1-phenylethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

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 theSalmonella typhimuriumstrains 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.

In vitro gene mutation study in mammalian cells

An in vitromammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of 1-phenylethanol (CAS No. 98-85-1) when administered to Chinese Hamster Ovary (CHO) cells.In the genotoxicity test, 1-phenylethanol was administered to CHO cells for 3 hrs at the dose levels of 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. pH and osmolality was not determined in the gene mutation test.Only the positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. One very diffuse colony were seen in one well out of four at 5 mM and in the presence with 4% S9 liver microsomal fraction. This diffuse colony is not regarded to be relevant since the single spot was only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the overall results of the tested concentrations of 1-phenylethanol, i.e. the test chemical did not show any evidence of diffuse or clear colonies present.When the mutation frequency was determined, a frequency of 4.53 x 10^-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of 1-phenylethanol and in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that 1-phenylethanol does not give rise to gene mutations when CHO cells are exposedin vitroto the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs.Based on the results of the current study, we conclude that 1-phenylethanol does not give rise to gene mutations when CHO cells are exposed to the test chemicalin vitroat 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or abscence of metabolic activation.

Link to relevant study records

Reference

Reference 1

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

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

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%

Target gene:

Histidine

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The purpose of this study was to assess toxic and genotoxic effects of 1-phenylethanol on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 0.5, 1, 2.5 or 5 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: 1-phenylethanol was easily dissolved in DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Preincubation period: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

OTHER EXAMINATIONS: Not applicable
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Rationale for test conditions:

No data

Evaluation criteria:

The cell line was observed for gene mutation

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Additional information on results:

Positive controls valid for study without S9

Remarks on result:

other: No mutagenic potential

Conclusions:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to 1-phenylethanol in the concentration of 0, 0.5, 1, 2.5 or 5 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with 1-phenylethanol. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to 1-phenylethanol. Therefore, it is considered that 1-phenylethanol in the concentration of 0, 0.5, 1, 2.5 or 5 mM does not cause genetic mutation(s) in the presence or abscence of metabolic activation.

Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of 1-phenylethanol (CAS No. 98-85-1) when administered to Chinese Hamster Ovary (CHO) cells.

In the genotoxicity test, 1-phenylethanol was administered to CHO cells for 3 hrs at the dose levels of 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. pH and osmolality was not determined in the gene mutation test.

Only the positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. One very diffuse colony were seen in one well out of four at 5 mM and in the presence with 4% S9 liver microsomal fraction. This diffuse colony is not regarded to be relevant since the single spot was only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the overall results of the tested concentrations of 1-phenylethanol, i.e. the test chemical did not show any evidence of diffuse or clear colonies present.

When the mutation frequency was determined, a frequency of 4.53 x 10^-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of 1-phenylethanol and in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that 1-phenylethanol does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs.

Based on the results of the current study, we conclude that 1-phenylethanol does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or abscence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Gene mutation in vivo:

In vivo micronucleus assay in mouse bone marrow was carried out to test the clastogenic nature of the test compound1-phenylethanol. The animals were given 1-phenylethanol in single oral doses up to the maximum tolerated dose of 750 mg/kg body weight (0, 187.5, 375 or 700 mg/kg body weight).Bone marrow was sampled 24 and 48 h after treatment. Under the experimental conditions used, there was no evidence of increased micronuclei frequencies at any dose or sampling time. The number of polychromaticerythrocytes containing either small or large micronuclei at each dose was not significantly increased above the concurrent negative (solvent) control frequencies and was always within the historical negative control range (0.3–3.3% based on > 300 experiments) at each sampling time. The ratio of PCE: NCE was not affected in treated animals; the slight depression after 48 h and increase after 24 h may reflect the normal variability rather than bone marrow toxicity. Oral administration of a single dose of 1-phenylethanol led to evident signs of toxicity such as staggering in the intermediate (375 mg/kg) and top (750 mg/kg) dose animals and to irregular respiration, piloerection, abdominal position and to a narcotic-like state in all animals of the highest dose group, clearly demonstrating the attainment of a maximum tolerated dose. These findings indicate that 1- phenylethanol is not clastogenic in vivo.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo

Remarks:

Type of genotoxicity: other: Micronucleus assay

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: OECD No. 474 (July 21, 1997) and EC Directive 2000/32, B. 12 (May 19, 2000)

Principles of method if other than guideline:

In vivo micronucleus test in mice was performed to evaluate the mutagenic nature of the test compound 1-phenylethanol

