

0

0

0

0

1

1

1.0

0.5

 

C2

1000

0

0

0

0

0

0

0.0

 

0.10

54

C1

1000

0

0

0

0

0

0

3

0.0

1.5

 

C2

1000

3

0

0

0

0

3

3.0

 

0.12

28

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

0.133

52

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

 

0.20

9

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

Positive Controls

 

 

Mitomycin C

(0.05 µg/mL)

72

C1

1000

21

0

0

0

0

21

42

21.0

21.0 ***

 

C2

1000

21

0

0

0

0

21

21.0

 

Colchicine

(0.5 µg/mL)

#

C1

1000

36

2

0

0

0

38

57

38.0

28.5 ***

 

C2

1000

19

0

0

0

0

19

19.0

 

With S9 mix (+S9): 3-h Treatment + 24-h Recovery

 

Vehicle Control

100

C1

1000

2

0

0

0

0

2

4

2.0

2.0

 

C2

1000

2

0

0

0

0

2

2.0

 

Test Material (mM)

 

 

0.025

95

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

 

0.05

91

C1

1000

2

0

0

0

0

2

3

2.0

1.5

 

C2

1000

1

0

0

0

0

1

1.0

 

0.10

73

C1

1000

2

0

0

0

0

2

3

2.0

1.5

 

C2

1000

1

0

0

0

0

1

1.0

 

0.15

50

C1

1000

0

0

0

0

0

0

0

0.0

0.0

 

C2

1000

0

0

0

0

0

0

0.0

 

0.20

25

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

 

0.25

49

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

 

0.30

18

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

 

0.40

1

C1

 

 

 

 

 

 

 

 

 

 

 

C2

 

 

 

 

 

 

 

 

 

 

Positive Controls

 

 

Cyclophosphamide

(3 µg/mL)

69

C1

1000

35

0

0

0

0

35

62

35.0

31.0 ***

 

C2

1000

27

0

0

0

0

27

27.0

PD: Population Doubling

Vehicle control: DMSO

C1: Culture 1

C2: Culture 2

#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment

Statistics: 2 x 2 contingency table:

*: p < 0.05

***: p < 0.001

Table 8. Historical Data (01 Jan 2017 to 01 October 2018) - Without Metabolic Activation (-S9)

Parameter

Frequency of Micronucleated Cells in 1000 Cells

Treatment Conditions

3 Hours Treatment + 24 Hours Recovery

24 Hours Treatment + 0 Hours Recovery

Control Items

Vehicle Control

Mitomycin C

(1 µg/mL)

Colchicine

(0.5 µg/mL)

Vehicle Control

Mitomycin C

(1 µg/mL)

Colchicine

(0.5 µg/mL)

n

41

39

37

42

38

38

Mean

1.9

125.7

25.0

2.1

34.4

24.4

SD

1.2

75.4

14.1

1.3

18.8

13.6

Lower CL 95%

1.5

101.3

20.3

1.7

28.2

19.9

Upper CL 95%

2.3

150.1

29.7

2.5

40.5

28.9

5thPercentile

0.0

29.5

8.5

0.5

10.5

6.5

Median

2.0

97.0

21.5

2.0

28.3

23.8

95thPercentile

4.0

266.0

57.0

4.5

71.0

54.0

Min

0.0

27.5

8.0

0.0

8.5

4.5

Max

4.0

283.0

57.0

5.0

73.5

59.5

 

Table 9. Historical Data (01 Jan 2017 to 01 October 2018) - With Metabolic Activation (+S9)

Parameter

Frequency of Micronucleated Cells in 1000 Cells

Treatment Conditions

3 Hours Treatment + 24 Hours Recovery

Control Items

Vehicle Control

Cyclophosphamide (6 µg/mL)

n

41

39

Mean

1.7

85.1

SD

1.1

56.1

Lower CL 95%

1.3

66.9

Upper CL 95%

2.0

103.3

5thPercentile

0.0

21.0

Median

1.5

71.5

95thPercentile

3.5

182.5

Min

0.0

12.0

Max

5.0

186.0

SD: Standard Deviation

CL: Confidence Limit

Max: Maximal Value

Min: Minimal Value

N: Number of Values

Conclusions:
The test material did not induce any chromosome damage, or damage to the cell division apparatus, in cultured somatic L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
Executive summary:

In a key Guideline OECD 487 in vitro micronucleus assay, the potential of the test material ((2E)-2-methyl-3-phenylacrylaldehyde) to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/- was evaluated.

