mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

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:

03 October 1990 to 01 November 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

EC Number:

240-539-0

EC Name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Cas Number:

16484-77-8

Molecular formula:

C10H11ClO3

IUPAC Name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Test material form:

solid: flakes

Remarks:

brown hard flakes

Details on test material:

- Storage conditions: Contained in an amber glass jar; this was stored at ambient temperature in the dark.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: No determinations of homogeneity or concentration were performed on prepared suspensions of the test material.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 4 - 5 weeks old
- Weight at study initiation: Animals weighed 19.2 - 23.6 g on arrival
- Housing: Housed in single-sex groups of two or five in high density polypropylene cages with stainless steel tops
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: All animals were acclimatised for at least four days prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 21 °C
- Humidity (%): 40 - 64 % R.H
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light: 12-hour dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5 % methyl cellulose
- Justification for choice of solvent/vehicle: The test material was found to form a doseable and apparently homogeneous suspension in 0.5 % methyl cellulose.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dosing suspensions were freshly prepared in 0.5 % methyl cellulose on the day of dosing, each concentration being individually formulated, and mixed prior to use.
- The dosage-volume was 10 mL/kg.

Duration of treatment / exposure:

Single administration.

Frequency of treatment:

The animals received the test material once.

Post exposure period:

Five male and five female mice per group were scheduled to be killed 24 hours after treatment; further lots of five males and five females from groups 1 and 4 were scheduled to be killed 48 and 72 hours after treatment.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Remarks:

Main micronucleus test

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Main micronucleus test

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Main micronucleus test

No. of animals per sex per dose:

Five / sex / group

Control animals:

yes, concurrent vehicle

Positive control(s):

Chlorambucil (5 animals per sex) in aqueous 10 % ethanol.
- Route of administration: Oral gavage
- Doses / concentrations: 30 mg/kg

Examinations

Tissues and cell types examined:

Animals were killed by cervical dislocation following carbon dioxide inhalation. Femurs from each animal were rapidly dissected out and cleaned of adherent tissue. The epiphyses were cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2.5 mL foetal calf serum using a syringe and needle.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- Preliminary toxicity test
Initially, the toxicity of the test material to dividing bone marrow erythrocytes was examined: Slide evaluation was restricted to determination of numbers of polychromatic and mature erythrocytes and calculation of the ratio of polychromatic to mature cells for each animal, and for each group. The selection of levels tested were based on an oral LD50 of approximately 800 mg/kg in the rat. Dosing was by the oral route, on one occasion and at a dosage-volume of 10 mL/kg. All animals were killed 72 hours after treatment. The experimental design was as follows: 62.5 125, 250 and 500 mg/kg bw (two males and two females per group).


DETAILS OF SLIDE PREPARATION: The recovered cells were centrifuged at 1 000 rpm for five minutes. The bulk of the supernatant fluid was discarded and the cell pellet re-suspended in the remaining fluid. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test or main micronucleus test respectively) prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually, using the Schmid (May-Grunwald and Giemsa) staining technique.
When air-dried, permanent mounts were made using DPX mountant, after clearing for five minutes in xylene.


METHOD OF ANALYSIS:
- Preliminary toxicity test
The slides were examined under the light microscope, and regions judged to be of adequate technical quality to permit scoring were selected under low magnification. At high magnification (x 1 000, oil immersion) a total of at least 2 000 erythrocytes per animal were examined. Each erythrocyte scored was classed as polychromatic or mature: Polychromatic cells stain blue/pink and the older cells stain red/pink. At least 1 000 cells of each type were scored from each animal where possible, but where there was an appreciable deviation from unity in the ratio of polychromatic to mature erythrocytes, scoring continued until a minimum of 2 000 of the predominant cell type were counted.
- Main micronucleus test
At least one slide from each animal was randomly coded by a person not subsequently involved in the scoring of the study. Care was taken to ensure that no unique slide identifications remained visible in order to eliminate bias. Slides were examined as detailed for the preliminary toxicity test, but in addition each erythrocyte scored was examined for the presence or absence of micronuclei. When examination had been completed, the data were decoded. The number of micronucleated cells per 1 000 erythrocytes was then calculated. The ratio of polychromatic to mature cells was also determined; a decrease in this may indicate inhibition of cell division following treatment, and the incidence of micronuclei in the mature cell population 24 hours after treatment reflects the pre-treatment situation, since most of these cells were produced before treatment. The frequency of micronuclei in polychromatic cells provides an index of induced genetic damage.