GLP compliance:

yes

Type of assay:

other: Mouse bone marrow micronucleus assay

Species:

mouse

Strain:

other: NMRI

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

Details on test animals and env conditions
TEST ANIMALS
- Source: Charles River Deutschland GmbH (Sulzfeld, Germany)
- Age at study initiation: 5–8 weeks old
- Weight at study initiation: 30g
- Assigned to test groups randomly: [no/yes, under following basis: ] Random
- Fasting period before study: No data available
- Housing: Makrolon cages type MI in fully air-conditioned rooms
- Diet (e.g. ad libitum): standard rat/mouse laboratory feed ad libitum
- Water (e.g. ad libitum): drinking-quality tap water ad libitum
- Acclimation period: Minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20–24⁰C
- Humidity (%): 30–70%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12/12-h light/dark cycle (light on 6:00–18:00 h)

IN-LIFE DATES: From: To: No data available

Route of administration:

oral: gavage

Vehicle:

Vehicles
- Vehicle(s)/solvent(s) used: Olive oil
- Justification for choice of solvent/vehicle: No data available
- Concentration of test material in vehicle: 0, 187.5, 375 or 700 mg/kg body weight
- Amount of vehicle (if gavage or dermal): 10 ml/ kg body weight
- Type and concentration of dispersant aid (if powder): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on exposure:

- Vehicle(s)/solvent(s) used: Olive oil
- Justification for choice of solvent/vehicle: No data available
- Concentration of test material in vehicle: 0, 187.5, 375 or 700 mg/kg body weight
- Amount of vehicle (if gavage or dermal): 10 ml/ kg body weight
- Type and concentration of dispersant aid (if powder): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Duration of treatment / exposure:

Duration of exposure: 24 or 48 hrs

Frequency of treatment:

Once

Post exposure period:

No data available

Remarks:

Doses / Concentrations:
0, 187.5, 375 or 700 mg/kg body weight
Basis:
actual ingested

No. of animals per sex per dose:

Total: 40
0 mg/Kg bw: 10 (5/dose at 24 and 48 hrs)
187.5 mg/Kg bw: 5/dose at 24 hrs
375 mg/Kg bw: 5/dose at 24 hrs
750 mg/Kg bw: 10 (5/dose at 24 and 48 hrs)

Positive control 1: 5/dose at 24 hrs
Positive control 2: 5/dose at 24 hrs

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive controls
Cyclophosphamide- for clastogenicity
Vincristine sulfate- for aneugenic activity
- Justification for choice of positive control(s): No data available
- Route of administration: Oral
Cyclophosphamide- Oral
Vincristine sulfate- intraperitoneally

- Doses / concentrations:
Cyclophosphamide- 20 mg/kg body weight
Vincristine sulfate- 0.15 mg/kg body weight

Tissues and cell types examined:

PCEs and NCEs isolated from bone marrow cells

Details of tissue and slide preparation:

Details of tissue and slide preparation
CRITERIA FOR DOSE SELECTION: In a range finding study for the determination of the
acute oral toxicity using male and female mice, deaths were observed at doses of 800 mg/kg body weight and above. At 750 mg/kg body weight, all animals survived, but clinical signs of toxicity such as piloerection, irregular respiration and staggering were observed.

Thus, this dose was considered to be the maximum tolerated dose and the dose levels selected for this study were 750, 375 and 187.5 mg/kg body weight.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: Micronucleus suspension was dropped onto clean slides. Smears were prepared, air-dried and stained with May-Gruenwald and Giemsa solutions.

METHOD OF ANALYSIS: Microscopic analysis was performed

OTHER: The bone marrow was prepared.
After having sacrificed the animals by cervical dislocation, the femora were removed and freed from muscles. The epiphyses were cut off and the bone marrow was flushed out of the diaphysis into centrifuge tubes using a cannulated syringe filled with fetal calf serum (FCS; 37C; about 2 ml/femur). After thorough mixing, the suspension was centrifuged at 1,500 rpm for 5 min and the sediment was re-suspended with 50 µL fresh FCS.

Evaluation criteria:

For each animal, 2,000 polychromatic erythrocytes (PCEs) were scored and evaluated for the presence of small micronuclei (d < D/4) indicative of a clastogenic mode of action and large micronuclei (d ‡ D/4) indicative of an aneugenic mode of action

The number of normochromatic erythrocytes (NCEs) both with and without micronuclei in the fields containing 2,000 PCEs were recorded separately.