 

Based on the results of a preliminary cytotoxicity test, the test material was dissolved in the vehicle (dimethylsulfoxide (DMSO)) and tested in two independent experiments (Experiment 1 and Experiment 2) in the absence and presence of metabolic activation (±S9). Test concentrations used in the experiments were as follows:

 

Preliminary Cytotoxicity Test:

Absence of metabolic activation (-S9):

3h treatment + 24h recovery: 0.0164, 0.0410, 0.102, 0.256, 0.64, 1.6, 4, and 10 mM

24 h treatment + 0 h recovery: 0.0164, 0.0410, 0.102, 0.256, 0.64, 1.6, 4, and 10 mM

 

Presence of metabolic activation (+S9):

+S9 (3h treatment + 24h recovery): 0.0164, 0.0410, 0.102, 0.256, 0.64, 1.6, 4, and 10 mM

 

Experiment 1:

Absence of metabolic activation (-S9):

3h treatment + 24h recovery: 0.0156, 0.0313, 0.0625, 0.125, 0.167, 0.188, 0.25, and 0.375 mM

24 h treatment + 0 h recovery: 0.0078, 0.0156, 0.0313, 0.0469, 0.0625, 0.0938, 0.125, and 0.188 mM

 

Presence of metabolic activation (+S9):

3h treatment + 24h recovery: 0.0313, 0.0625, 0.125, 0.188, 0.25, 0.281, 0.375, and 0.75 mM

 

Experiment 2:

Absence of metabolic activation (-S9):

3h treatment + 24h recovery: 0.0125, 0.025, 0.05, 0.10, 0.133, 0.15, 0.20, and 0.30 mM

24 h treatment + 0 h recovery: 0.0125, 0.025, 0.05, 0.10, 0.12, 0.133 and 0.20 mM

 

Presence of metabolic activation (+S9):

3h treatment + 24h recovery: 0.025, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, and 0.40 mM

 

Cytotoxicity was evaluated at each dose level and assessed by determining the Population Doubling (PD) of cells. Subsequent to counting, cells were washed and fixed and cells from at least three dose levels of the test material treated cultures were dropped onto clean glass slides which were then airdried and stained in 5% Giemsa. Slides from vehicle and positive control cultures were also prepared using the same procedure. For each experiment (±S9), micronuclei were analyzed for three dose levels of the test material, for the vehicle, and for the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). The number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

In the preliminary cytotoxicity test, an emulsion was observed in the culture medium, at dose levels ≥ 4 mM following the 3-hour treatment period and at 10 mM at the end of the 24-hour treatment period. Following the 3-hour treatment in the absence of metabolic activation, a severe cytotoxicity was observed at concentrations ≥0.256 mM, as observed by a 100% decrease in the PD. Following the 24-hour treatment in the absence of metabolic activation, a slight to severe cytotoxicity was observed at concentrations ≥0.0410 mM, as observed by a 30 to 100% decrease in the PD. Following the 3-hour treatment in the presence of metabolic activation, a moderate to severe cytotoxicity was observed at concentrations ≥0.256 mM, as observed by a 47 to 100% decrease in the PD.

 

No precipitate or emulsion was observed in the culture medium at the end of the treatment period in experiments 1 and 2 in the absence and presence of metabolic activation.

 

In the absence of metabolic activation, following the 3-hour treatment, a moderate to severe cytotoxicity was induced at concentrations ≥ 0.10 mM, as observed by a 42 to 100% decrease in the PD. Following the 24-hour treatment, a moderate to severe cytotoxicity was induced at concentrations ≥ 0.10 mM, as observed by a 46 to 91% decrease in the PD. In the presence of metabolic activation, moderate to severe cytotoxicity was induced at concentrations ≥ 0.15 mM, as observed by a 45 to100% decrease in the PD.

 

In experiment 1, in the absence of metabolic activation, and subsequent to the 3-hour treatment, a slight increase in the frequency of micronucleated cells relative to the vehicle control, was observed at the concentration of 0.125 mM (p<0.05) together with a dose response trend (p<0.01). Considering the relatively low magnitude of this increase and since none of the analyzed concentrations showed frequencies of each replicate culture above the vehicle control historical range, these results did not meet the criteria for a clear positive or a clear negative response. In experiment 2, subsequent to the 3-hour treatment, no statistically significant increase in the frequency of micronucleated cells relative to the vehicle control (p>0.05) was observed at any of the analyzed concentrations. No dose response relationship was demonstrated by the linear regression (p>0.05) and none of the analyzed concentrations demonstrated frequencies of each replicate culture above the vehicle control historical range. Since the slight increase observed in the first experiment was mainly due to one out the two replicate cultures, and not observed in the second experiment despite the recommended level of cytotoxicity being reached, this observation was considered to not be biologically relevant and the overall results were considered to demonstrate a negative response.

