2-(2,6-diethyl-4-methyl-phenyl)propanediamide

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:

11 August 2003 to 21 August 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guidelines S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guidelines S2B: Genotoxicity. A Standard Battery for Genotoxicity Testing of Pharmaceuticals.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

452-330-3

EC Name:

-

Cas Number:

314020-40-1

Molecular formula:

C14H20N2O2

IUPAC Name:

2-(2,6-diethyl-4-methyl-phenyl)propanediamide

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: Powder, yellowish
- Storage condition of test material: In the dark at ambient temperature

Method

Target gene:

S. typhimurium: Histidine synthesis

E. coli: Tryptophan synthesis

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Top agar consisting of 0.6 % w/v agar and 0.5 % w/v sodium chloride in deionised water. Prior to testing, the molten top agar was prepared by adding sterile 0.5 mM histidine/0.5 mM biotin stock solution (10 mL solution: 100 mL) agar).
0.5 mL S9 mix (or S9 buffer) was then added to the number of aliquots of one strain required for one concentration, followed by 0.1 mL of the appropriate concentration of the test substance preparation. Finally 2.0 mL top agar was then added to each aliquot, and the resulting mixture poured onto the surface of a prepared pre-labelled Vogel Bonner plate (9 cm diameter vented Petri-dish prepared with 25 mL Vogel Bonner minimal medium and containing 1.5 % w/v agar and 2 % w/v glucose) and allowed to gel.
- Periodically checked for genotype stability: Yes. The overnight culture from each new frozen culture was screened for the deep-rough characters, DNA repair deficiency and Ampicillin resistance. The presence of the uvrB deletion was confirmed by testing the sensitivity of each culture to mitomycin C (10 µL of a 10 µg/mL solution) in the same manner as sensitivity to crystal violet was tested.
- Periodically "cleansed" against high spontaneous background: Yes. When fresh frozen stocks were prepared, the strains were tested for amino acid requirement.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli, other: E. coli WP2P uvr A and E. coli WP2P

Details on mammalian cell type (if applicable):

- Type and identity of media: Top agar consisting of 0.6 % w/v agar and 0.5 % w/v sodium chloride in deionised water. Prior to testing, the molten top agar was prepared by adding sterile tryptophan solution (10 mL 0.5 mM stock: 100 mL agar).
0.5 mL S9 mix (or S9 buffer) was then added to the number of aliquots of one strain required for one concentration, followed by 0.1 mL of the appropriate concentration of the test substance preparation. Finally 2.0 mL top agar was then added to each aliquot, and the resulting mixture poured onto the surface of a prepared pre-labelled Vogel Bonner plate (9 cm diameter vented Petri-dish prepared with 25 mL Vogel Bonner minimal medium and containing 1.5 % w/v agar and 2 % w/v glucose) and allowed to gel.
- Periodically checked for genotype stability: Yes. The overnight culture from each new frozen culture was screened for the deep-rough characters, DNA repair deficiency and Ampicillin resistance. The presence of the uvrA mutation was confirmed by testing the sensitivity of each culture to mitomycin C (10 µL of a 10 µg/mL solution) in the same manner as sensitivity to crystal violet was tested.
- Periodically "cleansed" against high spontaneous background: Yes. When fresh frozen stocks were prepared, the strains were tested for amino acid requirement.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

5000, 2500, 1000, 500, 200 and 100 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:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Details on positive control substance assignment 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: Plates were incubated inverted at 37 °C for three days in the dark.

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 and evidence that the test was valid: i.e. there was a background lawn on the solvent control plates and on the plates for (at least) the lower concentrations of test substance, and that the positive controls had responded as expected. All plates were counted using an automated colony counter (Cardinal® automated counter linked to the Ames Study Manager system [Perceptive Instruments]) adjusted appropriately to permit the optimal counting of mutant colonies.

Evaluation criteria:

Test data from individual experiments are considered valid if:
a) the concurrent solvent control data are acceptable
b) the positive control data show acceptable increases

Failure of one or more tester strain/S9 combinations does not invalidate the data for the remainder of a concurrent experiment.

A positive response in a (valid) individual experiment is acheived when one or both of the following are met:
a) a significant, dose related increase in the mean number of revertants is observed; or
b) a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) is observed at one or more concentrations.

