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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 November 1992 to 07 December 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Deviations:

yes

Remarks:

see below

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1984

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

The limit dose of 2000 mg/kg, promulgated by the OECD and subsequently ratified in 92/49/EEC after the release of the guidelines, was used.

GLP compliance:

yes

Type of assay:

other: Mouse micronucleus test

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

Details on test material:

- Appearance: Tan or brown solid
- Storage conditions: In the dark at ambient temperature

Test animals

Species:

mouse

Strain:

Swiss

Remarks:

OF1 strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 6 weeks old
- Weight at study initiation: Mean body weight of 29.2 to 33.4 g for the males and 24.4 to 30.0 g for the females
- Assigned to test groups randomly: Yes, allocated randomly to groups according to sex
- Fasting period before study: No
- Housing: The animals were housed in polycarbonate cages (33.5 x 18.7 x 13.0 cm) and each cage contained 5 mice of the same sex and group (3 for the supplementary animals). Each cage contained autoclaved sawdust.
- Diet: ad libitum pelleted diet
- Water: ad libitum access to filtered tap water contained in bottles
- Acclimation period: 5 day acclimation period

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 50 ± 20 % (relative)
- Air changes: Ventilation was provided in the form of filtered and non-recycled fresh air
- Photoperiod: 12 h/12 h light/dark cycle

IN-LIFE DATES
- From: 12 November 1992
- To: 19 November 1992

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1 % aqueous hydrogel of methylcellulose
- Justification for choice of solvent/vehicle: Not specified
- Concentration of test material in vehicle: 100 mg/mL
- Dose volume: 20 mL/kg
- Lot/batch no. of vehicle: 92126 provided by Prolabo, Paris

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was suspended in the vehicle at a concentration of 100 mg/mL and homogenised using a magnetic stirrer before and during administration. The preparations were made immediately before use.

Duration of treatment / exposure:

Animals were sacrificed either after 24 or 48 hours following treatment with the test material and vehicle control. Animals were sacrificed 24 hours after treatment with the positive control.

Frequency of treatment:

Single administration

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Two groups each consisting of 5 males and 5 females were treated with the test material at 2000 mg/kg. An additional group of 3 males and 3 females was also treated with the test material at 2000 mg/kg.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide was used as the positive control
- Route of administration: Oral gavage
- Doses / concentrations: 50 mg/kg dosed at a concentration of 2.5 mg/mL
- Preparation: The test material was dissolved in distilled water immediately before use

Examinations

Tissues and cell types examined:

Bone marrow smears prepared from the femurs of the mice

Details of tissue and slide preparation:

PREPARATION OF SMEARS
At the time of sacrifice, all the animals were sacrificed after CO2 inhalation in excess. The femurs of the mice were removed and the bone marrow eluted out using foetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with May-Grünwald-Giemsa. Two slides/animal were prepared, but only one was used for scoring.

ANALYSIS
All slides were scored under a high powered microscope (x 1000). For each animal, the micronuclei were counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Evaluation criteria:

The following criteria were used as an aid for determining a positive response:
- A statistically significant increase in the number of MPE for at least one of the sampling times when compared to the vehicle group;
- This increase should double the number of MPE for the historical data.

The results were considered negative if the above criteria were not fully met.

Statistics:

At each sampling time, the mean number of micronucleated polychromatic erythrocytes (MPE) and the PE/NE ratio from the treated groups were compared to the simultaneous vehicle groups. The intergroup comparison was performed using:
- For MPE, the chi-squared test;
- For the PE/NE ratio, the Student's "t" test, in which p = 0.05 was used as the lowest level of significance.

Biological and statistical significances were considered during the evaluation.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The administration of 2000 mg/kg induced no mortality and no clinical signs. Consequently, 2000 mg/kg, being the maximum dose specified in the guidelines, was selected for the cytogenetic study.

RESULTS OF DEFINITIVE STUDY
No clinical signs and no mortality were observed after treatment with the test material. The MPE and PE/NE ratio data are summarised Table 1. In the 2 vehicle control groups, the mean values of micronucleated polychromatic erythrocytes were within the range of the historical data. In all groups treated with the test material, the mean values of micronucleated polychromatic erythrocytes were similar to those of their respective vehicle groups at each sampling time, and no statistically significant differences were observed. Moreover, the PE/NE ratio was not statistically significantly different from that of the respective vehicle control groups. As no mortality occurred in the study, the supplementary animals were not needed and no smears were prepared for these animals.
The treatment with CPA induced a statistically significant increase (p<0.001) in the number of MPE, which demonstrated the sensitivity of this in vivo test under the experimental conditions.

Any other information on results incl. tables

Table 1: Summary of MPE and PE/NE ratio data

Group	Dose (mg/kg)	MPE/PE		PE/NE Ratio		Time of sacrifice (hours)
		Mean	SD	Mean	SD
Vehicle	-	1.5	1.0	0.6	0.2	24
Test Material	2000	2.4	1.2	0.7	0.3
Positive Control	50	43.1***	7.1	0.9	0.5
Vehicle	-	1.2	0.7	0.7	0.2	48
Test Material	2000	1.0	0.6	0.7	0.2

Five animals per sex per group

PE = polychromatic erythrocytes

NE = normochromatic erythrocytes

MPE /PE = micronucleated polychromatic erythrocytes / 1000 polychromatic erythrocytes

SD = standard deviation

***p<0.001

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test material did not induce cytogenetic damage to the bone marrow cells of mice when treated by the oral route at 2000 mg/kg in the micronucleus test.

Executive summary:

The potential for the test material to induce cytogenetic damage to the bone marrow cells of mice was investigated in accordance with the standardised guidelines OECD 474 and EU method B12 under GLP conditions.

Following a preliminary study to define the dose to be used, the animals of the treated groups received a single administration of the test material at the limit dose of 2000 mg/kg by oral gavage. Two groups each comprising 5 males and 5 females (and one additional group composed of 3 males and 3 females) were dosed. The vehicle was 1 % aqueous methylcellulose and 2 vehicle groups were also treated. The animals of the positive control group (1 group, comprising 5 males and 5 females) received a single administration of cyclophosphamide at 50 mg/kg.

The animals of the treated and vehicle control groups were sacrificed 24 and 48 hours after treatment and the animals of the positive control group were sacrificed 24 hours after treatment. Bone marrow smears were then prepared.

For each animal, the micronuclei were counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

There were no clinical signs of toxicity after treatment with the test material. At the two sampling times, the number of MPE and the PE/NE ratio in mice exposed to the test material did not differ statistically from the concurrent vehicle control values. As no mortality occurred in the study, the supplementary animals were not needed and no smears were prepared for these animals.

In the two vehicle control groups, the mean values of micronucleated polychromatic erythrocytes (MPE) were within the range of the historical data. Cyclophosphamide induced a highly significant increase (p<0.001) in MPE, indicating the sensitivity of the test system under the experimental conditions.

Under the conditions of the study, the test material did not induce cytogenetic damage to the bone marrow cells of mice when treated by the oral route at 2000 mg/kg in the micronucleus test.