N-ethyl-p-methoxy-α-methylphenethylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Gene mutation toxicity study (Plate incorporation method) was performed to determine the mutagenic nature of the test chemical

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-methoxybenzyl alcohol

EC Number:

203-273-6

EC Name:

4-methoxybenzyl alcohol

Cas Number:

105-13-5

Molecular formula:

C8-H10-O2

IUPAC Name:

(4-methoxyphenyl)methanol

Test material form:

liquid

Details on test material:

- Name of test material: 4-methoxybenzyl alcohol
- IUIPAC name: 4-methoxybenzyl alcohol
- Molecular Formula: C8H10O2
- Molecular Weight: 138.166 g/mol
- Substance type: Organic
- Physical state: Liquid
- Purity: 99.9%

Specific details on test material used for the study:

- Name of test material: 4-methoxybenzyl alcohol
- IUPAC name: 4-methoxybenzyl alcohol
- Molecular Formula: C8H10O2
- Molecular Weight: 138.166 g/mol
- Substance type: Organic
- Physical state: Liquid
- Purity: 99.9%

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Sprague-Dawley rat liver S9 mix

Test concentrations with justification for top dose:

0, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl suphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 activation Migrated to IUCLID6: 3µg/plate for TA100 and 5µg/plate for TA1535

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 activation Migrated to IUCLID6: 80µg/plate for TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 activation Migrated to IUCLID6: 0.5µg/plate for TA102

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 activation Migrated to IUCLID6: 0.2µg/plate for TA98

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA): 1µg/plate for TA100; 2µg/plate for TA1535 and TA 1537

Remarks:

with S9 activation

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 activation Migrated to IUCLID6: 5µg/plate for TA98

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1,8-Dihydroxyanthraquinone (DAN): 10µg/plate for TA102

Remarks:

with S9 activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): No data

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): Triplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met :
The test material should have induced a reproducible, dose related and statistically(Dunnett’s method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett’s method of linear regression

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Prliminary dose range finding study was performed to determine the doses for the main study. The doses for the preliminary study were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 or 5000 g/plate. The plate incorporation test was performed by mixing 0.1mL bacterial culture (TA100), 2 mL of molten, trace histidine supplemented, top agar, 0.1 mL of the test material formulation, 0.5mL of S9 mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar. Ten doses of the test material and vehicle control (DMSO) were tested. After 48 hrs incubation at 37deg C, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Table 1 Spontaneous Mutation Rates (Concurrent Negative Control)

Number of Revertant (Mean number of colonies per plate)
Base pair substitution type			Frameshift type
TA100	TA1535	TA102	TA98	TA1537
92	34	386	17	12
120             (107)	34          (34)	374         (380)	16          (18)	13       (11)
110	34	380	22	9

Table 2  Test Result: Without Metabolic Activation Plate incorporation method

Test Period		From: 07 October 2003					To: 10 October 2003
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		TA102		TA98		TA1537
-	0	142 132 125	(133) 8.5#	35 34 31	(33) 2.1	327 335 380	(347) 28.6	21 12 22	(18) 5.5	16 16 11	(14) 2.9
-	100	102 114 130	(115) 14.0	29 36 37	(34) 4.4	286 368 364	(339) 46.2	22 20 16	(19) 3.1	9 11 19	(13) 5.3
-	333	100 111 103	(105) 5.7	29 39 33	(34) 5.0	308 343 362	(338) 27.4	17 17 13	(16) 2.3	19 20 11	(17) 4.9
-	1000	121 123 100	(115) 12.7	38 32 38	(36) 3.4	340 345 295	(327) 27.5	20 17 16	(18) 2.1	20 23 21	(21) 1.5
-	2500	111 119 98	(109) 10.6	40 33 39	(37) 3.8	347 328 366	(347) 19.0	18 7 22	(16) 7.8	17 13 20	(17) 3.5
-	5000	98 123 100	(107) 13.9	38 40 C	(39) 1.4	131 302 303	(312) 16.5	13 9 17	(13) 4.0	25 13 15	(18) 6.4
Positive control	Name Concentration (µg/plate)	ENNG		ENNG		MMC		4NQO		9AA
		3		5		0.5		0.2		80
S9-Mix -	No. colonies per plate	483 470 547	(500) 41.2	792 873 1036	(900) 124.3	2350 2645 2248	(2414) 206.2	158 180 166	(168) 11.1	698 1050 1225	(991) 268.4

EENG    N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO  4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

MMC  Mitomycin C

C         Contaminated

#         Standard deviation

Table 3       Test Result: With Metabolic Activation Plate incorporation method

Test Period		From: 07 October 2003					To: 10 October 2003
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		TA102		TA98		TA1537
+	0	100 128 132	(123) 11.7#	15 15 33	(21) 10.4	393 369 350	(371) 21.5	26 39 28	(31) 7.0	17 24 20	(20) 3.5
+	100	136 134 117	(129) 10.4	15 16 17	(16) 1.0	388 324 343	(352) 32.9	41 29 22	(31) 9.6	13 20 15	(16) 3.6
+	333	93 139 120	(117) 23.1	17 9 17	(14) 4.6	380 377 326	(361) 30.3	32 32 21	(28) 6.4	13 15 22	(17) 4.7
+	1000	109 130 130	(123) 12.1	19 13 13	(15) 3.5	399 362 363	(375) 21.1	36 25 33	(31) 5.7	15 22 19	(19) 3.5
+	2500	114 94 114	(107) 11.5	23 9 16	(16) 7.0	372 399 404	(392) 17.2	38 30 34	(34) 4.0	27 20 22	(23) 3.6
+	5000	113 110 115	(113) 2.5	16 22 21	(20) 3.2	386 418 302	(369) 59.9	36 34 34	(35) 1.2	17 21 20	(19) 2.1
Positive control	Name Concentration (µg/plate)	2AA		2AA		DAN		BP		2AA
		1		2		10		5		2
S9-Mix +	No. colonies per plate	1411 1499 1649	(1520) 120.3	320 371 333	(341) 26.5	691 878 727	(765) 99.2	165 256 239	(220) 48.4	327 264 356	(316) 47.0

2AA     2-Aminoanthracene

BP        Benzo(a)pyrene

DAN      1,8-Dihydroxyanthraquinone

#            Standard deviation

Applicant's summary and conclusion

Conclusions:

The test chemical was considered to be non mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation in the Plate incorporation assay and hence is likely to be non-mutagenic.

Executive summary:

Gene mutation toxicity study (Plate incorporation method) was performed to determine the mutagenic nature of the test chemical. The method was designed to meet the requirements of the OECD Guidelines for testing of the Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment (plate incorporation) was determined in a preliminary toxicity assay and was 100 to 5000µg/plate. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Based on the observations made, the test chemical was considered to be non mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 with and without metabolic activation in the Plate incorporation assay and hence is likely to be non-mutagenic.