Methyl (R)-2-(4-hydroxyphenoxy)propionate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 December 1999 to 17 March 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro chromosome aberration study in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

Methyl (R)-2-(4-hydroxyphenoxy)propionate

EC Number:

411-950-4

EC Name:

Methyl (R)-2-(4-hydroxyphenoxy)propionate

Cas Number:

96562-58-2

Molecular formula:

C10H12O4

IUPAC Name:

methyl (2R)-2-(4-hydroxyphenoxy)propanoate

Test material form:

solid: flakes

Details on test material:

- Appearance: White flakes
- Storage conditions of test material: At room temperature and protected from light

Method

Species / strain

Species / strain / cell type:

lymphocytes: cultured human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Two healthy donors; collected into heparinised sterile tubes
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood
- Modal number of chromosomes: Stable karyotype with 46 chromosomes
- Normal (negative control) cell cycle time: Average cell cycle time of 12-14 hours

MEDIA USED
- Type and identity of media: To prepare each culture, 0.5 mL of heparinised whole blood was added to 5 mL of RPMI 1640 medium containing 20 % foetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and phytohaemagglutinin (PHA: a mitogen to stimulate the lymphocytes to divide). The cultures were then placed at 37 °C for 48 hours.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Experiments without S9 mix: 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 μg/mL
- Experiments with S9 mix: 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 μg/mL for the first experiment and 156.25, 312.5, 625, 1250, 2500 and 3750 μg/mL for the second experiment

In the culture medium, the dose-level of 5000 μg/mL showed no precipitate. At this dose-level, the pH was about 7.4 (as for vehicle control) and the osmolality equal to 416 mOsm/kg H2O (459 mOsm/kg H2O for the vehicle control).
This dose-level was therefore selected as the highest dose-level for treatment in the first experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test material was freely soluble in the vehicle (DMSO) at 1000 mg/mL
The test material was dissolved in the vehicle at a concentration of 500 mg/mL for the first experiment and 375 mg/mL for the second experiment. The preparations were made immediately before use.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
In case of treatment with S9 mix, the final concentration of S9 fraction in the culture medium was 1.5 % (i.e. 15 % S9 mix). The treatment volume was 55 μL/5.5 mL culture medium.

- First experiment
Lymphocyte cultures were exposed to the test or control materials, both in the absence and presence of S9 mix, for 3 hours then rinsed. One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. Harvest time was 20 hours from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.
As this experiment gave clear positive results without S9 mix and equivocal results with S9 mix, an additional experiment, with S9 mix, was performed.

- Second experiment
Cells were exposed to the test or control materials for 3 hours and then rinsed. One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. Harvest times were 20 hours and 44 hours from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later.

NUMBER OF REPLICATIONS: Duplicate cultures per dose level

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Analysis of 200 metaphases/dose-level (with 44 to 46 chromosomes) was made, with 100 metaphases/culture whenever possible. Only 50 metaphases/culture were analysed when at least 10 % cells with structural chromosome aberrations were observed. The following structural aberrations were recorded for each metaphase: gaps, chromatid and chromosome breaks and exchanges and others (multiple aberrations and pulverisations).

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity of the test material was evaluated using the mitotic index (number of cells in mitosis/1000 cells examined), which indicates whether a material induces mitotic inhibition.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

Evaluation criteria:

A cell having one or more structural chromosome aberrations was recorded as a single cell with structural chromosome aberrations. Therefore the total frequency of cells with structural chromosome aberrations was not necessarily equivalent to the total number of aberrations.

ACCEPTANCE CRITERIA
This study was considered valid if the following criteria were met:
- The frequency of cells with structural chromosome aberrations in the vehicle controls is consistent with historical data;
- The frequency of cells with structural chromosome aberrations in the positive controls is significantly higher than that of the controls and consistent with the historical data.

