5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile

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:

1996-03-06 to 1996-03-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted to the standards of the time: no preincubation study was conducted and the strains selected do not conform to current best practice.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

421-300-1

EC Name:

-

Cas Number:

138564-59-7

Molecular formula:

C12H9N3O2S

IUPAC Name:

5-methyl-2-[(2-nitrophenyl)amino]thiophene-3-carbonitrile

Test material form:

solid: particulate/powder

Details on test material:

97.7%
Item code QA405K
Lilly lot no: 356MO1

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor and 356M01

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the dark at ambient temperature
- Solubility and stability of the test substance in the solvent/vehicle: Soluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: DMSO was used to dissolve and dilute

FORM AS APPLIED IN THE TEST (if different from that of starting material)
33, 100, 333, 1000, 3333, and 10000 ug/plate for the toxicity test.
For the two mutation tests, the concentrations were 3, 10, 33, 100, 333, and 100 ug/plate for the first test, and 2.5, 7.5, 25, 75, 250 and 750 ug/plate for the second test.

Method

Target gene:

his G 46 in TA 1535 and TA 100
his C 3076 in TA 1537
his D 3052 in TA 1538 and TA 98

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Preparation of supernatant fluid from rat liver using S9 mix

Test concentrations with justification for top dose:

33, 100, 333, 1000, 3333, and 10000 ug/plate for the toxicity test. The top dose was the maximum solubility of the test susbtance in the vehicle.
For the two mutation tests, the concentrations were 3, 10, 33, 100, 333, and 100 ug/plate for the first test, and 2.5, 7.5, 25, 75, 250 and 750 ug/plate for the second test.

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: 2-aminoanthracene (2-AAN), sodium azide (NaN3)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Preincubation period: 16 h
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 2 days

SELECTION AGENT (mutation assays):
An Aroclor 1254 induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity

NUMBER OF REPLICATIONS:
3 for each control and test concentration with and without S9 mix

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
colonies were counted using a Biotran III automated counter (New Brunswick Incorporated, NJ, USA) at maximum sensitivity ie colonies of 0.1 mm or more in diameter counted.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

The particular damage induced to DNA by a mutagen may cause reversions to occur which are observable in certain strains of Salmonella typhimurium, but not in others. It is necessary, therefore, to use a variety of bacterial strains in order to test for a broad range of chemical mutagens. At the present time, available data suggest that the use of the 5 strains used in this project permits the detection of a wide spectrum of mutagens.

It is well recognised, however, that many chemicals which may be reactive in a mammalian cell, following metabolic activation, are quite inactive in bacterial cells.
Extracts of mammalian cells are combined, therefore, with the bacterial indicator cells in a tissue mediated assay to increase the relevance of the test in assessing the mutagenicity of chemicals to man.

Evaluation criteria:

A test was considered acceptable if for each strain:
i) the bacteria demonstrated their typical responses to crystal violet,
ampicillin and u. v. light.
ii) on at least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and
TA 1538, 5-35.
iii) on at least 2 of the positive control plates there were x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, x 1.5 the mean vehicle control mutant numbers per plate. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required on at least 2 of the positive control plates.
iv) no toxicity or contamination was observed in at least 4 dose levels.
v) in cases where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number.

Where these criteria were met, a significant mutagenic response was recorded if there was:
i) for S. typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98, at least a doubling of the mean concurrent vehicle control values at some concentration of the test substances and, for S. typhimurium strain TA 100, a 1.5-fold increase over the control value. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
ii) a dose related response, although at high dose levels this relationship could be inverted because of, for example, (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.
iii) a reproducible effect in independent tests.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All tests were acceptable according to the study criteria except the second mutation assay with Salmonella typhimurium TA 1537, the culture used was contaminated; This was repeated.

There was no toxicity to the bacteria. Precipitation of the test material occurred at 1000 µg per plate and above in the presence and absence of S9 mix.

All strains of S. typhimurium were sensitive to crystal violet, whereas only the plasmid-containing strains, TA 98 and TA 100, were resistant to ampicillin. The strains were also tested for sensitivity to u. v. light emitted over a period of 10 s from a CAMAG u.v. lamp set at 254 nm. Increased sensitivity to u.v. light was demonstrated. These results are consistent with the known properties of these bacteria.

Any other information on results incl. tables

Table 1 : toxicity screen

Strain	Dose level µg/plate	Liver S9	mean revertant colony counts	SD	individual revertant colony counts
TA100	solvent	-	135	-	135
	33	-	123	-	123
	100	-	105	-	105
	333	-	122	-	122
	1000	-	114	-	114 P
	3333	-	129	-	129 P
	10000	-	120	-	120 P
	solvent	+	114	-	114
	33	+	128	-	128
	100	+	120	-	120
	333	+	123	-	123
	1000	+	142	-	142 P
	3333	+	134	-	134 P
	10000	+	121	-	121 P

