2,6-difluorobenzonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 March 1992 to 2 October 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(see field "Principles of method if other than guideline" for further information)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

(see field "Principles of method if other than guideline" for further information)

Principles of method if other than guideline:

Protocol Deviations:
- SOP TDS 203, 204, 205 and 207 were used in the experiments
- By mistake the temperature of the waterbath was not constantly measured during the overnight incubation (29 September 1992 to 30 September 1992) of the strains for the second mutation test. The temperature was measured once on 30 September 1992 at the end of the incubation period. At that moment the temperature of the waterbath was 37ºC.
- The test to determine the characteristics of the Salmonella typhimurium strains used in the first mutation study was done on 26 April 1991.

The above mentioned deviations are not considered to have interfered with the integrity of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,6-difluorobenzonitrile

EC Number:

217-589-7

EC Name:

2,6-difluorobenzonitrile

Cas Number:

1897-52-5

Molecular formula:

C7H3F2N

IUPAC Name:

2,6-difluorobenzonitrile

Details on test material:

- Identification: Diflubenil
- Physical state: Yellowish paste

Method

Target gene:

S. typhimurium: Histidine synthesis

Species / strain

Species / strain / cell type:

other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Details on mammalian cell type (if applicable):

- Type and identity of media: To tubes containing 2 ml histidine-deficient agar supplemented with a histidine/biotin mixture to support initial growth, maintained at a temperature of 52 ºC the following were added: 0.1 ml of an overnight growth culture of each of five strains, 0.1 ml of a solution of test material in DMSO and either 0.5 ml of the S9-mix or 0.5 ml cofactors mix. The components were mixed and rapidly poured onto a selective agar plate to gel.
- Properly maintained: yes. After solidification of the agar, the plates were turned upside down and incubated in the dark at 37ºC for 48-72 hours. S9-mix, cofactors mix and the test material solution were checked for sterility.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

625, 1250, 2500 and 5000 µg/plate both in the presence and absence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

the vehicle of the test material (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: (details on positive control substance assigned is presented in Table 1 in the field "Any other information on materials and methods incl. tables")

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 hours

NUMBER OF REPLICATIONS: Test concentrations were performed in triplicate

EVALUATION PROCEDURE: Following the total incubation period the plates were examined for the lack of microbial contamination. Each culture was also examined for the number of spontaneous revertants. Revertant colonies were counted automatically with an Artek 880 colony counter.

Evaluation criteria:

A positive response in the assay system is considered to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the solvent control, together with evidence of a dose-response relationship. This response has to be greater than the laboratory solvent historical solvent control range. The positive response has to be reproducible in an independent experiment.

Results and discussion

Test results

Species / strain:

other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Tables 2 and 3 show the individual plate counts, the standard deviation and the mean number of revertant colonies of the five tester strains exposed to the test material together with the solvent control (DMSO) and positive controls.

In the first mutation study no increase in revertant colony counts was observed for any of the five tester strains, both in the presence and absence of the S9 -mix.

In the second mutation assay a two-fold increase in colony counts was observed with the test material at concentrations of 2500 µg/plate and 5000 µg/plate with strain TA 1535 in the absence of the S9 -mix only. This increase in the number of revertants fell within the laboratory historical solvent control range (10 ±4). All other strains showed no increase of revertant colony counts, compared to the solvent control, neither in the presence or absence of the S9 -mix.

