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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-06-08 to 2015-07-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Asahi Glass Co., Ltd. (10, Goikaigan, Ichihara-shi, Chiba 290-8566, Japan); Lot# 150330
- Expiration date of the lot/batch: 2015-12-30
- Purity test date: 2015-05-14

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigeration (actual range 5.4 - 8.1°C [test substance storage room] and 4.2 - 8.2°C [mutagenicity test room], in a dark place, in a sealed container
- Stability under test conditions: Stable under test conditions
- Solubility and stability of the test substance in the solvent/vehicle: visual inspection at preparation of the test substance detected no reaction to the vehicle (discoloration, exothermic reaction, foaming etc.)

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear and colourless solution

OTHER SPECIFICS:
- other information:
Molecular weight: 130.46
Purity: 99.98%
Impurities: unknown components (0.02%)

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:
other: tester strains checked for amino-acid requirement, deep rough character [rfa], UV sensitivity, and drug resistance
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Kikkoman Biochemifa Company (Lot# RAA201501A)
- method of preparation of S9 mix : The S9 was prepared from liver homogenates from Slc:SD rats (male, 7 weeks of age, body weight range 195-227 g) to which phenobarbital (PB) and 5,6-benzoflabone (BF) had been intraperitoneally administered for enzyme induction (PB 0.03 g/Kg on Day 1, PB 0.06 g/Kg on Day 2, PB 0.06 g/Kg + BF 0.08 g/Kg on Day 3, and PB 0.06 g/Kg on Day 4).
- concentration or volume of S9 mix and S9 in the final culture medium :

Composition of 1 mL of S9 mix
S9: 0.1 mL
Magnesium Chloride: 8 µmol
Potassium Chloride: 33 µmol
Glucose-6-phosphate: 5 µmol
Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH): 4 µmol
Nicotinamide adenine dinucleotide, reduced form (NADH): 4 µmol
Sodium-phosphate buffer (pH 7.4): 100 µmol

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not specified
Test concentrations with justification for top dose:
Dose-finding test: ±S9: 5, 15, 50, 150, 500, 1500, and 5000 µg/plate
In both the non-activated and activated assays, the average number of revertants in the test substance group was less than twice that in the corresponding negative control group in all tester strains and the number of revertants did not increase in a dose-related manner. No inhibition of cell growth or precipitation of the test substance was observed in any of the tester strains. On the basis of the above results, 5000 µg/plate was selected as the highest dose of the test substance and total of 6 doses were set in a twofold dilution series (5-step serial dilutions) for all tester strains in both the non-activated and activated assays.
Main test: ±S9: 156, 313, 625, 1250, 2500, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The solubility of the test substance in dimethyl sulfoxide (DMSO) was confirmed by the test facility. As a result, the test substance was quickly dissolved in DMSO at 50 mg/mL without exothermic reaction, foaming, or discoloration. DMSO was therefore selected as the vehicle as well as the negative control.

