3-phenylpropan-1-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-20 to 2017-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

July 2016

Deviations:

yes

Remarks:

please refer to 'Principles of method if other than guideline'

Principles of method if other than guideline:

A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

3-phenylpropan-1-ol

EC Number:

204-587-6

EC Name:

3-phenylpropan-1-ol

Cas Number:

122-97-4

Molecular formula:

C9H12O

IUPAC Name:

3-phenylpropan-1-ol

Method

Species / strain

Species / strain / cell type:

lymphocytes: human (primary culture)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (27 years old) for Experiment I and from a male donor (24 years old) for Experiment II.
- Suitability of cells: The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Cell culture conditions: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

Experimtent I (4 h exposure): 8.8, 15.5, 27.1, 47.4, 83.0, 145, 254, 445, 778, 1362 µg/mL, with and without S9 mix
Experiment II (40 h exposure): 8.8, 15.5, 27.1, 47.4, 83.0, 145, 254, 445, 778, 1362 µg/mL, with S9 mix

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine (without metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (pulse exposure), 20 h (continuous exposure, without S9 mix)
- Expression time (cells in growth medium): 16 h recovery period followed by another 20 h (4 h group), and 20 h (20 h exposure group)
- Fixation time (start of exposure up to fixation or harvest of cells): 40 h

SPINDLE INHIBITOR: Cytochalasin B

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide.

NUMBER OF CELLS EVALUATED: 500 cells per culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: see below
- Any supplementary information relevant to cytotoxicity:
To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI (Cytokinesis-block proliferation index) of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
Cytostasis % = 100 – 100 [(CBPI(test item) – 1) / (CBPI(solvent control) – 1)]

CBPI = (Mononucleate cells x 1) + (Binucleate cells x 2) + (Multinucleate cells x 3)

Rationale for test conditions:

according to guideline

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

Statistics:

Statistical significance was confirmed by the Chi Square Test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 778 µg/mL onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: no precipitation of the test item in the culture medium was observed

RANGE-FINDING/SCREENING STUDIES:
Dose selection was based on a preliminary test for cytotoxicity (Experiment I).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Mitomycin C (78 experiments): micronucleated cells mean 12.48 (95 % Ctrl limit): 1.44 - 23.52
Demecolcine (81 experiments): micronucleated cells mean 3.72 (95 % Ctrl limit): 1.43 - 6.01
Cyclophosphamide (165 experiments): micronucleated cells mean 5.16 (95 % Ctrl limit): 0.84 - 9.49
- Negative (solvent/vehicle) historical control data:
with metabolic activation: 96 experiments: micronucleated cells mean 0.62 (95 % Ctrl limit): 0.15 - 1.3
without metabolic activation: 78 experiments: micronucleated cells mean 0.60 (95 % Ctrl limit): 0.08 - 1.12

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Any other information on results incl. tables

Table 1: Cytotoxicity indicated as cytokinesis-block proliferation indexand cytostasis; exposure period 4 hrs without S9 mix, Experiment I

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
				c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	0.5 %	-	4 / 40 hrs	79	374	47	1.94	90	365	45	1.91	1.92
Pos. control^##	0.8 µg	-	4 / 40 hrs	147	339	14	1.73	157	330	13	1.71	1.72	21.7
Test item	445 µg	-	4 / 40 hrs	97	356	47	1.90	112	346	42	1.86	1.88	4.7
²	778 µg	-	4 / 40 hrs	59	396	45	1.97	71	367	62	1.98	1.98	n.c.
²	1362 µg	-	4 / 40 hrs	48	426	26	1.96	107	373	20	1.83	1.89	3.5

*          c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#                DMSO

^##         MMC

n.c.     Not calculated as the CBPI is equal or higher than the solvent control value

 

Table 2: Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 4 hrs with S9 mix, Experiment I

