1-phenylethanol

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:

22-03-2018 - 20-04-2018

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:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of test chemical 1-phenylethan-1-ol to induce gene muta¬tions in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-phenylethanol

EC Number:

202-707-1

EC Name:

1-phenylethanol

Cas Number:

98-85-1

Molecular formula:

C8H10O

IUPAC Name:

1-phenylethan-1-ol

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report):Methyl benzyl alcohol
- Molecular formula (if other than submission substance):C8H10O
- Molecular weight (if other than submission substance):122.166 g/mol
- Substance type:Organic- Physical state:Liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report):1-phenylethan-1-ol
- Molecular formula (if other than submission substance):C8H10O
- Molecular weight (if other than submission substance):122.166 g/mol
- Substance type:Organic- Physical state:Liquid
-Purity ; 99.8%

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other:

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 metabolic activation system

Test concentrations with justification for top dose:

0.0 (NC), 0.0158, 0.050, 0.158, 0.501, 1.582 mg/plate .

Vehicle / solvent:

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

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

RO water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); positive controls are used as per OECD 471 point number 25 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9);

Remarks:

S9 is also charecterised with mutagen that requires metabolic activation by microsomal enzyme such as DMSO,Aflatoxin B1,2-Aminoanthracene ,Benzo (a) Pyrene,2 amino3,4 dimethylimindazo (4,4 Aquinoline)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- 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): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

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:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding negative control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control such an increase is not considered biologically relevant.

Statistics:

No data

Results and discussion

Test results

Species / strain:

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

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 precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations 0.0 (NC), 0.002, 0.005, 0.0158, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. (Concentration of the substance is as per OECD guideline 471 point number 20.)

In the pre-experiment, the concentration range of the test item was 0.002 – 5.0 mg/plate based on the solubility and precipitation test. There was no reduction in colony count but reduction in background lawn was observed in treated concentration 5 mg/plate (T8) and no reduction in colony count as well as in background lawn in treated concentrations 1.582 (T7) mg/plate – 0.002 (T1) mg/plate both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.0158, 0.050, 0.158, 0.501, 1.582 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

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

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)	R	Without metabolic activation (-S9)		With metabolic activation (+S9)
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	119	22	125	24
	R2	115	20	123	22
	R3	123	23	120	20
T1 (0.002)	R1	103	15	111	20
	R2	119	17	106	18
	R3	107	17	103	18
T2 (0.005)	R1	107	16	113	19
	R2	105	16	109	20
	R3	104	17	113	19
T3 (0.0158)	R1	107	20	117	18
	R2	111	18	109	20
	R3	115	21	112	21
T4 (0.050)	R1	112	20	117	19
	R2	115	21	114	20
	R3	109	19	109	22
T5 (0.158)	R1	114	21	117	21
	R2	109	21	113	20
	R3	117	20	120	22
T6 (0.501)	R1	110	21	115	21
	R2	116	21	119	21
	R3	112	21	120	22
T7 (1.582)	R1	115	21	118	21
	R2	117	22	121	23
	R3	115	21	120	21
T8 (5)	R1	109	23	119	23
	R2	117	19	123	22
	R3	120	20	119	23
PC	R1	1152	1020	1464	1230
	R2	1140	1002	1424	1152
	R3	1122	978	1480	1194

NC           =     Negative control

PC            =     Positive control             

R              =     Replicate

T              =     Test concentration (T8: Highest, T1: Lowest)

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL I)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	8	15	24	125	266
	R2	7	16	22	123	284
	R3	8	14	20	120	276
T1 (0.0158)	R1	4	11	18	117	224
	R2	4	10	20	109	228
	R3	5	10	21	112	218
T2 (0.050)	R1	5	11	19	117	224
	R2	5	12	20	114	234
	R3	5	13	22	109	226
T3 (0.158)	R1	6	12	21	117	240
	R2	5	12	20	113	236
	R3	5	13	22	120	250
T4 ((0.501)	R1	6	14	21	115	246
	R2	7	14	21	119	254
	R3	6	13	22	120	262
T5 (1.582)	R1	7	14	21	118	276
	R2	8	13	23	121	264
	R3	6	15	21	120	278
PC	R1	174	446	1230	1464	1242
	R2	185	414	1152	1424	1374
	R3	167	346	1194	1480	1248

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	7	16	22	119	271
	R2	9	14	20	115	265
	R3	8	13	23	123	289
T1 (0.0158)	R1	4	10	20	107	215
	R2	4	11	18	111	220
	R3	4	10	21	115	226
T2 (0.050)	R1	4	11	20	112	217
	R2	4	12	21	115	229
	R3	5	10	19	109	231
T3 (0.158)	R1	5	12	21	114	233
	R2	5	10	21	109	241
	R3	6	13	20	117	244
T4 ((0.501)	R1	5	12	21	110	251
	R2	6	13	21	116	253
	R3	6	13	21	112	261
T5 (1.582)	R1	6	14	21	115	248
	R2	7	15	22	117	268
	R3	8	13	21	115	274
PC	R1	176	1160	1020	1152	1872
	R2	182	1236	1002	1140	1640
	R3	165	1232	978	1122	1688

NC= Negative Control,T=Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                  2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]:TA 102                                        Sodium azide [10μg/plate]: TA 1535, TA 100                                                 