Table2: 1st repeat Plate incorporation with metabolic activation

substance	dose level µg/plate	TA 1535 mean ± SD	TA 1537 mean ± SD	TA 1538 mean ± SD	TA 98 mean ± SD	TA 100 mean ± SD
DMSO	100µl	12 ± 2	12 ± 5	14 ± 5	20 ± 4	104 ± 12
TCN intermediate	3	7 ± 2	18 ± 5	25 ± 1	22 ± 5	113 ± 21
	10	6 ± 3	14 ± 2	20 ± 3	35 ± 3	120 ± 14
	33	4 ± 2	10 ± 2	17 ± 7	31 ± 2	102 ± 10
	100	12 ± 2	10 ± 2	20 ± 4	31 ± 6	106 ± 9
	333 (P)	13 ± 3	16 ± 2	23 ± 4	27 ± 6	102 ± 12
	1000 (P)	9 ± 3	9 ± 4	16 ± 4	27 ± 8	125 ± 6
Positive Controls	Compound	2AAN	2AAN	2AAN	2AAN	2AAN
	Dose level	2µg	2µg	0.5µg	0.5µg	0.5µg
	mean ± SD	120 ± 15	78± 4	204± 12	186± 18	350± 38

Table 3: 2nd repeat plate incorporation with metabolic activation

substance	dose level µg/plate	TA 1535 mean ± SD	TA 1537 mean ± SD	TA 1538 mean ± SD	TA 98 mean ± SD	TA 100 mean ± SD
DMSO	100µl	13 ± 4	14 ± 4	12 ± 4	33 ± 3	147 ± 10
TCN intermediate	2.5	13 ± 2	30 ± 9	14 ± 4	31 ± 9	125 ± 16
	7.5	7 ± 3	22 ± 3	7 ± 3	30 ± 7	133 ± 8
	25	8 ± 4	17 ± 2	15 ± 3	36 ± 2	125 ± 18
	75	10 ± 8	14 ± 1	19 ± 3	36 ± 8	145 ± 6
	250	11 ±2	11 ± 4	17 ± 6	34 ± 5	132 ± 1
	750 (P)	12 ± 4	12 ± 5	12 ± 7	30 ± 2	139 ± 16
Positive Controls	Compound	2AAN	2AAN	2AAN	2AAN	2AAN
	Dose level	2µg	2µg	0.5µg	0.5µg	0.5µg
	mean ± SD	145 ± 15	115± 16	218± 26	197± 20	301± 23

Table 4: 1st repeat plate incorporation without metabolic activation

substance	dose level µg/plate	TA 1535 mean ± SD	TA 1537 mean ± SD	TA 1538 mean ± SD	TA 98 mean ± SD	TA 100 mean ± SD
DMSO	100µl	8 ± 4	8 ± 4	14 ± 5	18 ± 4	98 ± 4
TCN intermediate	3	7 ± 4	9 ±3	12 ± 2	18 ± 9	96 ± 18
	10	7 ± 3	9 ±6	10 ±4	20 ± 7	102 ± 9
	33	8 ± 1	5 ± 3	12 ± 1	20 ± 1	102 ± 9
	100	9 ± 3	10 ± 1	9 ± 2	19 ± 2	94 ± 5
	333 (P)	13 ±2	9 ± 4	12 ± 1	18 ± 2	131 ± 6
	1000 (P)	11 ± 2	6 ± 4	11 ± 5	16 ± 3	118 ± 14
Positive Controls	Compound	NaN3	9AA	2NF	2NF	NaN3
	Dose level	1µg	80µg	1µg	1µg	1µg
	mean ± SD	209 ± 26	816± 80	233± 38	222± 29	541± 60

Table 5 2nd repeat without metabolic activation

substance	dose level µg/plate	TA 1535 mean ± SD	TA 1537 mean ± SD	TA 1538 mean ± SD	TA 98 mean ± SD	TA 100 mean ± SD
DMSO	100µl	12 ± 3	15 ± 2	7 ± 2	24 ± 2	126 ± 4
TCN intermediate	2.5	10 ± 3	19 ± 3	9 ± 3	18 ± 3	132 ± 7
	7.5	11 ± 7	19 ± 4	6 ± 3	24 ± 2	130 ± 13
	25	9 ± 1	19 ± 2	7 ± 4	21 ± 5	140 ± 15
	75	7 ± 5	13 ± 3	8 ± 3	14 ± 6	139 ± 3
	250 (P)	8 ±2	14 ± 2	8 ± 3	27 ± 3	142 ± 9
	750 (P)	6 ± 2	10 ± 2	6 ± 3	24 ± 4	131 ± 10
Positive Controls	Compound	NaN3	9AA	2NF	2NF	NaN3
	Dose level	1µg	80µg	1µg	1µg	1µg
	mean ± SD	181 ± 17	837± 158	229± 9	198± 10	287± 6

Key (P) = precipitate; NaN3 = sodium azide, 9AA = 9 -aminoacridine, 2NF= 2 -nitrofluorene, 2AAN= 2 -aminoanthracene. SD = standard deviation

Applicant's summary and conclusion

Conclusions:

This study found that the test substance does not induce mutagenicity when tested according to OECD guidelines.

Executive summary:

This study was carried out in accordance with OECD guideline 471. The test substance was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at concentrations ranging from 2.5 to 1000ug per plate.

The tests were conducted on agar plates in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity. Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix.

The results obtained in both experiments were similar. No mutagenic activity was observed in any of the 5 bacterial strains, in either activation conditions.

Precipitation of the test material was observed at doses of 250ug per plate and above. There was no toxicity to the bacteria. It was concluded that the test substance was not mutagenic to Salmonella typhimurium when tested in dimethylsulphoxide up to the limit of its solubility.