EVALUATION CRITERIA
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the χ2 test, in which p = 0.05 was used as the lowest level of significance.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

EXPERIMENTS WITHOUT S9 MIX
- Precipitate: No precipitate was observed at the end of the treatment period at 5000 μg/mL
- Cytotoxicity: The test material was completely toxic at the highest dose-level (100 % decrease in the mitotic index). At the lower dose-levels, the test material was slightly to strongly toxic as shown by the 10 to 98 % decrease in the mitotic index.
- Chromosomal aberration analysis: The dose-levels selected for analysis of chromosomal aberrations were: 312.5, 625 and 1250 μg/mL; the highest dose-level of 1250 μg/mL inducing 62 % decrease in the mitotic index. A clear and significant increase in the frequency of cells with chromosomal aberrations, when compared to vehicle control culture, was noted at 625 and 1250 μg/mL (frequencies of 10 % with p<0.001 and 14.7 % with p<0.001, respectively).
As this first experiment was clearly positive, no other experiment without S9 mix was performed.

EXPERIMENTS WITH S9 MIX
- Precipitate: No precipitate was observed at the end of the treatment period at 5000 μg/mL
- Cytotoxicity: In the first experiment, the test material was markedly to strongly toxic at dose-levels ≥2500 μg/mL as shown by the 48 to 90 % decrease in the mitotic index. In the second experiment, a slight to moderate toxicity was induced at almost all dose-levels: 27 to 67 % and 17 to 64 % decreases in the mitotic index at the 20- and 44-hour harvest times, respectively.
- Chromosomal aberration analysis:
Analysis of metaphases was performed at the following dose-levels:
1250, 2500 and 3750 μg/mL for the 20 hour harvest time in both experiments
2500 μg/mL for the 44-hour harvest time in the second experiment
In the first experiment, a significant increase (p<0.05) in the frequency of cells with chromosomal aberrations was noted at 1250 μg/mL (3 % compared to 0 % for the vehicle control culture) but not at the higher dose-levels.
In the second experiment, at the 20-hour harvest time, a dose-related but not significant increase in the frequency of cells with chromosomal aberrations, when compared to vehicle control cultures, was noted at 2500 and 3750 μg/mL (frequencies of 3 and 5 %, respectively). These increases were non statistically significant but the frequency of cells with chromosomal aberrations was out of the vehicle historical data and the non-significance was attributed to the high vehicle control frequency observed at the 20 hour-harvest time.
At the 44-hour harvest time, a significant increase in the frequency of cells with chromosomal aberrations was noted at 2500 μg/mL (3 % compared to 0 % for vehicle control).

VALIDITY
The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls remained consistent with acceptance criteria. The study was therefore considered valid.

Any other information on results incl. tables

Table 1: Mitotic Index

Experiment		Dose Level (µg/mL)
		0	78.125	156.25	312.5	625	1250	2500	3750	5000	Positive control
1 without S9 (3 h treatment, 20 h harvest)	Mean Mitotic Index (%)	8.10	6.50	7.25	7.00	5.50	3.10	0.30	0.20	0.00	3.20
	% of the Control	100	80	90	86	68	38	4	2	0	40
1 with S9 (3 h treatment, 20 h harvest)	Mean Mitotic Index (%)	6.30	5.05	7.25	5.10	6.45	6.70	3.15	3.25	0.65	2.75
	% of the Control	100	80	115	81	102	106	50	52	10	44
2 with S9 (3 h treatment, 20 h harvest)	Mean Mitotic Index (%)	4.10	-	2.45	3.00	4.20	3.90	1.35	2.45	-	0.45
	% of the Control	100	-	60	73	102	95	33	60	-	11
2 with S9 (3 h treatment, 44 h harvest)	Mean Mitotic Index (%)	2.95	-	2.45	2.20	2.30	3.10	1.30	1.05	-	-
	% of the Control	100	-	83	75	78	105	44	36	-	-

 

