1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one 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:

31 July 2003 to 18 September 2003

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:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide

EC Number:

222-148-7

EC Name:

1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide

Cas Number:

3371-33-3

Molecular formula:

C17H19NO4.BrH

IUPAC Name:

1-(3,5-dihydroxyphenyl)-2-{[1-(4-hydroxyphenyl)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: Source of the test substance was not reported. Batch No: 0003A/RCC 0122A.
- Expiration date of the lot/batch: October 2004
- Purity test date: 10 October 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark (ambient humidity).
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Solutions were freshly prepared for each experiment. The stability of the test substance can be assumed.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Solutions were freshly prepared for each experiment.
- 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 TA1537 (his C3076)(no R factor) detects frameshift mutations
Strain TA98 (his D3052) R factor pKM101 detects frameshift mutations
Strain TA100 (hisG46) R factor pK,101 detects basepair substitutions
Strain TA 1535 (his G46) (no R factor) detects basepair subsitutions
Strain TA 102 (hist G428) R factor pKM101, pAQ1 detects basepair substitutions.

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

100, 300,1000, 3000, 5000 µg/plate with and without metabolic activation (experiment 1)
500, 1000, 1500 µg/plate with and without metabolic activation (experiment 2, only with strain TA 1537)
All concentrations are given as free base equivalent.

Vehicle / solvent:

- Vehicle(s) used: DMSO and dist. water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

in experiment 2, two independent experiments were conducted, one with DMSO as solvent and as solvent control and one with distilled water as solvent and as solvent control.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene

Remarks:

without S9: 2-nitrofluorene, sodium azide, mitomycin C, 9-aminoacridine; with S9: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

EXPOSURE DURATION: 48 hrs

NUMBER OF REPLICATIONS: 3 each per concentration level and positive control. 6 per vehicle control


DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn, microcolonies

Evaluation criteria:

A reproducible, 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. Since the results were unequivocal, no detailed statistical analysis was performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 98 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA 98 without metabolic activation, TA 102 with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

No precipitation was observed up to the top concentration tested. There was, however, a concentration-dependent and strain-specific bacteriotoxicity, as seen by a reduced background lawn and/or a decrease of absolute revertants.

 

Mutagenicity

The test item did not increase consistently the number of revertant colonies in the tester strains S. typhimurium TA 1535, TA 98 and TA 102 in the concentration range from 100 to 5000 µg/plate (free base) compared to the negative control in the standard plate test. There was no substantial difference in the mutation frequency in the presence and absence of metabolic activation.

There was a slight mutagenic response (up to 1.6-fold) observed in S. typhimurium TA 100 (nonactivation) at 1000 µg/plate. A marked increase of revertants (up to 22.7-fold) was seen in TA 1537 (with metabolic activation) at 500 µg/plate and above, using the standard plate test, although no dose response was observed. The effect was more pronounced in the absence of metabolic activation. This positive result in TA 1537 was confirmed in a repeat assay.

All revertant counts were within the historical background data. The positive controls caused the expected mutagenic response showing the sensitivity and validity of the assay.

Table 1: Mutagenic activity of the test item. Mean values Experiment 1 (Plate incorporation). Without metabolic activation

Compound (µg/plate)	Mean Revertants/Plate
	S. typhimurium
	TA 1535	TA 1537	TA 98	TA 100	TA 102
DMSO	10	11	32	77	301
100	10	90	26	84	289
300	11	153	38	91	315
1000	16	250	17 T	127	308
3000	18	1 T	0 T	51 T	343
5000 T	0	0	0	0	231
NaN3 NaN3 5	538	-	-	703	-
9-AA 50	-	238	-	-	-
2-Nf 10	-	-	488	-	-
MMC 0.5	-	-	-	-	737

Table 2: Mutagenic activity of the test item. Mean values Experiment 1 (Plate incorporation). With metabolic activation

Compound (µg/plate)	Mean Revertants/Plate
	S. typhimurium
	TA 1535	TA 1537	TA 98	TA 100	TA 102
DMSO	15	13	34	82	285
100	15	15	35	89	289
300	12	23	40	90	334
1000	15	49	39	76	232
3000	16	31T	50	89	219 T
5000	14	18 T	31	68 T	50 T
9-AA 4	257	215	797	985	-
9-AA 10					1131
P: Precipitation; T: Toxic; -: Not used; Underlined values are regarded as increased
Historical Range	8 - 15	5 -21	32 - 54	49 - 119	285 - 418

Table 3: Mutagenic activity of the test item. Mean values Experiment 2 (Plate incorporation). Without metabolic activation

Compound (µg/plate)	Mean Revertants/Plate
	S. typhimurium TA 1537
	DMSO	Dist. Water
Control	9	8
500	144	126
1000	164	151
1500	147	137
9-AA 50	187	186

Table 4: Mutagenic activity of the test item. Mean values Experiment 2 (Plate incorporation). With metabolic activation

Compound (µg/plate)	Mean Revertants/Plate
	S. typhimurium TA 1537
	DMSO	Dist. Water
Control	11	9
500	19	30
1000	27	34
1500	27	44
9-AA 4	172	169
Historical Range	5 - 21

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the test substance, when tested up to bacteriotoxic concentrations, caused no mutations in the tester strains S. typhimurium TA 1535, TA 98 and TA 102 in the presence and absence of metabolic activation. However, there was a weak increase of revertants in TA 100 and a reproducible mutagenic response in S. typhimurium TA 1537.
Based on the results of this study, it was concluded that the test substance was mutagenic.