 

In experiments 1 and 2, in the absence of metabolic activation, and subsequent to the 24-hour treatment, no statistically significant increase in the frequency of micronucleated cells relative to the vehicle control (p>0.05) was observed at any of the analyzed concentrations. No dose response relationship was demonstrated by the linear regression (p>0.05) and none of the analyzed concentrations showed frequencies of each replicate culture above the vehicle control historical range. While the recommended level of cytotoxicity was not reached, considering the narrow concentration spacing used in both experiments and the 46% decrease in the PD obtained at the highest tested concentration in the experiment 2, the available results were considered sufficient to permit a reliable interpretation. Overall, the results were determined to have met the criteria for a negative response.

 

In experiments 1 and 2, in the presence of metabolic activation, no statistically significant increase in the frequency of micronucleated cells relative to the vehicle control (p>0.05) was observed at any of the analyzed concentrations. No dose response relationship was demonstrated by the linear regression (p>0.05) and none of the analysed concentrations produced frequencies of micronucleated cells in either replicate culture above the vehicle control historical range. These results were determined to have met the criteria for a negative response.

 

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria and the positive control cultures demonstrated clear statistically significant increases in the frequency of micronucleated cells.

Based on the results observed, the test material did not induce any chromosome damage, or damage to the cell division apparatus in cultured somatic L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Endpoint:
in vitro gene mutation study in mammalian cells
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo cytogenetic or gene mutation studies in mammalian cells are available for 2-methyl-3-phenyl-2-propenal. However, an in vivo bone marrow micronucleus test in mice and a gene mutation test in Drosophila melanogaster found no evidence of genotoxic activity. Overall, the available data indicate that 2-methyl-3-phenyl-2-propenal does not possess genotoxic potential.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Comparable to OECD Guideline 474. Deficiencies were: only four animals tested per dose; bone marrow taken only once, six hours after final treatment; only 1000 erythrocytes scored per animal
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
4 animals/dose (guideline recommends ≥ 5/sex/dose); bone marrow taken once, 6 h after final treatment (guideline recommends taking twice, 18-24 and 36-48 h after final treatment); 1000 erythrocytes scored (guideline recommends ≥ 2000)
GLP compliance:
not specified
Type of assay:
micronucleus assay
Specific details on test material used for the study:
Source: ICN-K &K (Plainview, NY, USA)
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: lvanovas GmbH (Kisslegg)
- Age at study initiation: 10 to 14 weeks
- Weight at study initiation: no data available
- Assigned to test groups randomly: no data available
- Fasting period before study: no data available
- Housing: no data available
- Diet (e.g. ad libitum): standard chow (Altromin) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data available
- Humidity (%): no data available
- Air changes (per hr): no data available
- Photoperiod (hrs dark / hrs light): no data available

IN-LIFE DATES: no data available
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: no data available
- Concentration of test material in vehicle: no data available
- Purity: no data available
Details on exposure:
intraperitoneal injections
Duration of treatment / exposure:
single injections at 0 and 24 h
Frequency of treatment:
once daily, for 2 days
Post exposure period:
6 hours
Remarks:
Doses / Concentrations:
438, 292 or 146 mg/kg bw/day
Basis:
no data
No. of animals per sex per dose:
probably 2 [4 animals/dose tested]
Control animals:
other: yes: concurrent negative controls (possibly given vehicle or no treatment)
Positive control(s):
no data
- Justification for choice of positive control(s): no data available
- Route of administration: no data available
- Doses / concentrations: no data available
Tissues and cell types examined:
bone marrow micronucleated polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: no data available

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): treatment at 0 and 24 hours, sampling at 30 hours

DETAILS OF SLIDE PREPARATION: stained with May-Gruenwald and Giemsa stains. 1000 polychromatic erythrocytes analysed for each animal on coded slides.

