N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenylpropionamide]

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1983-03-26)

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenylpropionamide]

EC Number:

245-442-7

EC Name:

N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenylpropionamide]

Cas Number:

23128-74-7

Molecular formula:

C40H64N2O4

IUPAC Name:

N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanamide]

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 98: rfa-, uvrB-, R-factor; TA 100: rfa-, uvrB-, R-factor; TA 1535: rfa-, uvrB-; TA 1537: rfa-, uvrB

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Preliminary toxicity test: concentrations ranging from 0.08 to 5000 µg/plate.
First test (with and without microsomal activation) 20, 78, 313, 1250, 5000 µg/plate
Second test (with and without microsomal activation): 500, 1000, 2000, 40000, 8000 µg/plate

Vehicle / solvent:

ethanol

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: for details see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: triplicates per strain and dose each in 2 independent experiments

POSITIVE CONTROLS:
Without S9 mix:
TA 98: daunorubicin-HCl, 5 and 10 µg/0.1 ml phosphate buffer;
TA 100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 µg/0.1 ml phosphate buffer
TA 1535: sodium azide, 2.5 and 5.0 µg/0.1 ml bidistilled water
TA 1537: 9(5)-aminoacridine hydrochloride monohydrate, 50 and 100 µg/0.1 ml dimethylsulfoxide
With S9 mix:
TA 98, TA 100 and TA 1537: 2-aminoanthracene, 5 µg/0.1 ml dimethylsulfoxide
TA 1535: cyclophosphamide, 250 µg/0.1 ml bidistilled water

Evaluation criteria:

A substance is to be considered as mutagenic when one or both of the following criteria are fulfilled:
1.) doubling of spontaneous mutation rate for strains TA 98, TA 1535 and TA 1537
2.) a mutation rate of at least 1.5 for strain TA 100
a dose response-relationship should be demonstrable

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

not applicable

Positive controls validity:

valid

Test results 3

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:

not applicable

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:

not applicable

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At concentrations of 313 µg/plate and above substance precipitates in soft agar

RANGE-FINDING/SCREENING STUDIES:
- 5000 µg/plate was selected at highest dose from cytotoxicity experiments. Up to 5000 µg/plate no cyctotoxic effect was seen in pretest

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of plate incorporation test (test 1) with and without metabolic activation

	Plate Incorporation test 1
Substance	With/ or without S9-mix	Test substance concentration [µg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
			Salmonella typhymurium
			Base-pair substitution type		Frameshift type
			TA 1535	TA 100	TA 1537	TA 98
vehicle only	-	0	16	116	4	31
test substance	-	20	13	99	6	33
test substance	-	78	13	103	4	28
test substance	-	313	11	107	5	29
test substance	-	1250	18	99	5	26
test substance	-	5000	11	85	6	19
Sodium Azide	-	2.5	548
Sodium Azide		5	788
Daunorubicin	-	5				536
Daunorubicin		10				189
4-Nitroquinoline- N-oxid	-	0.125		593
4-Nitroquinoline- N-oxid		0.25		1000
9-Aminoacridine- hydrochloride	-	50			110
9-Aminoacridine- hydrochloride	-	100			977
vehicle only	+	0	25	110	14	42
test substance	+	20	16	115	15	57
test substance	+	78	34	120	15	60
test substance	+	313	25	106	12	44
test substance	+	1250	28	127	12	63
test substance	+	5000	20	119	15	47
Cylophosphamide	+	250	597
2-Aminoanthracene				606	161	1250

Table 2: Results of plate- incorporation test with and without metabolic activation

	Plate Incorporation test 2
Substance	With/ or without S9-mix	Test substance concentration [µg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
			Salmonella typhymurium
			Base-pair substitution type		Frameshift type
			TA 1535	TA 100	TA 1537	TA 98
vehicle	-	0	18	135	9	27
test substance	-	500	14	109	7	29
test substance	-	1000	13	129	5	26
test substance	-	2000	12	132	6	26
test substance	-	4000	22	118	7	24
test substance	-	8000	16	105	9	22
Sodium Azide	-	2.5	461
Sodium Azide	-	5	699
4-Nitroquinoline- N-oxide	-	0.125		683
4-Nitroquinoline- N-oxide	-	0.25		1184
9-Aminoacridine- hydrochloride	-	50			1553
9-Aminoacridine- hydrochloride	-	100			2608
Daunorubicin- HCl	-	5				160
vehicle	+	0	20	145	19	45
test substance	+	500	16	127	17	42
test substance	+	1000	20	113	20	40
test substance	+	2000	18	123	19	34
test substance	+	4000	19	117	20	41
test substance	+	8000	25	96	20	54
2-Aminoanthracene	+	5		586	165	1171
Cyclophosphamid		250	983

Applicant's summary and conclusion

Executive summary:

A test of bacterial gene mutagenicity for the substance following the OECD guideline 471(1983) was conducted. Following strains were tested: TA 98, TA 100, TA 1535 and TA 1537. Test concentrations ranged from 20 to 8000 μg/ plate. Two experiments were conducted both in absence and presence of S9-mix. For the first experiment, the test concentration ranged from 20 to 5000 µg/plate. For the second experiment, the test concentration was in the range of 500 to 8000 µg/plate. No increase in revertant colonies was noticed, thus the test substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used with or without metabolic activation. Consequently the substance is considered to be nonmutagenic in this bacterial reverse mutation assay.