1-(3,5-Dihydroxyphenyl)-1-oxo-2-(N-1-(4-methoxyphenyl)isopropylamino)ethane Hydrobromide

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:

1999-01-12 to 1999-02-25-1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

The study was performed only with strain TA98 and strain TA100

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

yes

Remarks:

The study was performed only with strain TA98 and strain TA100

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-(3,5-dihydroxyphenyl)-2-{[1-(4-methoxyphenyl)propan-2-yl]amino}ethan-1-one hydrobromide

EC Number:

928-779-0

Cas Number:

1178555-23-1

Molecular formula:

C18-H21-N-O4 x HBr

IUPAC Name:

1-(3,5-dihydroxyphenyl)-2-{[1-(4-methoxyphenyl)propan-2-yl]amino}ethan-1-one hydrobromide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- - Source and lot/batch No.of test material:
No details on the source of the test material were provided. Batch number: 323.
- Expiration date of the lot/batch:
16 December 1999 (allocated by testing facility, 1 year after reciept of the test substance).
- Purity test date: No details reported.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark.
- Stability under test conditions: Stable The stability of the test substance in the vehicle was not indicated.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No details reported.
- Preliminary purification step (if any):No details reported.
- Final dilution of a dissolved solid, stock liquid or gel:No details reported.
- Final preparation of a solid: No details reported.

Method

Target gene:

Strain TA98 characterises the histidine mutation 'hisD3053/R-factor8' for frameshift mutations (* R-factor = plasmid pKM101 to increase error prone DNA repair).
Strain TA100 characterises the histidine mutation 'hisG46/R-factor' for base pair substitutions.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate (first experiment; TA98 and TA100 with and without metabolic activation)
500, 1000, 2000, 3000, 4000 and 5000 µg/plate (second experiment; TA100 with and without metabolic activation, TA98 without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (test substance, positive control substance methylmethanesulfonate, 2-aminoanthracene); Saline (positive control substance daunomycine)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 2-aminoanthracene and daunomycine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 each per concentration level and control

DETERMINATION OF CYTOTOXICITY
- Method: Bacterial background lawn, microcolonies

Evaluation criteria:

The assessment and interpretation of the results follows the OECD Guideline No. 471, i. e. a concentration-dependent increase in the number of revertants of at least one tester strain over the vehicle control value and/or outside the historical control range is indicative of genotoxic activity.

A test substance is considered negative (not mutagenic) in the test if: The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
A test substance is considered positive (mutagenic) in the test if: It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.

Statistics:

no formal hypothesis testing done

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item did not precipitate in the top agar. Precipitation of the test item was not observed on the plates at the start or at the end of the incubation period in both tester strains.
COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Any other information on results incl. tables

The negative and strain-specific positive control values were within the testing laboratory's background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Strain TA98

In the absence of S9 -mix, the test item showed negative responses over the entire dose range, i. e. no dose related, two-fold, increase in the number of revertants. In the presence of S9- mix, the test item induced up to 2.4- and 2.2 fold, dose related, increases in the number of revertant colonies compared to the solvent control in the first and second experiment respectively.

Strain TA100

In the absence of S9 -mix, the test item induced up to 3.9- and 5.5 -fold, dose related, increases in the number of revertant colonies compared to the solvent control in the first and second experiment respectively. In the presence of S9 -mix, the test item induced up to 1.9- and 2.2 -fold, dose related, increases in the number of revertant colonies compared to the solvent control in the first and second experiment respectively.

Table 1: Mutagenic response of the test item in the Salmonella typhimurium reverse mutation assay. Experiment 1

Dose (µg/plate)	Mean number of revertant (HIS+) colonies/3 replicate plates (+- S.D.) with two strains of Salmonella typhimurium
	TA98	TA100
	Without S9 -mix
positive control	589 +/- 29	1089 +/- 99
solvent control	17 +/- 5	66 +/- 7
3	12 +/- 3	77 +/- 17
10	13 +/- 4	68 +/- 4
33	12 +/- 2	67 +/- 17
100	13 +/- 2	77 +/- 15
333	15 +/- 3	90 +/- 3
1000	11 +/- 3 *	121 +/- 12
3330	MC**	259 +/- 63*
5000	0 +/- 0***	MC**

Dose (µg/plate)	Mean number of revertant (HIS+) colonies/3 replicate plates (+- S.D.) with two strains of Salmonella typhimurium
	TA98	TA100
	With S9 -mix
positive control	1132 +/- 191	1175 +/- 122
solvent control	19 +/- 1	78 +/- 10
3	17 +/- 4	101 +/- 2
10	19 +/- 3	74 +/- 9
33	17 +/- 5	82 +/- 8
100	17 +/- 3	87 +/- 6
333	21 +/- 5	86 +/- 13
1000	31 +/- 1	93 +/- 11
3330	37 +/- 6	148 +/- 12
5000	45 +- 6	75 +- 16

Solvent control; 0.1 ml dimethylsulphoxide

* Bacterial background lawn slightly reduced

** Bacterial background lawn extremely reduced

*** Bacterial background lawn absent

MC: Microcolonies

Table 2: Mutagenic response of the test item in the Salmonella typhimurium reverse mutation assay. Experiment 2

Dose (µg/plate)	Mean number of revertant (HIS+) colonies/3 replicate plates (+- S.D.) with two strains of Salmonella typhimurium
	TA98	TA100
	Without S9 -mix
positive control	-	1224 +/- 106
solvent control	-	62 +/- 3
500	-	104 +/- 7
1000	-	116 +/- 13
2000	-	230 +/- 45
3000	-	338 +/- 16
4000	-	34 +/- 24*

Dose (µg/plate)	Mean number of revertant (HIS+) colonies/3 replicate plates (+- S.D.) with two strains of Salmonella typhimurium
	TA98	TA100
	With S9 -mix
positive control	1315 +/- 72	1200 +/- 153
solvent control	22 +/- 3	69 +/- 6
500	33 +/- 1	99 +/- 8
1000	37 +/- 3	89 +/- 10
2000	47 +/- 8	116 +/- 13
3000	43 +/- 5	144 +/- 7
4000	48 +/- 2	153 +/- 9
5000	42 +/- 3	-

* Bacterial background lawn moderately reduced

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the test substance is mutagenic in the Salmonella typhimurium reverse mutation assay (Strains TA98 and TA100).