METHOD OF ANALYSIS: no data available
Evaluation criteria:
no data available
Statistics:
no data available
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
other: possibly negative control
Negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: no data available
- Solubility: no data available
- Clinical signs of toxicity in test animals: no data available
- Evidence of cytotoxicity in tissue analyzed: no data available
- Rationale for exposure: no data available
- Harvest times: no data available
- High dose with and without activation: no data available

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no data available
- Ratio of PCE/NCE (for Micronucleus assay): 1.7, 2.0 and 2.2 per thousand (for animals given 438, 292 and 146 mg/kg bw/day respectively). 2.0 per thousand for negative control.
- Appropriateness of dose levels and route: no data available
- Statistical evaluation: no data available
Conclusions:
Interpretation of results: negative
In an in vivo bone marrow micronucleus test, similar to that recommended by OECD Guideline 474, no consistent evidence of mutagenic activity was observed in mice following intraperitoneal injections of α-methylcinnamaldehyde at up to 438 mg/kg bw/day for 2 days.
Executive summary:

A publication briefly describes an in vivo micronucleus test carried out on groups of mice, conducted using a protocol similar to OECD Guideline 474.

Groups of four mice were given two intraperitoneal injections (at 0 and 24 hours) of α-methylcinnamaldehyde at 146, 292 or 438 mg/kg bw/day. Six hours after the final injection, mice were killed, and bone-marrow smears were prepared. Following fixation and staining, 1000 polychromatic erythrocytes were analysed from each animal, and the number of micronucleated polychromatic erythrocytes was recorded.

Treatment with α-methylcinnamaldehyde did not significantly induce increased numbers of micronuclei. Under the conditions of this assay, no convincing evidence of mutagenic activity was demonstrated for α-methylcinnamaldehyde.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In a key Guideline OECD 471 bacterial reverse mutation test (Charles River Laboratories Hungary Kft, 2020; Klimisch score = 1), 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 S. 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.

In good-quality weight of evidence studies, 2-methyl-3-phenyl-2-propenal exhibited no mutagenic activity, either in the presence or absence of rat, mouse or hamster liver metabolic activating fractions (S9), in six strains of Salmonella typhimurium (TA98, TA102, TA104, TA1535, TA1537 and TA1538) (Dillon et al. 1992a,b, 1998; Kato et al. 1989; Mortelmans et al. 1986; NTP, 1988; Wild et al. 1983). [Note: TA104 and TA1538 are additional to the current OECD Guideline’s recommended set of five tester strains.] For S. typhimurium strain TA100, results for mutagenicity have generally been negative (Kato et al. 1989; Mortelmans et al. 1986; Neudecker et al. 1983; Wild et al. 1983), but some weak/equivocal responses have been reported for this strain in the presence of metabolic activation (Dillon et al. 1992a,b; Dillon et al. 1998; NTP, 1988). In addition, mutagenic activity was not seen in Escherichia coli strain WP2uvrA/pKM101, with and without S9 (Kato et al. 1989) or in the SOS chromotest with E. coli PQ37 (Eder et al. 1993).

In a key Guideline OECD 487 in vitro micronucleus assay (Charles River Laboratories Evreux, 2020b; Klimisch Score = 1), the potential of the test material ((2E)-2-methyl-3-phenylacrylaldehyde) to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/- was evaluated.

 

Based on the results of a preliminary cytotoxicity test, the test material was dissolved in the vehicle (dimethylsulfoxide (DMSO)) and tested in two independent experiments (Experiment 1 and Experiment 2) in the absence and presence of metabolic activation (±S9). Test concentrations used in the experiments were as follows:

 

Preliminary Cytotoxicity Test:

Absence of metabolic activation (-S9):

3h treatment + 24h recovery: 0.0164, 0.0410, 0.102, 0.256, 0.64, 1.6, 4, and 10 mM

24 h treatment + 0 h recovery: 0.0164, 0.0410, 0.102, 0.256, 0.64, 1.6, 4, and 10 mM

 

Presence of metabolic activation (+S9):

+S9 (3h treatment + 24h recovery): 0.0164, 0.0410, 0.102, 0.256, 0.64, 1.6, 4, and 10 mM

 

Experiment 1:

Absence of metabolic activation (-S9):

3h treatment + 24h recovery: 0.0156, 0.0313, 0.0625, 0.125, 0.167, 0.188, 0.25, and 0.375 mM

24 h treatment + 0 h recovery: 0.0078, 0.0156, 0.0313, 0.0469, 0.0625, 0.0938, 0.125, and 0.188 mM

 

