Reaction mass of chlorodifluoroacetic acid and trifluoroacetic acid

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 07 july 2003 to 10 oct 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, OECD 471 compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Chlorodifluoroacetic acid

EC Number:

200-928-8

EC Name:

Chlorodifluoroacetic acid

Cas Number:

76-04-0

Molecular formula:

C2HClF2O2

IUPAC Name:

chloro(difluoro)acetic acid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix rat liver fraction treated with Aroclor 1254

Test concentrations with justification for top dose:

0, 312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: NaCl (0.9%)
- Justification for choice of solvent/vehicle: the most appropriate for the solubility of CDFA, and among recommended vehicles by the guideline.

Controls

Untreated negative controls:

no

Remarks:

vehicle served as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-Aminoacridine, 2-Nitroflurene, Mytomycin C, 4-Nitroquinoline-1oxide, 2-Anthramine

Remarks:

Details in test conditions

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for preliminary toxicity test and for the first experiment and in the second experiment without S9 mix; preincubation in the second experiment with S9 mix.

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: 48 to 72 hours at 37°C

NUMBER OF REPLICATES: 3

Evaluation criteria:

A reproductible 2-fold increase (for the TA 98, TA 100, TA 102, and E. coli WP2 uvrA strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

thinning of the bacterial lawn only in TA 100 strain

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY:
With a treatment volume of 100 µL/plate, the dose levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.
No noteworthy toxicity was noted towards the four satrins used, with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: the number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Any other information on results incl. tables

First experiment: Direct plate incorporation method

Strain	Compound	Dose level (µg/plate)	Metabolic activation	Mean revertant colony counts
TA 1535	0.9% NaCl	0	- S9 mix	14
	CDFA	312.5		15
		625		15
		1250		10
		2500		15
		5000		11
	NAN3	1		530
	0.9% NaCl	0	+ S9 mix	14
	CDFA	312.5		13
		625		13
		1250		14
		2500		14
		5000		13
	2AM	2		251
TA 100	0.9% NaCl	0	- S9 mix	97
	CDFA	312.5		98
		625		117
		1250		101
		2500		113
		5000		102
	NAN3	1		615
	0.9% NaCl	0	+ S9 mix	119
	CDFA	312.5		129
		625		119
		1250		110
		2500		114
		5000		101
	2AM	2		1750
TA 102	0.9% NaCl	0	- S9 mix	393
	CDFA	312.5		367
		625		426
		1250		375
		2500		400
		5000		368
	NAN3	1		1332
	0.9% NaCl	0	+ S9 mix	582
	CDFA	312.5		561
		625		571
		1250		519
		2500		588
		5000		472
	2AM	2		1768

 

Strain	Compound	Dose level (µg/plate)	Metabolic activation	Mean revertant colony counts
WP2 uvrA	0.9% NaCl	0	- S9 mix	30
	CDFA	312.5		31
		625		32
		1250		38
		2500		31
		5000		29
	NAN3	1		219
	0.9% NaCl	0	+ S9 mix	37
	CDFA	312.5		38
		625		33
		1250		25
		2500		32
		5000		32
	2AM	2		480
TA 1537	0.9% NaCl	0	- S9 mix	5
	CDFA	312.5		6
		625		7
		1250		9
		2500		5
		5000		4
	NAN3	1		280
	0.9% NaCl	0	+ S9 mix	7
	CDFA	312.5		8
		625		6
		1250		7
		2500		10
		5000		7
	2AM	2		108
TA 98	0.9% NaCl	0	- S9 mix	21
	CDFA	312.5		20
		625		21
		1250		21
		2500		22
		5000		30
	NAN3	1		211
	0.9% NaCl	0	+ S9 mix	28
	CDFA	312.5		27
		625		27
		1250		30
		2500		27
		5000		30
	2AM	2		1699

 

 

Second experiment : Direct plate incorporation method (without S9 mix) and preincubation method (with S9 mix)

Strain	Compound	Dose level (µg/plate)	Metabolic activation	Mean revertant colony counts
TA 1535	0.9% NaCl	0	- S9 mix	12
	CDFA	312.5		17
		625		12
		1250		13
		2500		13
		5000		14
	NAN3	1		607
	0.9% NaCl	0	+ S9 mix	12
	CDFA	312.5		15
		625		18
		1250		13
		2500		13
		5000		11
	2AM	2		147
TA 100	0.9% NaCl	0	- S9 mix	93
	CDFA	312.5		108
		625		107
		1250		128
		2500		123
		5000		123
	NAN3	1		640
	0.9% NaCl	0	+ S9 mix	119
	CDFA	312.5		132
		625		131
		1250		135
		2500		128
		5000		138
	2AM	2		1539
TA 102	0.9% NaCl	0	- S9 mix	399
	CDFA	312.5		394
		625		420
		1250		427
		2500		401
		5000		437
	NAN3	1		1342
	0.9% NaCl	0	+ S9 mix	483
	CDFA	312.5		460
		625		452
		1250		453
		2500		490
		5000		438
	2AM	2		2122

  

Strain	Compound	Dose level (µg/plate)	Metabolic activation	Mean revertant colony counts
WP2 uvrA	0.9% NaCl	0	- S9 mix	29
	CDFA	312.5		32
		625		24
		1250		20
		2500		19
		5000		32
	NAN3	1		468
	0.9% NaCl	0	+ S9 mix	42
	CDFA	312.5		39
		625		39
		1250		47
		2500		30
		5000		38
	2AM	2		221
TA 1537	0.9% NaCl	0	- S9 mix	6
	CDFA	312.5		14
		625		6
		1250		6
		2500		14
		5000		6
	NAN3	1		285
	0.9% NaCl	0	+ S9 mix	7
	CDFA	312.5		6
		625		8
		1250		6
		2500		10
		5000		9
	2AM	2		142
TA 98	0.9% NaCl	0	- S9 mix	26
	CDFA	312.5		29
		625		30
		1250		32
		2500		24
		5000		26
	NAN3	1		172
	0.9% NaCl	0	+ S9 mix	23
	CDFA	312.5		26
		625		29
		1250		25
		2500		22
		5000		16
	2AM	2		1675

 

Applicant's summary and conclusion

Conclusions:

Chlorodifluoroacetic acid did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli in the absence and presence of metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 102, TA 1535, TA 1537 of S. typhimurium and E. coli E. coli WP2 uvr A were exposed to Chlorodifluoroacetic acid at concentrations of 0 to 5000 µg/plate in the presence and absence of mammalian metabolic activation (rat liver).

The plate incorporation method was used for preliminary toxicity test and for the first experiment and in the second experiment without S9 mix. Pre-incubation was used in the second experiment with S9 mix.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of increase of mutant colonies. Chlorodifluoroacetic acid did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.