- Justification for percentage of solvent in the final culture medium: The test substance was dissolved and diluted with the vehicle (DMSO) to the prescribed concentrations. In the dose-finding test, a 50 mg/mL preparation was prepared, which was diluted in approximately threefold dilution series to prepare 15, 5, 1.5, 0.5, 0.15, and 0.05 mg/mL preparations. In the main test, a 50 mg/mL preparation was prepared, which was diluted in a twofold dilution series to prepare 25, 12.5, 6.25, 3.13, and 1.56 mg/mL preparations.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-Amino Anthracene (2-AA); 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2)
Remarks:
-S9: AF-2, NaN3, 9-AA; +S9: 2-AA
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate)
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1x10^9 cells/mL
- Test substance added in medium; in agar: plate incorporation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Each thawed tester strain suspension (12 µL) was inoculated into a 40 mL L-shaped tube containing 12 mL of the culture medium for subculture (nutrient broth medium), which was cooled with ice until the start of incubation, for 6 hours and 40 mins in both the dose-finding and main tests. The mixture was then incubated for 10 hours in a thermostatic shaking water bath set at 37°C, 40 mm in stroke, and a rate of 100 times/min. At termination of incubation, OD (660 nm) of the culture solution obtained was measured using a spectrophotometer to calculate the number of viable cells.
- Exposure duration: 10 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 49 hours
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: A culture solution in which the number of viable cells was above 1x10^9 cells/mL was used.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
Rationale for test conditions:
The tester strains used in the study were selected because they are widely accepted as the bacteria suitable for the investigating the genotoxic potential of chemicals. Study was conducted according to OECD Guideline 471 recommendations.
Evaluation criteria:
The test substance was judged to be positive for the potential to induce genetic mutation when the average number of revertants in the test substance group was twice or more than in the corresponding negative control group and a dose-related increase in the number of revertants was observed with good reproducibility in at least one tester strain. When reproducibility was not observed in the test results, a confirmation test was conducted to confirm reproducibility.
Statistics:
No statistical procedures were applied for evaluation of the test results.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the dose-finding test, in both the non-activated and activated assays, the average number of revertants in the test substance group was less than twice that in the corresponding negative control group in all tester strains and the number of revertants did not increase in a dose-related manner. No inhibition of cell growth or precipitation of the test substance was observed in an tester strain.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : See Table 1 and Table 2 for details

Ames test:
- Signs of toxicity : No
- Individual plate counts : See Table 1 and Table 2 for details
- Mean number of revertant colonies per plate and standard deviation : See Table 1 and Table 2 for details

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: Provided in Annex 2 of the study report
- Negative (solvent/vehicle) historical control data: Provided in Annex 2 of the study report

Any other information on results incl. tables

Table 1. Reverse Mutation test of HCFO-1233yd(Z) in Bacteria (Dose-finding Test)

±S9

Test substance dose (µg/Plate)

Number of Revertants/plate (Mean ± SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

Negative Control

85 87 87

(86 ± 1)

7 9 10

(9 ± 2)

23 18 15

(19 ± 4)

6 13 16

(12 ± 5)

7 5 6

(6 ± 1)

5

74 83 97

(85 ± 12)

8 10 8

(9 ± 1)

15 22 17

(18 ± 4)

17 12 10

(13 ± 4)

7 4 5

(5 ± 2)

15

83 86 72

(80 ± 7)

12 10 6

(9 ± 3)

17 19 20

(19 ± 2)

22 8 23

(18 ± 8)

8 5 5

(6 ± 2)

50

80 80 77

(79 ± 2)

6 7 7

(7 ± 1)

21 20 16

(19 ± 3)

3 11 11

(8 ± 5)

5 9 5

(6 ± 2)

150

97 89 78

(88 ± 10)

12 6 6

(8 ± 3)

12 20 16

(16 ± 4)

11 17 9

(12 ± 4)

6 7 5

(6 ± 1)

500

108 103 80

(97 ± 15)

11 10 7

(9 ± 2)

11 18 8

(12 ± 5)

9 11 10

(10 ± 1)

6 3 5

(5 ± 2)

1500

62 88 99

(83 ± 19)

5 5 5

(5 ± 0)

15 18 18

(17 ± 2)

12 22 10

(15 ± 6)

7 6 7

(7 ± 1)

5000

63 77 91

(77 ± 14)

6  7 4

(6 ± 2)

16 10 13

(13 ± 3)

22 10 13

(15 ± 6)

5 4 6

(5 ± 1)

+S9

Negative Control

97 105 132

(111 ± 18)

15 17 8

(13 ± 5)

23 25 28

(25 ± 3)

30 36 28

(31 ± 4)

7 8 8

(8 ± 1)

5

104 115 94

(104 ± 11)

15 8 11

(11 ± 4)

17 17 20

(18 ± 2)

18 36 29

(28 ± 9)

7 12 13

(11 ± 3)

15

99 103 97

(100 ± 3)

14 7 6

(9 ± 4)

13 13 16

(14 ± 2)

21 22 22

(22 ± 1)

