2,2-Difluoroethyl Acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 JUNE 2013 to 26 AUGUST 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2-difluoroethyl acetate

EC Number:

801-773-4

Cas Number:

1550-44-3

Molecular formula:

C4H6F2O2

IUPAC Name:

2,2-difluoroethyl acetate

Test material form:

liquid

Remarks:

clear and colorless.

Method

Target gene:

S. typhimurium strains: histidine requirement for growth.
E. coli strain: tryptophan requirement for growth.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: the S9 (lot No 3080, protein content 42.2 mg/mL) was purchased from Moltox (Boone, NC, USA) where it was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. Upon receipt at the testing facility, the S9 was stored at -60°C or colder until used.
- Method of preparation of S9 mix / Concentration or volume of S9 mix / S9 in the final culture medium: the S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM ß-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. 0.5 mL of the Sham mix was used in the experiments with metabolic activation
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
* Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.
* To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar.

Test concentrations with justification for top dose:

- Initial test - experiment B1: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate.
- Initial test - experiment B2: 50, 150, 500, 1500 and 5000 μg per plate.
- Confirmatory test - experiment B3: 50, 150, 500, 1500 and 5000 μg per plate.
- Justification for top dose: 5000 µg/plate (= 5 mg/plate) is the maximum test concentration recommended in OECD test guideline 471 for soluble non-cytotoxic substances.
See more details in the section "Any other information on results incl. tables" below.

Vehicle / solvent:

- Solvent used: DMSO (lot No SHBC3749V, purity 99.92%).
- Justification for choice of solvent: a solubility test was conducted to determine the vehicle. The test was conducted using water and DMSO to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents. DMSO was selected as the solvent of choice based on the solubility of the test material and compatibility with the target cells. The test material formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at the test facility.
Remark: All positive controls were diluted with DMSO except sodium azide, which was diluted with water.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO.

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

methylmethanesulfonate

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration:
1) Initial test: duplicate.
2) Confirmatory test: triplicate.
- Number of independent experiments:
1) Initial test: 2 consecutive experiments. The second experiment was carried with strain TA100 without S9 only due to an unacceptable vehicle control value in the first experiment. See more details in the results sections below.
2) Confirmatory test: 1 experiment.

METHOD OF TREATMENT/ EXPOSURE:
- Test material formulation: the plate-incorporation method was applied. The test substance was formulated in DMSO at the concentrations described in the field "Test concentrations with justification of top dose". The actual concentrations of the test substance in the test formulations were not determined. Therefore, the concentrations quoted in this report are nominal concentrations.
- Exposure conditions: on the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, the water used was sterile and deionized. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5% (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). One-half (0.5) mL of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50 μL of vehicle or test material dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test material aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The condition of the bacterial background lawn was evaluated for evidence of test material toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate.

METHODS FOR MEASUREMENTS OF GENOTOXICITY:
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and reported.

Rationale for test conditions:

Initial Test:
The initial assay (experiment B1) was used to establish the dose-range for the confirmatory assay and to provide a preliminary mutagenicity evaluation. Vehicle control, positive controls and eight dose levels of the test material were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. This initial test was repeated (experiment B2) with strain TA100 only due to an unacceptable vehicle control value in the first experiment. See more details in the results sections below.

Confirmatory Test:
The confirmatory mutagenicity assay was used to evaluate the mutagenic potential of the test material. Five dose levels of test material along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test material, vehicle control and positive controls were plated in triplicate.

Evaluation criteria:

For the test material to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test material. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0 times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.
A response was evaluated as negative, if it was neither positive nor equivocal.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Signs of toxicity: Neither precipitate nor toxicity was observed in initial (experiments B1 and B2) and confirmatory (experiment B3) tests.
- Genotoxicity results:
* In Experiment B1 (Initial test, see Table 1 in the field "Any other information on results incl. tables"), no positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation or with tester strains TA98, TA1535, TA1537 and WP2 uvrA in the absence of S9 activation. Due to an unacceptable vehicle control value, tester strain TA100 in the absence of S9 activation was not evaluated for mutagenicity but was retested in Experiment B2 based on the toxicity and precipitate profile observed.
* In Experiment B2 (Repeat of the Initial test, see Table 2 in the field "Any other information on results incl. tables"), no positive mutagenic response was observed with tester strain TA100 in the absence of S9 activation.
* In Experiment B3 (Confirmatory test, see Table 3 in the field "Any other information on results incl. tables"), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

HISTORICAL CONTROL DATA
See Table 4 in the field "Any other information on results incl. tables".

Any other information on results incl. tables

Sterility results:

No contaminant colonies were observed on the sterility plates for the vehicle control, the test material dilutions or the S9 and Sham mixes.

 

Tester strain titer results:

Experiment	Tester Strain
	TA98	TA100	TA1535	TA1537	WP2 uvrA
	Titer Value (x 10E9 cells per mL)
B1	0.1	0.8	1.7	2.1	2.4
B2	-	0.8	-	-	-
B3	0.9	1.1	1.5	2.1	2.8

 

Tabulated results:

 

