Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17/10/2012 - 11/12/2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Study performed according to Japanese guidelines including: "Procedures of Mutagenicity Test Using Microorganisms and Evaluation of Test Results" (February 8, 1999; the Office Memo by the Director of Chemical Risk Assessment Office of Industrial Safety and Health Department of Labour Standards Bureau, the Ministry of Labour) and "Reverse Mutagenicity Test on Bacteria" of "Mutagenicity Test" stipulated in the "Testing Methods for New Chemical Substances" (March 31, 2011; No. 0331-7, Pharmaceutical and Food Safety Bureau, the Ministry of Health, Labour and Welfare) and other relevant updates.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas
Specific details on test material used for the study:
Chemical name (Z)-1-Chloro-2,3,3,3-tetrafluoropropene
Other name 1224yd(Z)
Molecular formula C3HClF4
Molecular weight 148.49

Purity 96.7570% (GC area percentage)
Impurity (1) (E)-1-Chloro-2,3,3,3-tetrafluoropropene: 2.3672%
Impurity (2) 1,1,1,2-tetrafluoropropane: 0.1025%
Impurity (3) 2-Chloro-1,3,3,3-tetrafluoropropene: 0.0143%
Impurity (4) Unknown component: 0.7590%

Supplier ASAHI GLASS CO., LTD
Lot number 120517D-Fr8

Physicochemical properties
Boiling point 46.7°C (measuring pressure 0.21MPa)
Appearance at ordinary temperature Gas

Storage conditions:
The test substance was put into an airtight bomb and stored at room temperature
(cabinet No. 7 in the test substance storage room, permissible range: from 10 to
30°C).

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Dose Selection
a) Dose-range finding test
A total of six doses including 50.0 v/v% as the highest dose and five lower doses diluted
with a geometric progression of 2 were set.
b) Main test
The results of the dose-range finding test showed that the number of revertant colonies in
the test substance treatment groups was less than twice that in each negative control for
all tester strains with and without S9 mix. The bacterial growth inhibition was not
observed at any test condition.
Therefore, the highest dose was selected at 50.0 v/v% at which the precipitation of the
test substance was observed, and four lower doses of 25.0, 12.5, 6.25 and 3.13 v/v%
diluted with a geometric progression of 2 were employed for all tester strains with and
without S9 mix in the main test.
Vehicle / solvent:
Negative control substance ( dilution gas)
1) Name The Air (clean air) in the safety cabinet through a HEPA filter
2) Reason for selection of vehicle because the test substance was stable in air (information provided by the sponsor), the clean air was selected as a vehicle ( dilution gas).
Controls
Untreated negative controls:
other: Filtered fresh air
Remarks:
since the substance is a gas at room temperature, air was the diluent gas
Negative solvent / vehicle controls:
no
True negative controls:
other: Filtered fresh air
Remarks:
since the substance is a gas at room temperature, air was the diluent gas
Positive controls:
yes
Positive control substance:
sodium azide
furylfuramide
other: ICR 191; 2-Aminoanthracene
Details on test system and experimental conditions:
Medium and S9 Mix: Medium: Minimal glucose agar plate Tesmedia AN (Oriental Yeast) was used.
Soft agar: A solution containing 0.5 mM histidine and 0.5 mM biotin for S. typhimurium strains or 0.5 mM tryptophan for E. coli strain was added to a soft agar solution containing 0.6 w/v% agar (Bacto Agar, Lot No. 0082277, Difeo Laboratories) and 0.5 w/v% NaCl at a volume ratio of 1 to 10.

S9 mix: Rat liver S9: S9 (Oriental Yeast) prepared from the liver of 7-week-old male SD rats administered with phenobarbital (one time at 30 mg/kg and three times at 60 mg/kg) and 5,6-benzoflavone (one time at 80 mg/kg) was used. The S9 was cryopreserved in an ultra-deep freezer No. 3 in the Ames test room No. 1 below -80°C until use. S9 was thawed just before use.