Table 2: Summary of Chromosome Aberrations

Doses (µg/mL)	No. Cells Scored	Numerical Aberrations	Structural Aberrations		% Cells with Structural Aberrations
			Plus Gaps	Minus Gaps	Plus Gaps	Minus Gaps
Experiment 1 without S9 (3 h treatment, 20 h harvest)
0	200	0	3	3	1.5	1.5
312.5	150	0	8	8	5.3	5.3
625	150	1	19	15	12.0	10.0***
1250	150	0	27	25	14.7	14.7***
MMC 3	100	0	127	124	67.0	67.0***
Experiment 1 with S9 (3 h treatment, 20 h harvest)
0	200	0	0	0	0.0	0.0
1250	200	3	6	6	3.0	3.0*
2500	200	2	6	6	2.5	2.5
3750	200	2	3	3	1.5	1.5
CPA 50	100	0	73	67	41.0	40.0***
Experiment 2 with S9 (3 h treatment, 20 h harvest)
0	200	1	9	6	3.5	3.0
1250	200	3	4	0	2.0	0.0
2500	200	2	12	7	5.5	3.0
3750	200	4	26	14	11.0	5.5
CPA 50	100	0	111	88	59.0	53.0***
Experiment 2 with S9 (3 h treatment, 44 h harvest)
0	200	1	1	0	0.5	0.0
2500	200	0	9	6	4.5	3.0*

Statistical analysis χ2 test: *p<0.05; ***p<0.001

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material induced chromosome aberrations in cultured human lymphocytes with and without metabolic activation.

Executive summary:

The potential of the test material to induce chromosomal aberrations was investigated in accordance with the standardised guidelines OECD 473 and EU method B.10 under GLP conditions.

The test material was tested in two independent experiments, both with and without a metabolic activation system (S9 mix). Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used for the first experiment and dose-levels for scoring of chromosomal aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index. For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemagglutinin) and incubated at 37 °C in a humidified atmosphere of 5 % CO2/ 95 % air, for 48 hours. The test material was dissolved in DMSO. Without S9 mix 3 μg/mL of mitomycin C was used as the positive control and with S9 mix 50 μg/mL of cyclophosphamide was used.

In the culture medium, the dose-level of 5000 μg/mL showed no precipitate. At this dose-level, the pH and the osmolality were equivalent to those of the vehicle control.

In experiments without S9 mix, the treatment-levels were: 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 μg/mL. In the experiments with S9 mix, the treatment-levels were: 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 μg/mL for the first experiment, and 156.25, 312.5, 625, 1250, 2500 and 3750 μg/mL for the second experiment

For the first experiment, lymphocyte cultures were exposed to the test or control materials, with or without S9 mix, for three hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. As this experiment gave clear positive results without S9 mix and equivocal results with S9 mix, an additional experiment with S9 mix, was performed.

For the second experiment, cells were exposed to the test or control materials for three hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. One and a half hours before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis.

After hypotonic treatment the cells were fixed, spread on glass slides and stained.

In experiments without S9 mix, no precipitate was observed at the end of the treatment period at 5000 µg/mL. The test material was completely toxic at the highest dose-level. At the lower dose-levels, the test material was slightly to strongly toxic. The dose-levels selected for analysis of chromosomal aberrations were: 312.5, 625 and 1250 μg/mL. A clear and significant increase in the frequency of cells with chromosomal aberrations, when compared to vehicle control culture, was noted at 625 and 1250 μg/mL. As this first experiment was clearly positive, no other experiment without S9 mix was performed.

In experiments with S9 mix, no precipitate was observed at the end of the treatment period at 5000 μg/mL. In the first experiment, the test material was markedly to strongly toxic at dose-levels ≥2500 µg/mL. In the second experiment, a slight to moderate toxicity was induced at all dose-levels, but without clear evidence of a dose-relationship. Analysis of metaphases was performed at the following dose-levels: 1250, 2500 and 3750 μg/mL for the 20 hour harvest time in both experiments, and 2500 μg/mL for the 44-hour harvest time in the second experiment.

In the first experiment, a significant increase (p<0.05) in the frequency of cells with chromosomal aberrations was noted at 1250 μg/mL but not at the higher dose-levels. In the second experiment, at the 20-hour harvest time, a dose-related but not significant increase in the frequency of cells with chromosomal aberrations when compared to vehicle control cultures was noted at 2500 and 3750 μg/mL but the frequency of cells with chromosomal aberrations was out of the vehicle historical data and the non-significance was attributed to the high vehicle control frequency observed at the 20 hour-harvest time. At the 44-hour harvest time, a significant increase in the frequency of cells with chromosomal aberrations was noted at 2500 μg/mL.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls remained consistent with acceptance criteria. The study was therefore considered valid.

Under the conditions of this study, the test material induced chromosome aberrations in cultured human lymphocytes with and without metabolic activation.