A negative result in a (valid) individual experiment is achieved when:
a) there is no significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and
b) in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.

For a positive response in an individual experiment to be considered indicative of an unequivocal positive, i.e. mutagenic, result for that strain/S9 combination, then the observed effect(s) must be consistently reproducible.

Statistics:

All derived calculations were carried out by the computerised data analysis system.

Results and discussion

Test results

Test results 1

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98 and TA100 and E. coli WP2P uvrA and WP2P

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

other: S. typhimurium TA1537 and TA98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

other: S. typhimurium TA1535 and TA100 and E. coli WP2P uvrA and WP2P

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The results of the study were found to be comparable with the historical control.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Results with test material

Strain	Compound	Dose µg/plate	Mean revertants/plate		Treated/solvent		Individual revertant colony counts		Mean revertants/plate		Treated/solvent		Individual revertant colony counts
			-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
			Experiment 1						Experiment 2
TA 100	Test material	5000	95	194.7	1.1	1.5	97,97,91	219,223,142	94	259.7	1.0	1.3	88,93,101	276,278,225
		2500	91.3	165	1.0	1.3	84,91,99	163,164,168	85.7	245	0.9	1.2	83,84,90	237,273,225
		1000	78.7	138.3	0.9	1.0	77,51,108	152,135,128	95.3	214.3	1.0	1.0	72,106,108	219,224,200
		500	82	124.7	0.9	0.9	86,77,83	138,112,124	101.3	219.7	1.1	1.1	101,114,89	201,257,201
		200	103.7	125	1.2	0.9	101,110,100	110,125,140	96.3	212.3	1.0	1.0	100,101,88	226,219,192
		100	101	112	1.2	0.8	95,117,91	130,106,100	94.3	220	1.0	1.1	96,105,82	212,235,213
	DMSO		87.4	131.8			91,84,90,83,89	136,133,151,133,106	95.8	207.6			97,99,94,101,88	184,239,184,229,202
TA 1535	Test material	5000	4.0	8.7	0.8	1.5	4,4,M	10,6,10	3.3	8.7	0.6	1.0	4,2,4	9,11,6
		2500	7.0	11.3	1.3	2.0	7,4,10	10,12,12	6.0	8.7	1.0	1.0	7,4,7	9,12,5
		1000	4.0	9.7	0.8	1.7	6,1,5	4,23,2	5.7	7.0	1.0	0.8	7,6,4	4,11,6
		500	4.7	7.0	0.9	1.2	1,10,3	9,5,7	5.3	5.0	0.9	0.6	2,7,7	6,5,4
		200	7.0	10.7	1.3	1.8	10,7,4	15,11,6	6.0	8.3	1.0	1.0	10,7,1	10,9,6
		100	5.7	13.7	1.1	2.4	5,6,6	9,5,27	4.3	9.3	0.7	1.1	5,4,4	7,10,11
	DMSO		5.2	5.8			2,5,7,1,11	4,5,5,6,9	5.8	8.4			4,4,4,6,11	6,9,9,9,9
TA 1537	Test material	5000	8.3	27	0.8	3.5	6,12,7	21,32,28	4.0	31.3	0.4	4.2	2,6,4	27,29,38
		2500	3.3	19.7	0.3	2.5	4,4,2	18,26,15	6.3	17.7	0.6	2.4	5,9,5	9,17,27
		1000	8.0	11.7	0.7	1.5	6,6,12	18,13,4	8.7	9.7	0.9	1.3	9,11,6	13,4,12
		500	6.0	15	0.5	1.9	7,6,5	11,15,19	6.3	8.0	0.6	1.1	6,6,7	7,6,11
		200	8.7	9.3	0.8	1.2	13,6,7	5,6,17	8.7	6.7	0.9	0.9	10,1,15	11, 5, 4
		100	10.3	11.3	0.9	1.5	9,12,10	15,10,9	8	7.0	0.8	0.9	10,9,5	10,5,6
	DMSO		11	7.8			18,5,11,10,11	7,7,9,9,7	10	7.4			7,11,11,6,15	7,7,7,6,10
TA 98	Test material	5000	13.3	618.7	0.9	24.6	11,16,13	715,609,532	24.3	635.3	1.9	27.6	17,21,35	461,629,819
		2500	17.7	252.7	1.2	10	19,18,16	267,252,239	14.7	206	1.1	9.0	17,11,16	223,134,261
		1000	11	75	0.8	3.0	10,11,12	84,55,86	11.3	64.3	0.9	2.8	12,13,9	60,65,68
		500	10.7	51.7	0.7	2.1	12,9,11	44,60,51	18.7	48.3	1.4	2.1	12,27,17	49,47,49