Composition of S9 mix S9 mix was prepared just before use. One milliliter of S9 mix consisted of 8 μmol MgCh, 33 μmol KCl, 5 μmol Glucose-6-phosphate, 4 μmol NADPH, 4 μmol NADH, 100 μmol sodium-phosphate buffer (pH 7.4) and 0.1 mL of S9.

Pre-cultures of the Tester Strains Frozen stock culture of bacterial strains; 30 μL for TAlOO, 5 μL for WP2uvrA and 20 μL for TA98, TA1535 and TA1537, were respectively inoculated to 11 mL of Nutrient broth No. 2 (Lot No. 635187, OXOID) in L-tube (volume: 27 mL). The culture was incubated at 37±0.5°C for 9 hours with shaking at about 50 times/minute by a seesaw type of shaker (MONOSIN-IIA, TAITEC).

At the end of the incubation, the number of viable cells measured at OD 660nm was adjusted to 2.3-2.7xl0^9 cells/mL for S. typhimurium strains and to 3.8-4.2xl0^9 cells/mL for the E.coli strain.

Preparation of Test Substance Preparation Gas:
For preparation of the test substance preparation gas, the flowmeter (RK.2503F, KOFLOC), the bellows pump (BA-106F, IWAKI) and the glass syringe were used. a) Preparation of test substance preparation gas: The required amount of the test substance original gas and diluent gas was introduced into the gas sampling bag (analytic barrier bag, OMI ODOR-AIR SERVICE) for each concentration for preparation the test substance preparation gas. b) Timing of preparation: The test substance preparation gas were prepared just before use, kept in the safety cabinet at room temperature and used within 7 hours (the stability has been confirmed, detailed below).

Analysis of Test Substance Preparation Gas
a) Confirmation of Stability of test substance preparation gas
Analysis was conducted at the test facility (non-GLP) using gas chromatography (GC).
1) Preparation of analytical sample
50.0 v/v% and 0.100 v/v% of the test substance preparation gas were collected the
required amount from the analytic barrier bags containing the test substance
preparation gas immediately and 7 hours after. The collected test substance
preparation gas was diluted with the diluent gas precisely
and the analytical samples were prepared (n = 1 ).

Results of analysis
Concentrations of the test substance preparation gas in each analysis sample were
measured once using GC.
50.0 v/v% and 0.100 v/v% of the test substance preparation gas were stable for 7
hours after preparation at room temperature (storage condition). Concentrations of
the 50.0 v/v% and 0.100 v/v% dose were within the acceptable level (nominal
concentration ±10%). Therefore, these gas were judged to be stable for storage
condition.

Confirmation of Concentration of test substance preparation gas
All doses of the test substance preparation gases used in the test, concentration of the
test substance were measured immediately after preparation by GC.
1) Preparation of analytical sample
Immediately after preparation, the test substance preparation gas were collected the
required amount into the capacity known vacuum bottle with a vacuum state by the
pump from the analytic barrier bags containing the test substance preparation gas.
The collected test substance preparation gas was diluted with the diluent gas precisely, and the analytical samples were prepared (n = 1 ).
The all dose of the test substance preparation gas of the dose-range finding test and
25.0, 12.5, 6.25 and 3 . 1 3 (v/v%) dose of the main test were carried out re-prepared
and re-measured, due to the out of the range of 100 ± 10% for the set value.

Results of analysis
Concentrations of the test substance preparation gas in each analysis sample were
measured once using GC. Concentrations of the all dose of the test substance
preparation gas were within the acceptable level (nominal concentration 100 ±10%).
c) Outline of Analytical Method
Analysis was conducted according to validation of the analytical method (non-GLP) at
the test facility.

1) Validation of Analytical Method
(a) Preparation of sample for measurement
(1) Sample gas for linearity
The standard stock gas for the validation of analytical method was diluted with
diluted gas to make 0.0984, 0.196 and 0.389 v/v% standard gas
(2) Sample solutions for accuracy and repeatability
About 0.1, 0.2 and 0.4 v/v% of the standard gas, as
well as the linearity sample were prepared (each concentration n=3).

