Propanamide, N-[4-(2,4-dihydroxyphenyl)-2-thiazolyl]-2-methyl-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide

EC Number:

807-621-3

Cas Number:

1428450-95-6

Molecular formula:

C13H14N2O3S

IUPAC Name:

N-[4-(2,4-dihydroxyphenyl)-1,3-thiazol-2-yl]-2-methylpropanamide

Test material form:

solid: particulate/powder

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9) prepared from the livers of Aroclor 1254-induced rats

Test concentrations with justification for top dose:

Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate with and without metabolic activation
Experiment 2: for strains TA98 and TA1535: 20.48, 51.2, 128, 320, 800 and 2000 µg/plate with and without metabolic activation; for strains TA100, TA102 and TA1537: 8.192, 20.48, 51.2, 128, 320 and 800 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that the test substance was soluble in DMSO at concentrations of at least 100 mg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation)
Experiment 2: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: two separate experiments using triplicate plates with and without metabolic activation

DETERMINATION OF CYTOTOXICITY
- Method: clearing of background lawn

Evaluation criteria:

The test substance was considered positive in this assay if all of the following criteria were met:
- When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p ≤ 0.01) which was concentration related.
- The positive trend/effects described above were reproducible.

Statistics:

Dunnett's test was used to compare the counts at each concentration with the control.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was observed in both experiments at 2000 µg/plate and above.

COMPARISON WITH HISTORICAL CONTROL DATA: All negative controls were in the range of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects of the test substance were observed in all tester strains, starting for TA102 and TA1537 at 320 µg/plate with metabolic activation and 500 µg/plate without metabolic activation, for TA100 at 500 µg/plate with and without metabolic activation and for TA98 and TA1535 at 800 µg/plate with metabolic activation and 1581 µg/plate without metabolic activation. The cytotoxic effects ranged from slight diminution of the back ground lawn to complete extermination of the test bacteria at higher concentrations.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation).

Bacterial Reverse Mutation Assay, mean revertants colonies/plate ± standard deviation
EXPERIMENT 1 (plate incorporation)
S9-Mix	Without
Test item (µg/plate)	T98	TA100	TA1535	TA1537	TA102
DMSO	23.8 ± 6.1	94.8 ± 3.6	14.0 ± 4.1	6.0 ± 2.5	275.4 ± 33.5
5	24.0 ± 7.0	103.7 ± 7.6	13.3 ± 3.1	6.7 ± 2.5	276.7 ± 11.7
15.81	20.3 ± 4.5	102.3 ± 4.0	16.3 ± 2.5	6.3 ± 2.5	280.3 ± 34.3
50	19.7 ± 1.2	80.0 ± 6.0	22.0 ± 5.0 *	7.3 ± 1.5	275.3 ± 10.3
158.1	19.3 ± 3.8	84.7 ± 8.5	16.0 ± 4.6	7.3 ± 2.9	270.7 ± 11.0
500	16.3 ± 4.5	80.3 ± 5.5 S	15.7 ± 4.5	4.7 ± 0.6 S	217.7 ± 35.2 S
1581	5.3 ± 3.5 S	T	5.0 ± 1.0 V	T	T
5000	P, T	P, T	P, T	P, T	P, T
2NF	905.0 ± 37.0	---	---	---	---
NaN3	---	614.3 ± 58.6	549.7 ± 82.0	---	---
AAC	---	---	---	158.7 ± 31.2	---
MMC	---	---	---	---	850.3 ± 133.3
S9-Mix	With
Test item (µg/plate)	TA98	TA100	TA1535	TA1537	TA102
DMSO	30.4 ± 7.6	98.6 ± 9.0	18.0 ± 5.0	13.2 ± 5.0	251.6 ± 10.1
5	34.7 ± 5.1	110.3 ± 10.8	19.3 ± 4.9	18.7 ± 4.7	237.7 ± 14.4
15.81	43.0 ± 11.3	113.7 ± 5.5	16.7 ± 3.1	17.0 ± 6.1	254.0 ± 14.7
50	36.7 ± 2.5	106.7 ± 11.7	21.0 ± 9.5	16.0 ± 1.0	247.0 ± 13.1
158.1	39.0 ± 9.2	93.7 ± 12.4	19.0 ± 2.0	13.0 ± 2.6	228.7 ± 21.1
500	26.0 ± 5.3	73.7 ± 12.7 S	11.7 ± 2.9	8.3 ± 4.0 S	181.3 ± 23.2 S
1581	15.0 ± 2.6 S	T	5.3 ± 4.0 V	T	T
5000	P, T	P, T	P, T	P, T	P, T
BaP	297.7 ± 110.2	---	---	---	---
AAN	---	1081.7 ± 192.8	265.7 ± 3.8	198.3 ± 24.4	1387.3 ± 79.5
P: precipitation of the test substance observed S: slight thinning of background bacterial lawn V: very thin background bacterial lawn T: toxic, no revertant colonies   Positive controls: 2NF = 2-nitrofluorene NaN3 = sodium azide AAC = 9-aminoacridine MMC = Mitomycin C BaP = benzo[a]pyrene AAN = 2-aminoanthracene
* p<0.05

Table 2. Test results of experiment 2 (pre-incubation).

