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Diss Factsheets

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Octyl (R)-2-(4-chloro-2-methylphenoxy)propionate
EC Number:
EC Name:
Octyl (R)-2-(4-chloro-2-methylphenoxy)propionate
Cas Number:
Molecular formula:
octyl (2R)-2-(4-chloro-2-methylphenoxy)propanoate
Constituent 2
Reference substance name:
Mecoprop-P n-octyl ester
Mecoprop-P n-octyl ester
Constituent 3
Reference substance name:
Preventol B5
Preventol B5
Constituent 4
Reference substance name:
R-(+)-2-(4-chloro-2-methylphenoxy)-propionic acid, octyl ester
R-(+)-2-(4-chloro-2-methylphenoxy)-propionic acid, octyl ester
Details on test material:
- Stability under test conditions: The stability of teh test substance in the formulation was cofirmed for at least 2 hours at room temperature.

Test animals

Details on test animals or test system and environmental conditions:
- Strain: Crl:/ NMRI BR
- Source: Charles River, Germany
- Age at study initiation: approx. 6-12 weeks of age
- Weight at study initiation: males 36-38 g, females 28-34 g
- Housing: singly in Type II cages
- Diet and water: ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 22
- Humidity (%): 40-55
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The formulations were prepared on the day of testing and used within the time range for which stability has been analytically verified. The administration volume used for vehicle, positive control and test compound treated animals was 10mL/kg.

Groups of male and female mice were administered twice intraperitoneally with an interval of 24 hours with the test substance at 500, 1000, or 2000 mg/kg or solvent, whereas the positive control was administered only once. Animals were sacrificed 24 hours after last treatment and bone marrow smears were prepared.
Duration of treatment / exposure:
48 hours; animals were dosed twice with an interval of 24 hours, 24 hours after the second administration animals were sacrificed.
Frequency of treatment:
twice, sparated by 24 hours
Post exposure period:
24 hours after the second administration animals were sacrificed.
Doses / concentrations
Doses / Concentrations:
500, 1000, and 2000 mg/kg
actual ingested
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (dissolved in saline solution)


Tissues and cell types examined:
Bone marrow smears of at least one intact femur from each sacrificed animal was prepared 24 hours after the second administration. After staining of the preparations (May-Grünwald), normally 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes occurring per 2000 polychromatic erythrocytes were also recorded. In addition the number of normochromatic erythrocytes showing micronuclei was also established.
Details of tissue and slide preparation:
The selection of the test item doses was based on a pilot test. For this purpose three males and three females received two intraperitoneal injections separated by 24 hours at a dose of 2000 mg/kg. Based on these findings, 2000 mg/kg was chosen as high dose for the main test, as also recommended in the guideline.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Animals were dosed twice with an interval of 24 hours. The animals were sacrificed and bone marrow smears prepared 24 hours after the second administration.

Schmid's method was used to produce the smears (Schmid, W., The micronucleus-test for cytogenetic analysis. In: Holländer, A. (ed.), Chemical Mutagens, Principles and Methods for their Detection, Plenum Press New York 1976; Vol. 4:31-53).

Coded slides were evaluated using a light microscope at a magnification of about 1000x. Normally, 2000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. The number of normochromatic erythrocytes per 2000 polychromatic ones was noted. In addition the number of normochromatic erythrocytes showing micronuclei was also established.
Evaluation criteria:
A test was considered positive if there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the solvent control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of historical solvent controls
The comparisons of dose and control groups were performed using the nonparametric Dunn’s test and a quasi-binominal model with a log-link function to estimate relative risks and corresponding one-sided confidence intervals. In addition, the assay sensitivity was verified using a nonparametric rank-based one-sided Wilcoxon test. Moreover, historical reference intervals were calculated or data ranges were presented, respectively. A variation was considered statistically significant if its error probability was below 5%. In addition, standard deviations (SD) were calculated for all the means.

Results and discussion

Test results
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
After two intraperitoneal administrations the animals showed dose dependently the following compound-related symptoms: loss of weight, roughened fur, spasm, apathy, and twitching. Symptoms were recorded until sacrifice. These symptoms demonstrate relevant systemic exposure of males and females to the test item. There was no substance-induced mortality.
No symptoms were recorded for the control groups (solvent and positive control). No animals died in these groups.

The ratio of polychromatic to total erythrocytes in males and females was not altered by the treatment with the test item or the positive control.

No biologically relevant or statistically significant variations existed for males and females between the solvent control and the groups treated intraperitoneally with the test item, with respect to the incidence of micronucleated polychromatic erythrocytes. Similarly, there was no biologically relevant or statistically significant variation between the solvent control and the test item groups in the number of micronucleated normochromatic erythrocytes.
The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei in comparison to the solvent control.

Applicant's summary and conclusion

Executive summary:

The micronucleus test according to OECD TG 474 was employed to investigate Mecoprop-P n-octyl ester in male and female mice for a possible clastogenic or aneugenic effect in bone-marrow erythroblasts. The known clastogen cyclophosphamide, served as positive control.

Male and female mice received two intraperitoneal administrations at doses of 0 (vehicle control), 500, 1000 or 2000 mg/kg, each separated by 24 hours. Males and females of the positive control received a single intraperitoneal treatment with 20 mg/kg cyclophosphamide. The femoral marrow of all groups was prepared 24 hours after the last administration.

Males and females treated twice with the test item in doses up to 2000 mg/kg showed symptoms of toxicity after administration, starting at 500 mg/kg. These symptoms demonstrate relevant systemic exposure of males and females to the test item. However, all males and females survived until the end of the test.

There was no altered ratio between polychromatic and total erythrocytes.

After two intraperitoneal treatments of males and females with doses up to 2000 mg/kg no indications of a mutagenic effect of the test item were found. Cyclophosphamide, the positive control, had a clear mutagenic effect, as shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The ratio of polychromatic to total erythrocytes was not altered.

In conclusion, there was no indication of a mutagenic effect of Mecoprop-P n-octyl ester in the micronucleus test on male and female mice when administered intraperitoneally.