2',3-bis[[3-[3,5-di-tert-butyl-4-hydroxyphenyl]propionyl]]propionohydrazide

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:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(adopted 1981)

Deviations:

yes

Remarks:

, strain TA102 or E. coli is lacking (OECD TG 471, adopted 1997)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2',3-bis[[3-[3,5-di-tert-butyl-4-hydroxyphenyl]propionyl]]propionohydrazide

EC Number:

251-156-3

EC Name:

2',3-bis[[3-[3,5-di-tert-butyl-4-hydroxyphenyl]propionyl]]propionohydrazide

Cas Number:

32687-78-8

Molecular formula:

C34H52N2O4

IUPAC Name:

3-(3,5-di-tert-butyl-4-hydroxyphenyl)-N'-[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoyl]propanehydrazide

Method

Target gene:

HIS

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from Aroclor1254-induced rats

Test concentrations with justification for top dose:

25, 75, 225, 675 and 2025 µg per plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details on test system and conditions

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Each Petri dish contained: 1) approx. 20 mL of minimum agar (Agar, Difco Laboratories, Detroit, Michigan, U.S.A., plus salts (Vogel-Bonner Medium E) and glucose), 2) 0.1 mL of the solution of the test substance or the vehicle and 0.1 mL of a bacterial culture (in nutrient broth, Difco Laboratories, Detroit, Michigan, U.S.A., 0.8% plus 0.5% NaCl) in 2.0 mL of soft agar. The soft agar was composed of: 100 mL of 0.6% agar solution with 0.6% NaCl and 10 mL of a solution of l-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and +biotin 0.5 mM (Fluka, Buchs, Switzerland). In the experiments in which the substance was metabolically activated, 0.5 mL of an activation mixture was added also. One mL activation mixture contained: 0.3 mL S9 fraction of liver from rats induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A.) and 0.7 mL of a solution of co-factors.

DURATION
The plates were incubated for about 48 hours at 37°C in darkness.

NUMBER OF REPLICATIONS
3 plates per dose

DETERMINATION OF CYTOTOXICITY
No data

POSITIVE CONTROLS
TA 98: daunorubicin-HCl (5 and 10 µg/plate)
TA 100: 4-nitroquinoline-N-oxide (0.125 and 0.25 µg/plate)
TA 1535: N-Methyl-N'-nitro-N-nitrosoguanidine (3 and 5 µg/plate)
TA 1537: 9(5)aminoacridine hydrochloride monohydrate (50 and 100 µg/plate)
TA 1538: 2-nitrofluorene (5 and 10 µg/plate)
The activation mixture was tested with Strain TA 1535 and cyclophosphamide (250 µg/plate)

Evaluation criteria:

When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 675 and 2025 µg/plate the substance precipitated in soft agar

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments without microsomal activation.

Concentration (µg/plate)	TA98	TA100	TA1535	TA1537	TA1538
0	11	136	8	4	12
25	20	129	10	3	8
75	18	158	10	7	11
225	13	145	10	6	6
675	16	156	7	2	12
2025	17	159	7	6	9
Daunorubicin-HCl	19 (control)
5	134
10	293
4-NQO		156 (control)
0.125		650
0.250		>1000
N-methyl-N´-nitro-N-nitrosoguanidine			8 (control)
3			349
5			1470
9(5) Aminoacridine-HCl				6 (control)
50				85
100				520
2-Nitrofluorene					11 (control)
5					660
10					823

Table 2: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments with microsomal activation.

Concentration (µg/plate)	TA98	TA100	TA1535	TA1537	TA1538
0	26	141	4	3	15
25	24	136	7	4	15
75	21	141	12	5	19
225	26	128	7	5	16
675	24	112	7	3	16
2025	21	130	8	5	17
Cyclophosphamide
0			13
250			214

Applicant's summary and conclusion

Conclusions:

The test substance showed no mutagenic potential in bacterial cells.

Executive summary:

The mutagenic potential of the test material was assessed in an Ames assay with TA98, TA100, TA1535, TA1537 and TA1538 as tester strains. The bacteria were treated with 25, 75, 225, 675, and 2025 µg test substance per plate, with and without microsomal activation. Adequate positive controls and a solvent control were also applied. In the experiments performed with and without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with the test substance revealed no marked differences. The slight increase in the number of back-mutant colonies in the experiment on Strain TA 1535 with microsomal activation is attributed to variations in the rate of spontaneously occurring back-mutants.