OTHER: Animals were inspected daily throughout the acclimatisation period and the dosing period for signs of ill-health or reaction to treatment. Any deviation from normal was recorded. All animals were weighed on the day of treatment and again immediately before termination, and bodyweights were recorded. In addition, the animals in the preliminary toxicity test were weighed immediately prior to dosing and daily thereafter until termination.

Statistics:

The frequencies of micronucleated cells per 1 000 polychromatic erythrocytes scored were subjected to statistical analysis by the Mann-Whitney procedure (Mann and Whitney, 1942). A computer-based version of this test was employed and significance was determined by reference to tabulated values of R1.
Data from males and females within each group were compared using a two-tailed test. Where there was no significant difference within the group, the sexes were pooled for further analysis. For each sampling time (24, 48 or 72 hours), each treated group was compared with concurrent vehicle controls using a one-tailed test.

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
- Clinical signs of toxicity in test animals: All animals survived to scheduled termination. Three of the mice treated at 500 mg/kg showed an unstable gait after dosing and two of these also showed a hunched posture. One male treated at 62.5 mg/kg showed a hunched posture and rales two hours after dosing and one female treated at this dosage showed piloerection. All the mice had recovered within six hours of dosing and showed no further adverse reactions to treatment.
- Evidence of cytotoxicity in tissue analysed: No evidence of toxicity to the bone marrow (as shown by a reduction in the ratio of polychromatic to mature erythrocytes) was observed for any group treated with the test material. There was no evidence of toxicity of the test material (as evidenced by depression in bone marrow proliferation) to the bone marrow of the treated mice.
- After consideration of these data, the highest test material dosage selected for the main micronucleus test was 500 mg/kg.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: Occasional incidences of slight weight loss were noted in groups treated with the test material and in the vehicle control group. In view of the low incidence and absence of a strict relationship to dosage, no major significance can be attached to these results. All ten mice given chlorambucil, the positive control agent, lost weight during the 24 hour period before termination. No real indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any group treated with the test material. A few hours after dosing, eight of the mice treated with the test material at 500 mg/kg showed adverse reactions to treatment including underactivity (5 mice), hunched posture (2) and rales (1). Two of these animals were also prone and were sacrificed in extremis approximately two hours after dosing. All of the surviving mice had recovered by the day after dosing and no further adverse reactions to treatment were observed.
- Induction of micronuclei (positive control): Chlorambucil treatment produced a range of micronucleated cells per 1 000 polychromatic erythrocytes of 44.0 - 87.7 with a mean of 71.0. Statistical analysis showed that animals treated with chlorambucil had a significantly higher frequency of micronucleated polychromatic cells than vehicle control animals (p < 0.01). This increase in chromosomal damage after exposure to a known mutagen demonstrates the sensitivity of the test system.
The recorded incidence of micronuclei per 1 000 mature erythrocytes varied between 0.0 and 3.9 throughout all groups. These findings demonstrate the normal status of the animals used in the study: In particular, the low incidence in animals killed 24 hours after treatment shows the absence of any pre-treatment abnormality in the bone marrow.
- Incidence of micronucleated cells (Test material): Among mice killed 24 hours after treatment, the mean incidence of micronucleated polychromatic erythrocytes (per 1 000 polychromatic cells scored) was 1.7 for the vehicle control group, with a range of 0.0 - 3.0. Corresponding values for animals given the test material at 20, 100 or 500 mg/kg were closely similar: 2.4, 2.0 or 1.6 with ranges of 0.0-5.9, 0.0 - 5.0 and 0.0 - 2.8 respectively. Among mice killed after 48 or 72 hours, the mean incidences of micronucleated polychromatic erythrocytes in vehicle control groups were 1.3 (range 0.0 - 2.9) and 2.3 (range 1.0 - 3.5) respectively. Corresponding values for animals given test material at 500 mg/kg were 1.0 (range 0.0 - 2.7) and 1.7 (range 0.0 - 4.0) respectively. Thus, mean values for groups treated with the test material were closely similar to mean control group values at all termination times.
Statistical analysis confirmed that there was no significant difference in the incidence of micronucleated polychromatic erythrocytes between the vehicle control group and any group treated with test material, at any termination time (p > 0.05).
- Ratio of PCE/NCE: The ratio of polychromatic to mature erythrocytes was 0.7 in the vehicle control group at 24 hours. Ratios for groups given the test material at 20, 100 or 500 mg/kg and terminated 24 hours later were 0.7, 0.7 or 0.8 respectively. Forty eight hours after treatment, the ratio for the vehicle control group was 0.8, and in animals given the test material at 500 mg/kg it was 0.7. Seventy two hours after treatment, the ratio of polychromatic to mature erythrocytes for the vehicle control group was 0.6 and in animals treated with the test material it was 0.7. Ratios for all treatment groups were therefore closely similar to those of their respective vehicle control groups. In animals treated with chlorambucil, the ratio between polychromatic and mature erythrocytes was reduced (0.5).