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
				c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	0.5 %	+	4 / 40 hrs	70	366	64	1.99	73	342	85	2.02	2.01
Pos. control^##	15.0 µg	+	4 / 40 hrs	279	208	13	1.47	250	238	12	1.52	1.50	50.7
Test item	445 µg	+	4 / 40 hrs	79	343	78	2.00	97	344	59	1.92	1.96	4.5
²	778 µg	+	4 / 40 hrs	130	334	36	1.81	61	402	37	1.95	1.88	12.3
²	1362 µg	+	4 / 40 hrs	147	332	21	1.75	114	373	13	1.80	1.77	23.2

*             c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#                     DMSO

^##            CPA

 

Table 3: Number of micronucleated cells; exposure period 4 hrs without S9 mix, Experiment I

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	0.5 %	-	4 / 40 hrs	8	1	0	9	7	0	0	7	16	0.80
Pos. control^##	0.8 µg	-	4 / 40 hrs	136	15	1	152	144	17	2	163	315	15.75
Test item	445 µg	-	4 / 40 hrs	8	0	0	8	3	0	0	3	11	0.55
²	778 µg	-	4 / 40 hrs	3	1	0	4	4	0	0	4	8	0.40
²	1362 µg	-	4 / 40 hrs	3	1	0	4	12	3	0	15	19	0.95

^#                                            DMSO

^##                   MMC

 

Table 4:Number of micronucleated cells; exposure period 4 hrs with S9 mix, Experiment I

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	0.5 %	+	4 / 40 hrs	4	0	0	4	5	1	0	6	10	0.50
Pos. control^##	15.0 µg	+	4 / 40 hrs	52	6	1	59	54	3	0	57	116	5.80
Test item	445 µg	+	4 / 40 hrs	4	1	0	5	4	0	0	4	9	0.45
²	778 µg	+	4 / 40 hrs	5	0	0	5	5	0	0	5	10	0.50
²	1362 µg	+	4 / 40 hrs	4	2	0	6	5	1	0	6	12	0.60

#             DMSO

##           CPA

 

 

Table 5:Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 20 hrs without S9 mix, Experiment II

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
				c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	0.5 %	-	20 / 40 hrs	40	397	63	2.05	68	380	52	1.97	2.01
Pos. control^##	75 ng	-	20 / 40 hrs	251	226	23	1.54	279	206	15	1.47	1.51	49.6
Test item	254 µg	-	20 / 40 hrs	109	359	32	1.85	98	367	35	1.87	1.86	14.6
²	445 µg	-	20 / 40 hrs	126	340	34	1.82	132	342	26	1.79	1.80	20.4
²	778 µg	-	20 / 40 hrs	200	292	8	1.62	250	245	5	1.51	1.56	44.1

*             c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#                     DMSO

^##            Demecolcine

 

Table 6: Number of micronucleated cells; exposure period 20 hrs without S9 mix, Experiment II

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	0.5 %	-	20 / 40 hrs	8	0	0	8	2	1	0	3	11	0.55
Pos. control^##	75 ng	-	20 / 40 hrs	36	7	3	46	19	9	3	31	77	3.85
Test item	254 µg	-	20 / 40 hrs	7	0	0	7	3	0	0	3	10	0.50
²	445 µg	-	20 / 40 hrs	3	0	0	3	2	0	0	2	5	0.25
²	778 µg	-	20 / 40 hrs	3	0	0	3	2	0	0	2	5	0.25

^#                     DMSO

^##                   Demecolcine

Applicant's summary and conclusion

Conclusions:

The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Executive summary:

The test item (dissolved in DMSO) was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group, two parallel cultures were analyzed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis. The highest treatment concentration in this study, 1362 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test. In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix after continuous treatment, moderate cytotoxicity of 44.1 % cytostasis was observed at the highest evaluated concentration (778 µg/mL). Due to a steep cytotoxic gradient caused by the test item the exact requested among of cytotoxicity was not met. The next higher tested and highest applied concentration (1362 µg/mL), however, which was separated by a smaller factor than requested by the guideline, showed clear cytotoxic effects and were not evaluable for cytogenetic damage (no cells available). In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. Demecolcine (75 ng/mL), MMC (0.8 µg/mL) and CPA (15.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.