Statistics:

Wilcoxon’s rank test (one-sided) was performed for comparison of the relative frequencies of micronucleated cells in each animal of the dose groups with those of the negative control groups.

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No mutagenic potential

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Dose range is not properly mentioned but includes study at 750 and 800 mg/Kg bw
- Solubility: No data available
- Clinical signs of toxicity in test animals: Deaths were observed at doses of 800 mg/kg body weight and above. At 750 mg/kg body weight, all animals survived, but clinical signs of toxicity such as piloerection, irregular respiration and staggering were observed.
- Evidence of cytotoxicity in tissue analyzed: No data available
- Rationale for exposure: No data available
- Harvest times: No data available
- High dose with and without activation: No data available
- Other: No data available

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay): PCEs and NCEs were observed
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE:NCE was not affected in treated animals; the slight depression after 48 h and increase after 24 h may reflect the normal variability rather than bone marrow toxicity.
- Appropriateness of dose levels and route:
Dose level: 0, 187.5, 375 or 700 mg/kg body weight
Route: Oral
- Statistical evaluation: Wilcoxon’s rank test (one-sided) was performed for comparison of the relative frequencies of micronucleated cells in each animal of the dose groups with those of the negative control groups.

Conclusions:

The test compound 1-phenylethanol is not clastogenic in nature when study was performed using mouse bone marrow cells.

Executive summary:

In vivo micronucleus assay in mouse bone marrow was carried out to test the clastogenic nature of the test compound1-phenylethanol. The animals were given 1-phenylethanol in single oral doses up to the maximum tolerated dose of 750 mg/kg body weight (0, 187.5, 375 or 700 mg/kg body weight).Bone marrow was sampled 24 and 48 h after treatment. Under the experimental conditions used, there was no evidence of increased micronuclei frequencies at any dose or sampling time. The number of polychromaticerythrocytes containing either small or large micronuclei at each dose was not significantly increased above the concurrent negative (solvent) control frequencies and was always within the historical negative control range (0.3–3.3% based on > 300 experiments) at each sampling time. The ratio of PCE: NCE was not affected in treated animals; the slight depression after 48 h and increase after 24 h may reflect the normal variability rather than bone marrow toxicity. Oral administration of a single dose of 1-phenylethanol led to evident signs of toxicity such as staggering in the intermediate (375 mg/kg) and top (750 mg/kg) dose animals and to irregular respiration, piloerection, abdominal position and to a narcotic-like state in all animals of the highest dose group, clearly demonstrating the attainment of a maximum tolerated dose. These findings indicate that 1- phenylethanol is not clastogenic in vivo.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical 1-phenylethan-1-ol – (CAS No. 98-85-1). The studies are as mentioned below:

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 theSalmonella typhimuriumstrains 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.

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of 1-phenylethanol (CAS No. 98-85-1) when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, 1-phenylethanol was administered to CHO cells for 3 hrs at the dose levels of 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. pH and osmolality was not determined in the gene mutation test. Only the positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. One very diffuse colony were seen in one well out of four at 5 mM and in the presence with 4% S9 liver microsomal fraction. This diffuse colony is not regarded to be relevant since the single spot was only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the overall results of the tested concentrations of 1-phenylethanol, i.e. the test chemical did not show any evidence of diffuse or clear colonies present. When the mutation frequency was determined, a frequency of 4.53 x 10^-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of 1-phenylethanol and in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that 1-phenylethanol does not give rise to gene mutations when CHO cells are exposedin vitroto the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs. Based on the results of the current study, we conclude that 1-phenylethanol does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or abscence of metabolic activation.

In another study, Gene mutaton toxicity study was performed to determine the mutagenic nature of the test chemical 1-phenylethanol (CAS No. 98-85-1)

. The study was performed by the preincubation assay using S. typhimurium strains TA98, TA100, TA1535, or TA1537 in the presence and absence of 0% Aroclor 1254-induced male Sprague Dawley rat or Syrian hamster liver S9 mix. The test chemical was dissolved in DMSO and used at dose level of 0, 33, 100, 333, 1000, 2500, 3333, 6666 microgram/plate. Concurrent solvent and positive control chemicals were also included in the study. 1-phenylethanol was not mutagenic in S. typhimurium strains TA98, TA100, TA1535, or TA1537 when tested at concentrations up to 6666 µg/plate with a preincubation protocol in the presence or absence of 10% Aroclor 1254-induced male Sprague Dawley rat or Syrian hamster liver S9 mix. As per the CLP classification, the test material does not classify as a mutagen in vitro.