Bacterial Reverse Mutation Assay, mean revertants colonies/plate ± standard deviation
EXPERIMENT 2 (pre-incubation)
S9-Mix	Without
Test item (µg/plate)	T98	TA100	TA1535	TA1537	TA102
DMSO	22.2 ± 5.2	94.8 ± 14.6	17.6 ± 2.9	11.2 ± 4.2	233.4 ± 12.6
8.192	---	94.7 ± 14.0	---	10.0 ± 4.0	243.3 ± 8.1
20.48	24.3 ± 8.1	70.7 ± 8.7	22.7 ± 6.8	9.7 ± 3.5	236.7 ± 4.6
51.2	16.7 ± 4.9	87.7 ± 5.8	14.7 ± 5.8	9.0 ± 2.6	221.3 ± 0.6
128	20.3 ± 2.3	96.3 ± 17.2	16.0 ± 2.6	8.0 ± 3.6	201.0 ± 22.1
320	20.3 ± 3.2	87.0 ± 9.8	11.7 ± 3.8	10.3 ± 4.5	180.7 ± 15.0
800	10.3 ± 2.5	47.3 ± 6.7	11.3 ± 4.7	4.7 ± 1.5	102.7 ± 5.7 V
2000	5.3 ± 2.1 P, V	---	2.3 ± 0.6 P, V	---	---
2NF	735.7 ± 87.9	---	---	---	---
NaN3	---	671.7 ± 94.0	686.7 ± 24.1	---	---
AAC	---	---	---	100.3 ± 34.8	---
MMC	---	---	---	---	715.0 ± 99.3
S9-Mix	With
Test item (µg/plate)	TA98	TA100	TA1535	TA1537	TA102
DMSO	33.8 ± 6.1	120.0 ± 9.9	20.6 ± 4.5	17.0 ± 4.0	225.2 ± 23.6
8.192	---	114.0 ± 18.5	---	19.3 ± 1.5	261.3 ± 11.6 *
20.48	39.3 ± 4.7	112.7 ± 7.5	15.7 ± 0.6	18.7 ± 2.5	249.0 ± 15.6
51.2	39.0 ± 7.5	126.7 ± 14.0	17.7 ± 5.5	16.7 ± 1.2	259.0 ± 15.6 *
128	32.3 ± 6.7	125.7 ± 7.4	16.3 ± 3.5	21.0 ± 3.0	232.7 ± 13.7
320	28.3 ± 5.9	119.0 ± 5.6	12.7 ± 2.5	19.3 ± 0.6 S	229.7 ± 2.3 S
800	15.0 ± 3.5 S	T	2.3 ± 0.6 V	T	T
2000	P, T	---	P, T	---	---
BaP	339.0 ± 28.2	---	---	---	---
AAN	---	1414.7 ± 33.5	257.7 ± 10.4	260.0 ± 13.9	942.3 ± 199.4
P: precipitation of the test substance observed S: slight thinning of background bacterial lawn V: very thin background bacterial lawn T: toxic, no revertant colonies   Positive controls: 2NF = 2-nitrofluorene NaN3 = sodium azide AAC = 9-aminoacridine MMC = Mitomycin C BaP = benzo[a]pyrene AAN = 2-aminoanthracene
* p<0.05

Applicant's summary and conclusion

Conclusions:

It was concluded that W630 (Sample ID: 26480) did not induce mutation in five
histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of
Salmonella typhimurium when tested under the conditions of this study. These
conditions included treatments at concentrations up to 5000 μg/plate (the maximum
recommended concentration according to current regulatory guidelines) or toxic
concentrations, in the absence and in the presence of a rat liver metabolic activation
system (S-9).

Executive summary:

W630 (Sample ID: 26480) was assayed for mutation in five histidine-requiring strains

(TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in

the absence and in the presence of metabolic activation by an Aroclor 1254-induced

rat liver post-mitochondrial fraction (S-9), in two separate experiments.

All W630 (Sample ID: 26480) treatments in this study were performed using

formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO).

Experiment 1 treatments of all the tester strains were performed in the absence and in

the presence of S-9, using final concentrations of W630 (Sample ID: 26480) at 5,

15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, plus negative (vehicle) and positive

controls. Following these treatments, evidence of toxicity was observed at 500 and/or

1581 μg/plate and above in all strains in the absence and presence of S-9.

Experiment 2 treatments of all the tester strains were performed in the absence and in

the presence of S-9. For strains TA98 and TA1535, the maximum test concentration

was reduced to 2000 μg/plate and for strains TA100, TA1537 and TA102 the

maximum test concentration was reduced to 800 μg/plate, based on toxicity observed

in Experiment 1. Narrowed concentration intervals were employed covering the

ranges 20.48 – 2000 μg/plate or 8.192 – 800 μg/plate respectively, in order to

examine more closely those concentrations of W630 (Sample ID: 26480) approaching

the maximum test concentration. In addition, all treatments in the presence of S-9

were further modified by the inclusion of a pre-incubation step. Following these

treatments, evidence of toxicity was observed at 320 μg/plate and above in strain

TA1537 and TA102 in the presence of S-9; 800 μg/plate and above in strain TA98 in

the absence and presence of S-9 and strain TA1535 in the presence of S-9; and 800

and/or 2000 μg/plate in strain TA100 in the absence and presence of S-9, and strains

TA1535, TA1537 and TA102 in the absence of S-9.