Linearity
The linearity sample was measured by GC. The calibration curve was made by
using the concentration of the test substance on the horizontal line and the
detection value on the vertical line. The correlation coefficient of the regression
equation which was obtained from least square analysis was R=0.999. Therefore,
it was confirmed that the result satisfied the criteria for linearity (0.999 or more).

Accuracy and repeatability
The accuracy and repeatability sample was measured by GC. Accuracy and
repeatability were confirmed by the test substance concentrations calculated from
the regression formula obtained at the linearity.
Accuracies of about 0.1 v/v% standard gas were 3.5%, 2.6% and 2.2%, and
repeatability was 0.3%.
Accuracies of about 0.2 v/v% standard gas were 1 . 1 % , 2.1% and -3.1%, and
repeatability was 2.5%.
Accuracies of about 0.4 v/v% standard gas were -3.7%, -4.4% and -4.0%, and
repeatability was 0.3%.
It was confirmed that the results satisfied the criteria for accuracy and
repeatability (accuracy: within ±10%, repeatability: 5% or less).

Preparation of Positive Control Substance Solutions a) Preparation method and storage conditions NaN3 was dissolved in distilled water. AF-2, ICR-191 and 2AA were dissolved in DMSO. These positive control substance solutions were stored in the ultra-deep freezer No. 3 in the Ames test room No. 1 below -80°C. b) Timing of use The positive control substance solutions were thawed just before use.

Methods

Because the test substance was gas at room temperature, this study was performed by the gas exposure method with and without S9 mix. The positive control was performed by the plate-incorporation method with and without S9 mix. Duplicate plates were used for the negative control, the positive control and the test substance treatment groups. The study number, name of the tester strain, the presence or absence of S9 mix and the dose level were noted on each plate.

Procedures
a) Gas exposure method After 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.4) or S9 mix, and 0.1 mL of the bacterial culture were added to a test tube, the mixture was vortexed and poured onto a minimal glucose agar plate. The cover of plate was taken off in a safety cabinet. The plates were set on a plate holder upside down in each dose at the presence or absence of S9 mix. The plate holder was put into an analytic barrier bag whose edge was cut off. The opening of bag was sealed by a heat sealer. Air in the bag was vacuumed by a pump and then the dilution gas or the test substance preparation gas was injected. The bags were incubated at 37±0.5°C for 24 hours. After the incubation, the test substance preparation gas was removed in a safety cabinet, followed by an injection of fresh air to clear the inside of analytic barrier bag. The bag was cut open after the fresh air was removed to take the plates out. Then the cover was put on the plate. The plates were put into bags upside down in each dose at the presence or absence of S9 mix, followed by incubation at 37±0.5°C for 24 hours. The number of revertant colonies was counted after the incubation.

b) Plate-incorporation method 0.1 mL of the positive control substance solutions, 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.4) or S9 mix, and 0.1 mL of the bacterial culture were added to a test tube. Two milliliters of the soft agar were then added to each tube and the mixture was poured onto a minimal glucose agar plate. The number of revertant colonies was counted after incubation at 37±0.5°C for 48 hours.

Confirmation of Sterility S9 mix (0.5 mL) was mixed with 2 mL of the soft agar and tested with the same method of the negative control to examine contamination. Minimal glucose agar plates which 2 mL of the soft agar was poured onto were exposed to the highest dose of the test substance preparation gas by the gas exposure method to examine contamination.

Observation and Colony Counting
Observation: The precipitation of the test substance was observed macroscopically and the bacterial growth inhibition was observed by using a stereomicroscope at the end of the incubation.

Colony counting:
All plates were counted with a colony analyzer (CA-11D, SYSTEM SCIENCE). Square correction and miss counting correction were conducted when counting with the colony analyzer.