Applicant's summary and conclusion

Conclusions:

Under the conditions of study, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of the test material.

Executive summary:

The genotoxicity of the test material was assessed according to OECD Test Guideline 474 and in compliance with GLP.

The effect of the test material on chromosome structure in bone marrow cells was investigated following acute oral administration to mice. Chromosome damage was measured indirectly by counting micronuclei.

A preliminary toxicity test was conducted in which groups of two male and two female mice were treated with the test material at dosages of 62.5, 125, 250 and 500 mg/kg. In all cases the test material was dosed orally, suspended in aqueous 0.5 % (w/v) methyl cellulose (0.5 % MC). Three of the four mice treated at 500 mg/kg showed transient signs of reaction to treatment. No evidence of toxicity to the bone marrow (as shown by a reduction in the ratio of polychromatic to mature erythrocytes) was observed for any group treated with the test material.

In the main micronucleus test, male and female mice were given a single oral dose of the test material at 20, 100 or 500 mg/kg. Concurrent vehicle and positive control groups of mice were similarly dosed with 0.5 % MC or chlorambucil (30 mg/kg) respectively. Five males and five females from each group were scheduled to be killed 24 hours after treatment; further lots of five males and five females, given the test material at 500 mg/kg or the vehicle control, were scheduled to be killed 48 and 72 hours after treatment. Bone marrow smears on glass slides were made from each animal. These slides were then stained and prepared for examination.

A total of at least 2 000 erythrocytes per animal was then examined for the presence of micronuclei, using the light microscope. Calculated values of micronuclei per 1 000 polychromatic erythrocytes were analysed statistically using the Mann-Whitney U test. The ratio of polychromatic: mature cells was also calculated for each animal, as an indicator of gross toxicity.

No real indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any group treated with the test material. A few hours after dosing, eight of the mice treated with the test material at 500 mg/kg showed adverse reactions to treatment including underactivity (5 mice), hunched posture (2) and rales (1). Two of these animals were also prone and were sacrificed in extremis approximately two hours after dosing. All of the surviving mice had recovered by the day after dosing and no further adverse reactions to treatment were observed.

Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of the test material were similar to those in concurrent controls. This lack of treatment-related effect was apparent in both sexes, and was confirmed by statistical analysis. Statistically significant increases over controls were, however, seen in positive control group animals given chlorambucil at 30 mg/kg (p < 0.01). 

Under the conditions of study, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of the test material. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.