11 13 7

(10 ± 3)

50

105 112 98

(105 ± 7)

7 9 6

(7 ± 2)

21 21 12

(18 ± 5)

38 12 27

(26 ± 13)

6 8 5

(6 ± 2)

150

117 107 85

(103 ± 16)

12 16 11

(13 ± 3)

23 26 20

(23 ± 3)

32 34 21

(29 ± 7)

7 7 10

(8 ± 2)

500

96 122 118

(112 ± 14)

11 10 13

(11 ± 2)

11 11 26

(16 ± 9)

25 24 17

(22 ± 4)

11 8 7

(9 ± 2)

1500

111 93 114

(106 ± 11)

12 8 7

(9 ± 3)

12 14 13

(13 ± 1)

27 29 24

(27 ± 3)

12 8 8

(9 ± 2)

5000

114 96 92

(101 ± 12)

9 6 6

(7 ± 2)

15 17 16

(16 ± 1)

14 32 34

(27 ± 11)

10 8 11

(10 ± 2)

Positive Control S9 Mix (-)

Chemical

AF-2a

NaN3b

AF-2

AF-2

9-AAc

Dose

(µg/Plate)

0.01

0.5

0.01

0.1

80

Number of Revertants/

Plate

583 625 536

(581 ± 45)

289 274 281

(281 ± 8)

82 73 70

(75 ± 6)

406 382 387

(392 ± 13)

566 706 661

(644 ± 71)

Positive Control S9 Mix (+)

Chemical

2-AAd

2-AA

2-AA

2-AA

2-AA

Dose

(µg/Plate)

1

2

10

0.5

2

Number of Revertants/

Plate

2744 2647 2330

(2574 ± 217)

555 528 497

(527 ± 29)

1176 1158 1124 (1153 ± 26)

869 829 905

(868 ± 38)

496 605 551

(551 ± 55)

Negative Control: Dimethylsulfoxide (DMSO)

a AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide

b NaN: Sodium Azide

c 9-AA: 9-Aminoacridine hydrochloride hydrate

d 2-AA: 2-Aminoanthracene

Table 2. Reverse Mutation test of HCFO-1233yd(Z) in Bacteria (Main Test)

±S9

Test substance dose (µg/Plate)

Number of Revertants/plate (Mean ± SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

Negative Control

79 87 88

(85 ± 5)

11 7 7

(8 ± 2)

21 25 21

(22 ± 2)

22 15 13

(17 ± 5)

5 5 4

(5 ± 1)

156

80 84 98

(87 ± 9)

14 5 6

(8 ± 5)

20 17 23

(20 ± 3)

13 20 18

(17 ± 4)

5 10 6

(7 ± 3)

313

89 88 81

(86 ± 4)

7 5 8

(7 ± 2)

16 15 12

(14 ± 2)

12 20 21

(18 ± 5)

5 6 3

(5 ± 2)

625

85 82 86

(84 ± 2)

13 10 10

(11 ± 2)

12 12 15

(13 ± 2)

13 11 13

(12 ± 1)

3 5 7

(5 ± 2)

1250

76 101 85

(87 ± 13)

9 6 7

(7 ± 2)

26 21 12

(20 ± 7)

12 12 12

(12 ± 0)

6 4 3

(4 ± 2)

2500

82 74 75

(77 ± 4)

13 11 8

(11 ± 3)

16 15 12

(14 ± 2)

10 12 14

(12 ± 2)

3 6 5

(5 ± 2)

5000

74 86 73

(78 ± 7)

8 8 6

(7 ± 1)

17 17 13

(16 ± 2)

12 16 14

(14 ± 2)

3 4 6

(4 ± 2)

+S9

Negative Control

79 104 116

(100 ± 19)

9 7 11

(9 ± 2)

20 17 23

(20 ± 3)

31 22 35

(29 ± 7)

16 10 13

(13 ± 3)

156

107 109 97

(104 ± 6)

5 8 11

(8 ± 3)

25 24 18

(22 ± 4)