Table 1: Initial Test: experiment B1

	TA 98		TA 100		TA1535		TA 1537		WP2uvrA
	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Test material dose level (µg) per plate	Mean revertants per plate (standard deviation)
5000	22 (0)	25 (4)	-	97 (14)	15 (7)	14 (7)	9 (1)	8 (5)	23 (1)	29 (3)
1500	16 (4)	31 (3)	-	69 (16)	8 (2)	18 (4)	9 (1)	4 (1)	20 (4)	21 (8)
500	10 (1)	31 (3)	-	77 (4)	14 (11)	16 (6)	8 (2)	8 (2)	16 (4)	22 (7)
150	22 (3)	30 (6)	-	89 (1)	12 (2)	15 (1)	6 (3)	7 (2)	14 (1)	24 (1)
50	12 (1)	20 (1)	-	76 (1)	11 (3)	13 (2)	7 (4)	12 (2)	12 (8)	22 (10)
15	10 (1)	26 (2)	-	75 (6)	11 (1)	12 (9)	5 (2)	9 (4)	12 (8)	28 (1)
5.0	14 (5)	34 (11)	-	99 (16)	9 (7)	11 (1)	8 (5)	12 (4)	28 (1)	22 (6)
1.5	17 (0)	25 (2)	-	88 (11)	5 (1)	15 (1)	9 (0)	12 (5)	20 (1)	20 (4)
Negative control (DMSO)	18 (4)	20 (0)	-	95 (17)	10 (1)	18 (0)	7 (4)	11 (0)	18 (4)	22 (2)
Positive control	161 (4)	346 (57)	-	374 (77)	419 (23)	97 (1)	283 (64)	47 (2)	311 (6)	264 (73)

 

Table 2: Initial Test: experiment B2

	TA 100
	- S9
Test material dose level (µg) per plate	Mean revertants per plate (standard deviation)
5000	95 (6)
1500	96 (16)
500	109 (34)
150	107 (1)
50	117 (17)
Negative control (DMSO)	98 (18)
Positive control	693 (8)

 

Table 3: Confirmatory Test: experiment B3

	TA 98		TA 100		TA1535		TA 1537		WP2uvrA
	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Test material dose level (µg) per plate	Mean revertants per plate (standard deviation)
5000	10 (4)	28 (4)	100 (13)	113 (20)	12 (6)	11 (7)	8 (4)	9 (2)	22 (6)	22 (8)
1500	11 (4)	25 (3)	98 (24)	117 (19)	11 (6)	18 (5)	8 (2)	6 (2)	22 (2)	28 (3)
500	11 (3)	22 (4)	94 (12)	107 (6)	11 (2)	16 (9)	5 (3)	8 (5)	23 (10)	23 (5)
150	13 (3)	31 (4)	104 (3)	115 (25)	11 (3)	18 (5)	5 (4)	10 (6)	19 (5)	22 (2)
50	14 (4)	24 (6)	104 (7)	119 (21)	14 (1)	19 (7)	6 (3)	8 (3)	19 (7)	23 (7)
Negative control (DMSO)	21 (9)	28 (4)	98 (5)	104 (14)	12 (2)	19 (4)	4 (1)	10 (3)	23 (4)	30 (3)
Positive control	222 (74)	488 (35)	569 (30)	531 (189)	493 (44)	111 (12)	227 (44)	32 (1)	276 (24)	277 (18)

 

Historical control data:

 

Table 4: Historical negative and positive control values 2010 – 2012

Revertants per plate:

		- S9				+ S9
Strain	Control	Mean	SD	Min	Max	Mean	SD	Min	Max
TA 98	Negative	21	10	3	64	26	10	4	61
	positive	266	168	51	1871	428	196	54	3355
TA 100	Negative	102	21	47	251	115	23	41	247
	positive	645	160	232	1393	744	273	235	2301
TA 1535	Negative	12	5	1	45	12	5	1	50
	positive	523	174	20	1593	114	110	20	1472
TA 1537	Negative	7	4	0	29	8	4	0	28
	positive	503	384	17	3448	65	71	13	1272
WP2 uvrA	Negative	32	11	5	84	35	11	8	80
	positive	422	173	42	1796	223	88	44	969

SD = standard deviation; Min = minimum value; Max = maximum value;

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, 2,2-Difluoroethyl acetate did not cause mutagenic responses in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in Escherichia coli strain WP2 uvrA in either the presence or absence of metabolic activation.

Executive summary:

The mutagenic potential of 2,2-Difluoroethyl acetate was investigated in bacteria in an in vitro study performed according to OECD test guideline 471 (Ames test) under GLP compliance.

The histidine-requiring Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and the tryptophan-­requiring Escherichia coli strain WP2uvrA were tested in both the absence and presence of a liver fraction of Aroclor-induced rats for metabolic activation (S9-mix). The study was performed in two phases, using the plate incorporation method. The first phase (i.e. initial test with two subsequent experiments) was used to establish the dose range for the confirmatory test and to provide a preliminary mutagenicity evaluation. The second phase (i.e. confirmatory test) was used to evaluate and confirm the mutagenic potential of the test material. The following conditions were applied in the different experiments:

• Initial test (experiment B1):
  
  
  
  • Tested strains: all.
  
  
  • Tested concentrations: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate.
  
  
  • Metabolic activation: with and without.
  
  
  
  


• Initial test (experiment B2):
  
  
  
  • Tested strains: Salmonella typhimurium strain TA100.
  
  
  • Tested concentrations: 50, 150, 500, 1500 and 5000 μg per plate.
  
  
  • Metabolic activation: without.
  
  
  
  


• Confirmatory test (experiment B3):
  
  
  
  • Tested strains: all.
  
  
  • Tested concentrations: 50, 150, 500, 1500 and 5000 μg per plate.
  
  
  • Metabolic activation: with and without.
  
  
  
  

Negative controls (DMSO) and positive controls were run simultaneously. The plates were incubated at 37±2 °C for 48 to 72 hours. Subsequently, the revertant colonies were counted.

No positive mutagenic responses were observed at any concentration or with any tester strain in either the absence or presence of metabolic activation. No test substance precipitation or appreciable toxicity was observed. All validity criteria of the OECD test guideline 471 were met.

Under the conditions of this study, 2,2-Difluoroethyl acetate did not cause mutagenic responses in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA in either the presence or absence of Aroclor-induced rat liver S9.