Evaluation criteria:
JUDGEMENT CRlTERlA OF TEST RESULTS The test substance was judged to be positive when the number of revertant colonies increased to twice or more than that in the negative control and when the responses were dose-related and/or reproducible. The other cases were judged to be negative.

FACTORS AFFECTED RELIABILITY OF TEST There were no factors that might have affected the reliability of the test.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Remarks:
Test substance is a gas at room temperature - vehicle diluent gas is fresh air
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
not applicable
Remarks:
Test substance is a gas at room temperature - vehicle diluent gas is fresh air
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Remarks:
Test substance is a gas at room temperature - vehicle diluent gas is fresh air
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Remarks:
Test substance is a gas at room temperature - vehicle diluent gas is fresh air
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Remarks:
Test substance is a gas at room temperature - vehicle diluent gas is fresh air
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 2 Results of the main test

Test substance: HCFO-1224yd(Z)

Test period

From November 6, 2012 to November 8, 2012

With (+) or without (-) S9 mix

Test substance dose (v/v) %

Number of revertant colonies per plate

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9 mix

Negative control

116

10

35

32

8

106 (111)

17 (14)

41 (38)

19 (26)

12 (10)

3.13

118

13

31

29

13

132 (125)

16 (15)

25 (28)

26 (28)

12 (13)

6.25

122

15

28

18

11

102 (112)

17 (16)

31 (30)

15 (17)

7 (9)

12.5

99

17

42

17

8

109 (104)

12 (15)

38 (40)

14 (16)

7 (8)

25.0

98

13

45

32

10

86 (92)

18 (16)

36 (41)

28 (30)

6 (8)

50.0

69

17

26

28

8

73 (71)

11 (14)

20 (23)

22 (25)

10 (9)

+S9 mix

Negative control

98

14

33

32

20

91 (95)

16 (15)

33 (33)

36 (34)

8 (14)

3.13

122

15

32

26

12

117 (120)

13 (14)

34 (33)

28 (27)

10 (11)

6.25

88

14

38

29

10

104 (96)

15 (15)

28 (33)

31 (30)

10 (10)

12.5

93

13

26

24

18

86 (90)

14 (14)

20 (23)

28 (26)

8 (13)

25.0

90

12

22

29

15

92 (91)

8 (10)

34 (28)

29 (29)

11 (13)

50.0

82

11

32

19

13

87 (85)

16 (14)

27 (30)

14 (17)

7 (10)

Positive control

-S9 mix

Chemical

AF-2

NaN3

AF-2

AF-2

ICR-191

Dose(μg/plate)

0.01

0.5

0.01

0.1

0.5

Nrevertant colonies/ plate

402

73

139

753

133

454 (428)

77 (75)

161 (150)

723 (738)

180 (157)

Positive control +S9 mix

Chemical

2AA

2AA

2AA

2AA

2AA

Dose(μg/plate)

1

2

10

0.5

2

Nrevertant colonies/ plate

1954

316

635

360

331

2246 (2100)

228 (272)

538 (587)

372 (366)

181 (256)

[Notes]

• ( ): The mean number of colonies per plate.

• AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

• NaN3: Sodium azide

• 2AA: 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:
For both the dose-range finding test, and the main test, for all tester strains, the number of revertant colonies in the test substance treatment groups was less than twice that in each negative control with and without S9 mix. The bacterial growth inhibition was not observed at any test condition.

The mutagenicity of the test substance was judged to be negative because the number of revertant colonies in the test substance treatment groups was less than twice that in the negative control for all tester strains regardless of the presence or absence of S9 mix. The numbers of the revertant colonies in the positive controls were above twice that in the negative controls. The test results showed that the numbers of revertant colonies in the negative and positive controls were within the range of the historical data at the testing facility. It was also confirmed that the test system was free from bacterial contamination, which declares the test results to be valid.

It was concluded that HCFO-1224yd(Z) had no ability to induce mutations under the present test conditions.