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using CHO cells in the presence and absence of S9 metabolic activation system. The test chemical was soluble in DMSO and used at dose level of 0, 1000, 1500 or2000 microgram/L (without S9) and 0, 1000, 2000 or 3000 microgram/L (with S9). Concurrent solvent and positive control chemicals were also included in the study. In cytogenetic tests with CHO cells, 1-phenyletanol induced a significant increase in chromosomal aberrations in CHO cells, in the presence of S9, within a concentration range of 1000-3000 µg/ml, but not the absence of metabolic activation.

In the same study, sister chromatid exchange assay was performed to determine the mutagenic nature of the test chemical. The study was performed using CHO cells in the presence and absence of S9 metabolic activation system. Concurrent solvent and positive control chemicals were also included in the study. In cytogenetic tests with CHO cells, 1-phenylethanol did not induce sister chromatid exchange or cell cycle delay when tested over a concentration range of 33-1000 µg/ml with or without Aroclor 1254-induced male Sprague Dawley rat liver S9. As per the CLP classification, the test material does not classify as a mutagen in vitro.

Differential growth inhibition of tw oE. colicultures (DNA polymerase deficient strain P3478 and parent strain P3110) was evaluated as a rapid screening technique for evaluating the mutagenic nature of 1-phenylethanol. In a typical assay,1-phenylethanolwas applied to two plates containing the pol A⁺organism and two plates containing the pol A^-organism. The plates were then incubated for 16 hrs and the zones of inhibition were measured.

 1-phenylethanol did not cause growth inhibition due to DNA damage in two strains of Escherichia coli exposed to 50 µL/plate without S9 metabolic activation. As per the CLP classification, the test material does not classify as a gene mutant in vitro.

In yet another study, Reversion mutation assay was performed for the test chemical α-Methylbenzyl alcohol using the bacterium Escherichia coli strain Sd-4-73. The test compound α-Methylbenzyl alcohol was not mutagenic in an assay for reversion from streptomycin dependence to streptomycin independence in strain Sd-4-73 Escherichia coli and hence is not a gene mutant.

Gene mutation in vivo:

In vivo micronucleus assay in mouse bone marrow was carried out to test the clastogenic nature of the test compound1-phenylethanol. The animals were given 1-phenylethanol in single oral doses up to the maximum tolerated dose of 750 mg/kg body weight (0, 187.5, 375 or 700 mg/kg body weight).Bone marrow was sampled 24 and 48 h after treatment. Under the experimental conditions used, there was no evidence of increased micronuclei frequencies at any dose or sampling time. The number of polychromaticerythrocytes containing either small or large micronuclei at each dose was not significantly increased above the concurrent negative (solvent) control frequencies and was always within the historical negative control range (0.3–3.3% based on > 300 experiments) at each sampling time. The ratio of PCE: NCE was not affected in treated animals; the slight depression after 48 h and increase after 24 h may reflect the normal variability rather than bone marrow toxicity. Oral administration of a single dose of 1-phenylethanol led to evident signs of toxicity such as staggering in the intermediate (375 mg/kg) and top (750 mg/kg) dose animals and to irregular respiration, piloerection, abdominal position and to a narcotic-like state in all animals of the highest dose group, clearly demonstrating the attainment of a maximum tolerated dose. These findings indicate that 1- phenylethanol is not clastogenic in vivo.

In another study, 1-phenylethanol was included in a study of the effects of styrene and its metabolites on cell proliferation and histomorphology in different compartments of the lungs of female Crl Icr: CD1 mice. The animals were dosed intraperitoneally with a-methylbenzyl alcohol at 100 mg/kg, three times daily at 2-h intervals, for 3 days. The rate of DNA S-phase synthesis in lungs was assessed using 5-bromo-2-deoxyuridine (BrdU). Therewere no signs of clinical toxicity, effects on body weight, changes in absolute or relative lung weights, or gross appearance of the lungs. There was only a small (1.5-fold) increase in cell proliferation in the alveoli and no significant increase in large/medium bronchi or terminal bronchioles. The pattern and character of the severe morphological lesions following exposure to styrene were not seen following exposure to a-methylbenzyl alcohol. Hence 1-phenylethanol does not classify as gene mutant in vivo.

Based on the data available for the test chemical, 1-phenylethan-1-ol – (CAS No. 98-85-1) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the test chemical, 1-phenylethan-1-ol – (CAS No. 98-85-1) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.