21 31 41

(31 ± 10)

7 8 7

(7 ± 1)

313

107 109 106

(107 ± 2)

8 9 6

(8 ± 2)

30 20 30

(27 ± 6)

36 40 41

(39 ± 3)

6 7 10

(8 ± 2)

625

120 100 90

(103 ± 15)

9 11 11

(10 ± 1)

25 15 23

(21 ± 5)

25 26 20

(24 ± 3)

3 7 12

(7 ± 5)

1250

120 108 113

(114 ± 6)

11 10 12

(11 ± 1)

31 29 22

(27 ± 5)

35 30 30

(32 ± 3)

11 8 12

(10 ± 2)

2500

112 110 118

(113 ± 4)

10 13 8

(10 ± 3)

27 18 23

(23 ± 5)

20 32 30

(27 ± 6)

11 12 16

(13 ± 3)

5000

123 137 119

(126 ± 9)

11 7 11

(10 ± 2)

20 18 25

(21 ± 4)

33 25 30

(29 ± 4)

12 12 10

(11 ± 1)

Positive Control S9 Mix (-)

Chemical

AF-2a

NaN3b

AF-2

AF-2

9-AAc

Dose

(µg/Plate)

0.01

0.5

0.01

0.1

80

Number of Revertants/

Plate

580 555 541

(559 ± 20)

293 277 288

(286 ± 8)

66 75 60

(67 ± 8)

294 281 324

(300 ± 22)

665 721 481

(622 ± 126)

Positive Control S9 Mix (+)

Chemical

2-AAd

2-AA

2-AA

2-AA

2-AA

Dose

(µg/Plate)

1

2

10

0.5

2

Number of Revertants/

Plate

2743 2788 2722

(2751± 34)

540 531 567

(546± 19)

1215 1138 1209 (1187± 43)

836 824 787

(816± 26)

531 468 465

(488± 37)

Negative Control: Dimethylsulfoxide (DMSO)

aAF-2: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide

bNaN: Sodium Azide

c9-AA: 9-Aminoacridine hydrochloride hydrate

d2-AA: 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:
The test material was not considered to be mutagenic under the conditions of this bacterial reverse mutation assay.
Executive summary:

In a key Guideline OECD 471 study (Safety Research Institute for Chemical Compounds Co., Ltd, 2015), the potential of the test material (HCFO-1233yd(Z)) to induce genetic mutation in bacteria was evaluated in a bacterial reverse mutation assay using Salmonella typhimurium strains TA98, TA100, TA 1535, TA1537 and Escherichia coli strain WP2uvrA in the absence and presence of metabolic activation (±S9).

 

In the study, a dose-finding test was conducted initially where the highest dose of the test substance was set at 5000 µg/plate and a total of 7 doses (5, 15, 50, 150, 500, 1500, and 5000 µg/plate) were set in approximately threefold dilution series for both the non-activated and activated assays. On the basis of the results of the dose-finding test, 5000 µg/plate was selected as the highest dose of the test substance and total of 6 doses were set in a twofold dilution series (5-step serial dilutions) for all tested strains in both the non-activated and activated assays (Main test: ±S9: 156, 313, 625, 1250, 2500, and 5000 µg/plate).

 

In the dose-finding as well as the main test, in both the non-activated and activated assays, the average number of revertants in the test substance group was less than twice that in the corresponding negative control group in all tester strains and the number of revertants did not increase in a dose-related manner. No inhibition of cell growth or precipitation of the test substance was observed in any tested strain. Thus, reproducibility was obtained between the results of the dose-finding test and those of the main test.

 

In the dose-finding as well as the main test, all the average number of revertants in the negative control group of all tester strains were within the range of control values based on the historical control data of the test facility. The average number of revertants in the positive control group of each tester strain clearly increased and was twice or more that in the corresponding negative control group. These results confirmed that each tester strain had appropriate sensitivity to the mutagens.

 

Based on the results observed, the test material was not considered to be mutagenic under the conditions of this bacterial reverse mutation assay.