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate]   Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	7	16	27	119	251
	R2	7	16	28	120	263
	R3	8	15	26	118	270
T1 (0.0158)	R1	4	11	20	101	234
	R2	4	12	20	104	238
	R3	4	11	19	108	242
T2 (0.050)	R1	5	12	22	106	236
	R2	4	13	23	107	240
	R3	5	12	21	110	242
T3 (0.158)	R1	5	13	22	109	250
	R2	5	12	24	113	241
	R3	6	14	23	107	248
T4 ((0.501)	R1	6	13	24	116	245
	R2	5	15	25	114	252
	R3	6	15	26	117	256
T5 (1.582)	R1	7	16	26	117	262
	R2	8	15	27	120	266
	R3	6	15	25	119	258
PC	R1	162	340	1328	1448	1434
	R2	181	430	1360	1518	1432
	R3	183	376	1384	1480	1512

 

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	6	17	27	121	258
	R2	7	16	28	119	262
	R3	8	15	27	117	274
T1 (0.0158)	R1	4	10	21	104	230
	R2	5	11	21	102	227
	R3	4	11	20	105	244
T2 (0.050)	R1	5	12	19	111	242
	R2	5	10	23	107	246
	R3	4	12	21	109	262
T3 (0.158)	R1	5	13	23	112	256
	R2	6	16	22	115	260
	R3	4	14	22	117	248
T4 (0.501)	R1	6	15	25	114	264
	R2	5	14	23	113	259
	R3	6	13	24	116	264
T5 (1.582)	R1	7	15	25	119	265
	R2	7	16	26	120	256
	R3	6	14	27	115	268
PC	R1	182	1160	894	1128	1568
	R2	176	1224	928	1086	1608
	R3	180	1272	952	1164	1656

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate

PC= Positive control                                                                       2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100        
2-Aminoanthracene [10μg/plate]:TA 102                                              Sodium azide [10μg/plate]: TA 1535, TA 100,                                            

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 4 - MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.67	0.58	15.00	1.00	22.00	2.00	122.67	2.52	275.33	9.02
T1 (0.0158)	4.33	0.58	10.33	0.58	19.67	1.53	112.67	4.04	223.33	5.03
T2 (0.050)	5.00	0.00	12.00	1.00	20.33	1.53	113.33	4.04	228.00	5.29
T3 (0.158)	5.33	0.58	12.33	0.58	21.00	1.00	116.67	3.51	242.00	7.21
T4 (0.501)	6.33	0.58	13.67	0.58	21.33	0.58	118.00	2.65	254.00	8.00
T5 (1.582)	7.00	1.00	14.00	1.00	21.67	1.15	119.67	1.53	272.67	7.57
PC	175.33	9.07	402.00	51.07	1192.00	39.04	1456.00	28.84	1288.00	74.54

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	8.00	1.00	14.33	1.53	21.67	1.53	119.00	4.00	275.00	12.49
T1 (0.0158)	4.00	0.00	10.33	0.58	19.67	1.53	111.00	4.00	220.33	5.51
T2 (0.050)	4.33	0.58	11.00	1.00	20.00	1.00	112.00	3.00	225.67	7.57
T3 (0.158)	5.33	0.58	11.67	1.53	20.67	0.58	113.33	4.04	239.33	5.69
T4 (0.501)	5.67	0.58	12.67	0.58	21.00	0.00	112.67	3.06	255.00	5.29
T5 (1.582)	7.00	1.00	14.00	1.00	21.33	0.58	115.67	1.15	263.33	13.61
PC	174.33	8.62	1209.33	42.77	1000.00	21.07	1138.00	15.10	1733.33	122.46

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100 Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                  

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

 

 

TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATIONMETHOD
(TRIAL II)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.33	0.58	15.67	0.58	27.00	1.00	119.00	1.00	261.33	9.61
T1 (0.0158)	4.00	0.00	11.33	0.58	19.67	0.58	104.33	3.51	238.00	4.00
T2 (0.050)	4.67	0.58	12.33	0.58	22.00	1.00	107.67	2.08	239.33	3.06
T3 (0.158)	5.33	0.58	13.00	1.00	23.00	1.00	109.67	3.06	246.33	4.73
T4 (0.501)	5.67	0.58	14.33	1.15	25.00	1.00	115.67	1.53	251.00	5.57
T5 (1.582)	7.00	1.00	15.33	0.58	26.00	1.00	118.67	1.53	262.00	4.00
PC	175.33	11.59	382.00	45.30	1357.33	28.10	1482.00	35.04	1459.33	45.62

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.00	1.00	16.00	1.00	27.33	0.58	119.00	2.00	264.67	8.33
T1 (0.0158)	4.33	0.58	10.67	0.58	20.67	0.58	103.67	1.53	233.67	9.07
T2 (0.050)	4.67	0.58	11.33	1.15	21.00	2.00	109.00	2.00	250.00	10.58
T3 (0.158)	5.00	1.00	14.33	1.53	22.33	0.58	114.67	2.52	254.67	6.11
T4 (0.501)	5.67	0.58	14.00	1.00	24.00	1.00	114.33	1.53	262.33	2.89
T5 (1.582)	6.67	0.58	15.00	1.00	26.00	1.00	118.00	2.65	263.00	6.24
PC	179.33	3.06	1218.67	56.19	924.67	29.14	1126.00	39.04	1610.67	44.06

NC= Negative Control, T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Applicant's summary and conclusion

Conclusions:

The test chemical 1-phenylethan-1-ol did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Ames test was performed to investigate the potential of 1-phenylethan-1-ol – (CAS No. 98-85-1) to induce gene muta­tions in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.0158, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.0158, 0.050, 0.158, 0.501, 1.582 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with 1-phenylethan-1-ol – (CAS No. 98-85-1) at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data.

The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test item 1-phenylethan-1-ol – (CAS No. 98-85-1) did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.