Table 2: Salmonella/Microsome Mutation assay 1

Dose (µg/ plate)		TA 98		TA 100		TA 1535		TA 1537		TA 1538
		+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
0		31	38	94	85	4	9	8	20	18	24
		31	52	83	73	5	13	14	23	20	17
		32	32	91	87	8	12	20	14	NB	19
	Mean	31	41	89	82	6	11	14	19	18; 20	20
	SD	1	10	6	8	2	2	6	5		4
625		24	38	87	92	5	5	15	12	20	22
		20	35	66	100	5	7	6	16	18	31
		16	31	87	97	7	2	10	22	30	20
	Mean	20	35	80	96	6	5	10	17	23	24
	SD	4	4	12	4	1	3	5	5	6	6
1250		31	34	95	92	5	12	11	15	18	20
		24	47	69	100	7	6	13	16	17	24
		17	35	81	92	7	8	14	19	20	31
	Mean	24	39	82	95	6	9	13	17	18	25
	SD	7	7	13	5	1	3	2	2	2	6
2500		16	31	91	83	13	11	11	13	31	18
		22	35	66	100	6	16	16	18	30	24
		22	33	67	86	9	10	8	16	33	15
	Mean	20	33	75	90	9	12	12	16	31	19
	SD	3	2	14	9	4	3	4	3	2	5
5000		23	45	56	69	5	6	8	16	16*	21*
		21	32	34	81	4	2	10	12*	30	22
		22	38	62	45	7	6	13	14*	18*	32
	Mean	22	38	50	65	5	5	10	14*	21	25
	SD	1	7	14	18	2	2	3	2	8	6
Positive Control		2122	1952	217	919	327	35	240	227	2330	1757
		2183	1941	279	890	294	32	211	183	1882	1727
		2334	2364	217	979	290	30	273	231	2647	1824
	Mean	2213	2086	238	929	304	32	241	214	2286	1769
	SD	109	241	36	45	20	3	31	27	384	50

Mean: Average number of revertants per plate

SD: standard deviation

S9: liver homogenate from rats treated with Aroclor

* Toxic

NB: no background lawn (and therefore no colony forming)

Table 3: Salmonella/Microsome Mutation assay 2

Dose (µg/ plate)		TA 98		TA 100		TA 1535		TA 1537		TA 1538
		+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
0		18	30	90	93	6	5	7	6	64	63
		18	31	80	85	7	9	2	4	69	57
		27	41	93	85	5	9	9	9	51	54
	Mean	21	34	88	88	6	8	6	6	62	58
	SD	5	6	7	5	1	2	4	3	9	5
625		27	26	99	85	9	4	4	5	84	87
		26	28	98	76	9	6	6	9	66	51
		27	30	79	66	10	7	5	2	68	50
	Mean	27	28	92	76	9	6	5	5	73	63
	SD	1	2	11	10	1	2	1	4	10	21
1250		27	33	57	93	5	9	6	4	78	55
		24	17	56	78	12	11	6	8	85	41
		31	20	69	85	10	4	6	4	69	39
	Mean	27	23	62	85	9	8	6	5	77	45
	SD	4	9	7	8	4	4	0	2	8	9
2500		29	18	91	77	16	8	5	4	32	27
		18	29	76	99	16	8	7	8	88	7
		29	25	84	88	10	5	9	5	29	7
	Mean	25	24	84	88	14	7	7	6	50	14
	SD	6	6	8	11	3	2	2	2	33	12
5000		20	21	80	73	9	5	3	6	48	0
		29	18	74	78	14	9	3	5	32	0
		14	27	52	49	15	3	4	7	44	0
	Mean	21	22	69	67	13	6	3	6	41	0
	SD	8	5	15	16	3	3	1	1	8	0
Positive Control		681	1831	608	1390	726	112	265	170	1418	103*
		391	617	637	1318	640	104	299	166	1451	554
		1467	1703	581	1214	665	111	298	155	2426	757
	Mean	846	1384	609	1307	677	109	287	164	1765	554
	SD	557	667	28	88	44	4	19	8	573	757

Mean: Average number of revertants per plate

SD: standard deviation

S9: liver homogenate from rats treated with Aroclor

* Toxic

NB: no background lawn (and therefore no colony forming)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

At 2500 and 5000 µg/plate, the test material showed a two-fold increase of the number of revertants in strain TA 1535 in the absence of the S9-mix. Since this increase fell within the laboratory historical range of background revertant counts and because the effect was not reproducible, it was concluded that the test material showed no evidence of mutagenic potential both in the presence and in the absence of the S9-mix in the AMES bacterial system at the dose levels used.

Under the conditions of the study, the test material gave a negative (i.e. non-mutagenic) response in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 both in the presence and absence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and for classification and labelling purposes.

Executive summary:

The potential of the test material to cause gene mutation in bacterial strains was determined in accordance with standard guidelines OECD 471 and EU method B.14. Five strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) were treated in the presence and absence of a rat liver derived metabolic activation system (S9 mix). In two separate assays the test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used in the presence or in the absence of metabolic activation. It was therefore concluded that the test material showed no evidence of mutagenic potential both in the presence and in the absence of metabolic activation at the dose levels used.