Presence of metabolic activation (+S9):

3h treatment + 24h recovery: 0.0313, 0.0625, 0.125, 0.188, 0.25, 0.281, 0.375, and 0.75 mM

 

Experiment 2:

Absence of metabolic activation (-S9):

3h treatment + 24h recovery: 0.0125, 0.025, 0.05, 0.10, 0.133, 0.15, 0.20, and 0.30 mM

24 h treatment + 0 h recovery: 0.0125, 0.025, 0.05, 0.10, 0.12, 0.133 and 0.20 mM

 

Presence of metabolic activation (+S9):

3h treatment + 24h recovery: 0.025, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, and 0.40 mM

 

Cytotoxicity was evaluated at each dose level and assessed by determining the Population Doubling (PD) of cells. Subsequent to counting, cells were washed and fixed and cells from at least three dose levels of the test material treated cultures were dropped onto clean glass slides which were then airdried and stained in 5% Giemsa. Slides from vehicle and positive control cultures were also prepared using the same procedure. For each experiment (±S9), micronuclei were analyzed for three dose levels of the test material, for the vehicle, and for the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). The number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

In the preliminary cytotoxicity test, an emulsion was observed in the culture medium, at dose levels ≥ 4 mM following the 3-hour treatment period and at 10 mM at the end of the 24-hour treatment period. Following the 3-hour treatment in the absence of metabolic activation, a severe cytotoxicity was observed at concentrations ≥0.256 mM, as observed by a 100% decrease in the PD. Following the 24-hour treatment in the absence of metabolic activation, a slight to severe cytotoxicity was observed at concentrations ≥0.0410 mM, as observed by a 30 to 100% decrease in the PD. Following the 3-hour treatment in the presence of metabolic activation, a moderate to severe cytotoxicity was observed at concentrations ≥0.256 mM, as observed by a 47 to 100% decrease in the PD.

 

No precipitate or emulsion was observed in the culture medium at the end of the treatment period in experiments 1 and 2 in the absence and presence of metabolic activation.

 

In the absence of metabolic activation, following the 3-hour treatment, a moderate to severe cytotoxicity was induced at concentrations ≥ 0.10 mM, as observed by a 42 to 100% decrease in the PD. Following the 24-hour treatment, a moderate to severe cytotoxicity was induced at concentrations ≥ 0.10 mM, as observed by a 46 to 91% decrease in the PD. In the presence of metabolic activation, moderate to severe cytotoxicity was induced at concentrations ≥ 0.15 mM, as observed by a 45 to100% decrease in the PD.

 

In experiment 1, in the absence of metabolic activation, and subsequent to the 3-hour treatment, a slight increase in the frequency of micronucleated cells relative to the vehicle control, was observed at the concentration of 0.125 mM (p<0.05) together with a dose response trend (p<0.01). Considering the relatively low magnitude of this increase and since none of the analyzed concentrations showed frequencies of each replicate culture above the vehicle control historical range, these results did not meet the criteria for a clear positive or a clear negative response. In experiment 2, subsequent to the 3-hour treatment, no statistically significant increase in the frequency of micronucleated cells relative to the vehicle control (p>0.05) was observed at any of the analyzed concentrations. No dose response relationship was demonstrated by the linear regression (p>0.05) and none of the analyzed concentrations demonstrated frequencies of each replicate culture above the vehicle control historical range. Since the slight increase observed in the first experiment was mainly due to one out the two replicate cultures, and not observed in the second experiment despite the recommended level of cytotoxicity being reached, this observation was considered to not be biologically relevant and the overall results were considered to demonstrate a negative response.

 

In experiments 1 and 2, in the absence of metabolic activation, and subsequent to the 24-hour treatment, no statistically significant increase in the frequency of micronucleated cells relative to the vehicle control (p>0.05) was observed at any of the analyzed concentrations. No dose response relationship was demonstrated by the linear regression (p>0.05) and none of the analyzed concentrations showed frequencies of each replicate culture above the vehicle control historical range. While the recommended level of cytotoxicity was not reached, considering the narrow concentration spacing used in both experiments and the 46% decrease in the PD obtained at the highest tested concentration in the experiment 2, the available results were considered sufficient to permit a reliable interpretation. Overall, the results were determined to have met the criteria for a negative response.