		200	19.7	31.7	1.3	1.3	23,17,19	38,29,28	14.3	31.3	1.1	1.4	11,16,16	35,21,38
		100	15	25.3	1.0	1.0	16,13,16	24,26,26	17.7	28.7	1.4	1.2	26,9,18	39,28,19
	DMSO		14.6	25.2			16,17,10,19,11	23,17,23,28,35	13	23			10,13,13,12,17	28,27,17,15,28
WP2 (pKM101)	Test material	5000	53.7	55.7	0.8	0.6	39,55,67	49,56,62	39.7	56	1.0	0.9	47,46,26	56,57,55
		2500	63.3	78.7	0.9	0.9	66,73,51	66,95,75	37.7	57.3	0.9	0.9	30,49,34	39,86,47
		1000	64.3	75	0.9	0.8	72,47,74	65,82,78	46	71.3	1.1	1.1	43,50,45	73,79,62
		500	80.7	106	1.2	1.2	83,97,62	105,116,97	37	65.3	0.9	1.0	39,29,43	65,57,74
		200	104	92.3	1.5	1.0	101,116,95	89,105,83	39.7	64.3	1.0	1.0	41,35,43	73,62,58
		100	118.3	94.3	1.7	1.0	83,154,118	99,83,101	35.7	57	0.9	0.9	39,45,23	58,47,66
	DMSO		69.6	90			51,66,82,69,80	61,75,114,106,94	40.6	63.6			39,44,41,32,47	58,56,82,61,61
WP2 uvrA (pKM101)	Test material	5000	158.3	160.3	1.0	0.6	141,149,185	118,158,205	154.7	258	1.1	1.4	129,160,175	235,263,276
		2500	186	294	1.2	1.1	169,188,201	274,296,312	139.7	199.3	1.0	1.1	130,145,144	202,213,183
		1000	198.7	227	1.3	0.9	185,214,197	206,223,252	147.7	202.7	1.1	1.1	139,166,138	186,225,197
		500	217	270	1.4	1.0	256,218,177	242,283,285	137.7	174.3	1.0	0.9	140,117,156	166,183,174
		200	232	269	1.5	1.0	233,246,217	265,284,258	130	177	0.9	1.0	101,119,170	144,206,181
		100	238.3	248.3	1.5	0.9	272,237,206	261,216,268	138.7	185.7	1.0	1.0	140,146,130	198,173,186
	DMSO		156.8	263.4			173,149,147,135,180	259,219,304,267,268	137.8	184.2			128,125,151,161,124	168,157,190,209,197
M = Plate missing

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation All strains tested
negative with metabolic activation All strains tested except S. typhimurium TA1537 and TA98
positive with metabolic activation S. typhimurium TA1537 and TA98 strains

Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), response in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2P and WP2P uvrA in the absence of metabolic activation and in all strains, except for TA1537 and TA98, in the presence of metabolic activation. The test material gave a positive (i.e. mutagenic) response in S. typhimurium strains TA1537 and TA98 in the presence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.

Executive summary:

The potential of the test material to cause gene mutation in bacterial strains was determined in accordance with standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.1500. Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two Escherichia coli strains (WP2P and WP2PuvrA) were treated in the presence and absence of at 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 absence of metabolic activation, nor in strains TA1535, TA100, WP2P and WP2PuvrA in the presence of metabolic activation. In both assays, the test material induced reproducible, dose related increases in revertant colony numbers in strains TA1537 and TA98 in the presence of metabolic activation. The test material gave a negative (i.e. non-mutagenic), response in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2P and WP2P uvrA in the absence of metabolic activation and in all strains, except for TA1537 and TA98, in the presence of metabolic activation. The test material gave a positive (i.e. mutagenic) response in S. typhimurium strains TA1537 and TA98 in the presence of metabolic activation.