 

In experiments 1 and 2, in the presence of metabolic activation, no statistically significant increase in the frequency of micronucleated cells relative to the vehicle control (p>0.05) was observed at any of the analyzed concentrations. No dose response relationship was demonstrated by the linear regression (p>0.05) and none of the analysed concentrations produced frequencies of micronucleated cells in either replicate culture above the vehicle control historical range. These results were determined to have met the criteria for a negative response.

 

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria and the positive control cultures demonstrated clear statistically significant increases in the frequency of micronucleated cells.

 

Based on the results observed, the test material did not induce any chromosome damage, or damage to the cell division apparatus in cultured somatic L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

A bone marrow micronucleus test has also been conducted. No evidence of chromosome damage (as measured by increases in the incidence of micronuclei) was observed in the bone marrow of groups of four mice given intraperitoneal injections of 0, 146, 292 or 438 mg/kg bw on two occasions, one day apart (Wild et al. 1983).

 

No in vitro gene mutation studies in mammalian cells on 2‑methyl-3-phenyl-2-propenal are currently available. According to ECHA communication CCH-D-2114453633-49-01/F (attached in Section 13.2 of the dossier) received on December 14, 2018, an in vitro gene mutation study in mammalian cells (Annex VIII, Section 8.4.3.; test method: OECD TG 476 or TG 490) with the registered substance (provided that both studies requested under 1. and 2. have negative results) was requested by ECHA. The communication states that the registrant is required to submit the requested information in an updated registration dossier by 21 June 2021. This study (Study code: MLA01501) is currently on going at Gentronix Limited (United Kingdom). The registrant commits to update this section upon receipt of the final report from the CRO. Additionally, no evidence of heritable gene mutations was observed in the offspring of fruit flies (Drosophila melanogaster) fed 2-methyl-3-phenyl-2-propenal for 3 days (Wild et al. 1993). [The relevance of these findings to an understanding of mutagenic potential in mammalian systems is not fully established.]

 

In its evaluation of cinnamyl alcohol and related compounds (including 2-methyl-3-phenyl-2-propenal), JECFA noted a lack of direct mutagenic or genotoxic activity of 2-methyl-3-phenyl-2-propenal and considered estimated daily intake from its use as a food additive to be of no safety concern in Europe and the US (JECFA, 2001). Similarly, 2-methyl-3-phenyl-2-propenal is generally recognised as safe (GRAS) by the US Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) (Adams et al. 2004).

 

Overall, the available data indicate that 2-methyl-3-phenyl-2-propenal does not possess genotoxic potential.

 

References (not contained elsewhere in this IUCLID dossier)

Adams TB et al. (2004). The FEMA GRAS assessment of cinnamyl derivatives used as flavour ingredients. Fd Chem. Toxicol. 42, 157-185.

Dillon DM et al. (1992a). Detection of mutagenicity in Salmonella of some aldehydes and peroxides. Environ. Mol. Mutagen. 19, 15.

Dillon DM et al. (1992b). Optimal conditions for detecting bacterial mutagenicity of some aldehydes and peroxides. Mutat. Res. 271, 184.

Eder E et al. (1993). The possible role of α,β-unsaturated carbonyl compounds in mutagenesis and carcinogenesis. Tox. Lett. 67, 87-103.

JECFA (2001). Joint FAO/WHO Expert Committee on Food Additives. Safety evaluation of certain food additives and contaminants. WHO Fd. Add. Ser. 46.

Kato F et al. (1989). Mutagenicity of aldehydes and diketones. Mutat. Res. 216, 366. [Abstract only.]

Neudecker T et al. (1983). Effect of methyl and halogen substitutions in theαC position on the mutagenicity of cinnamaldehyde. Mutat. Res. 110, 1-8.

NTP (1988). National Toxicology Program. Alpha-Methyl cinnamaldehyde. Salmonella (981636).

Justification for classification or non-classification

2-Methyl-3-phenyl-2-propenal showed no convincing evidence of mutagenic potential in OECD Guideline 471 and good-quality AMES bacterial tests involving strains of Salmonella typhimurium and Escherichia coli , with and without added activation (S9). The test material did not induce any chromosome damage, or damage to the cell division apparatus, in cultured somatic L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system in an OECD Guideline 487in vitrocytogenicity / micronucleus study .

A SOS chromotest with E. coli was also negative. An in vivo bone marrow micronucleus test in mice and a gene mutation test in Drosophila melanogaster found no evidence of genotoxic activity.

Overall, according to the classification and labelling of the EC commission, 2-methyl-3-phenyl-2-propenal is not classified as a mutagen.