Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2, 1992 - September 9, 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1982
Qualifier:
according to guideline
Guideline:
other: EEC Annex V Committee (EEC 1984)
Qualifier:
according to guideline
Guideline:
other: JEPA/MITI joint directive (JEPA,MOHW,MITI 1987)
Qualifier:
according to guideline
Guideline:
other: Guideline of the JMOHW
Version / remarks:
JMOHW 1989
Qualifier:
according to guideline
Guideline:
other: US EPA TSCA
Version / remarks:
1987
GLP compliance:
yes
Type of assay:
other: micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
2-Ethylhexyl (R)-2-(2-methyl-4chlorophenoxy)propionate
EC Number:
630-324-3
Cas Number:
861229-15-4
Molecular formula:
C18H27ClO3
IUPAC Name:
2-Ethylhexyl (R)-2-(2-methyl-4chlorophenoxy)propionate
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: 5007
- sample No.:1505E
- Expiration date of the lot/batch: March 1993

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4°C in the dark

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River U.K. Limited, Margate,Kent, England
- Age at study initiation: approximately 35 days
- Weight at study initiation: 22-24 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: plastic disposable cages, sexes separated
- Diet: ad libitum, pelleted Biosure LAD 1 rodent diet
- Water: ad libitum, tap water
- Acclimation period:11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%):
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12 h/ 12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: methylcellulose
- Amount of vehicle (if gavage or dermal): 1%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspensions/emulsions of MCPP-P 2-EHE were prepared in aquous 1 % methylcellulose on the morning of the test.
Duration of treatment / exposure:
24 h, 48 h and 72 h
Frequency of treatment:
single
Doses / concentrationsopen allclose all
Dose / conc.:
320 mg/kg bw (total dose)
Remarks:
=16 mg/mL test item in suspension, preliminary test
Dose / conc.:
800 mg/kg bw (total dose)
Remarks:
=40 mg/mL test item in suspension, preliminary test
Dose / conc.:
1 600 mg/kg bw (total dose)
Remarks:
=80 mg/mL test item in suspension, preliminary test (Phase II)
Dose / conc.:
2 000 mg/kg bw (total dose)
Remarks:
=100 mg/mL test item in suspension, preliminary test
Dose / conc.:
2 500 mg/kg bw (total dose)
Remarks:
=125 mg/mL test item in suspension, preliminary test (Phase II)
Dose / conc.:
3 128 mg/kg bw (total dose)
Remarks:
=156.4 mg/mL test item in suspension, preliminary test (Phase II)
Dose / conc.:
5 000 mg/kg bw (total dose)
Remarks:
=250 mg/mL test item in suspension, preliminary test
Dose / conc.:
782 mg/kg bw (total dose)
Remarks:
=39.1 mg/mL test item in suspension, micronucleus test
Dose / conc.:
1 564 mg/kg bw (total dose)
Remarks:
=78.2 mg/mL test item in suspension, micronucleus test
Dose / conc.:
3 128 mg/kg bw (total dose)
Remarks:
=156.4 mg/mL test item in suspension, micronucleus test
No. of animals per sex per dose:
15 per sex and dose
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Route of administration: oral, gavage
- Doses / concentrations: prepared as a solution in 0.9% saline at a concentration of 0.6 mg/mL (dose: 12 mg/kg bw)

Examinations

Tissues and cell types examined:
both femurs dissected and bone marrow used for smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on resulted from preliminary toxicity test

SAMPLING TIMES: 5 males and 5 females from dose group and negative control sacrificed 24, 48 and 72 h after dosing; positive control group 24 h after dosing

DETAILS OF SLIDE PREPARATION: direct bone marrow smear was made onto slide containing a drop of calf serum, one smear from each femur, after air-drying smears were stained and fixed in methanol (>10 min). The smears were air-dried and stained for 10 min in 10% Giemsa (prepared by 1:9 dilution of Gurr´s improved R66 Giemsa with distilled water). Following rinsing in distilled water and differentiation in buffered distilled water (pH 6.8), the smears were air-dried and mounted with coverslips using DPX.

METHOD OF ANALYSIS: The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal.

Evaluation criteria:
Micronuclei are identified by the following criteria:
(i) large enough to discern morphological characteristics
(ii) should possess a generally rounded shape with a clearly defined outline
(iii) should be deeply stained and similar in colour to the nuclei of other cells-not black
(iv) should lie in the same focal plane as the cell
(v) lack internal structure i.e. they are pyknotic
(vi) there should be no micronucleus-line debris in the area surrounding the cell
The proportion of polychromatic erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated normochromatic erythrocytes observed during this assessment was also kept as recommended by Schmid (Schmid 1976). A positive response is normally indicated by a substantial, dose-related and statistically significant increase of micronucleated polychromatic erythrocytes compared to the incidence for the concurrent vehicle control group.
Statistics:
Non- parametric statistical methods, based on rank, are chosen for analysis of results. For comparison of an individual treated group with a concurrent control group, Wilcoxon´s sum of ranks test was used. For multiple group comparisons Kruskal-Wallis´ version of this test was used. Jonckheere´s test for trend was used to analyze for dose-effect relationships.

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male/female
Genotoxicity:
negative
Remarks:
at 24 h and 72 h sampling time
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mitomycin C caused large, highly significant increases in the frequency of mirconucleated polychromatic erythrocytes
Key result
Sex:
male/female
Genotoxicity:
negative
Remarks:
at 48 h sampling time after re-examination of slides
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mitomycin C caused large, highly significant increases in the frequency of mirconucleated polychromatic erythrocytes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 320, 800, 2000, 5000 mg/kg bw (Phase I); 1600, 2000, 2500, 3128 mg/kg bw (Phase II)
- Clinical signs of toxicity in test animals:
phase I: after dosing piloerection was observed in all animals; at 2000 mg/kg animals showed in addition hunched posture and lethargy until approximatedy 24 h after dosing; at 5000 mg/kg bw mortality occured (3 animals out of 4 died) and animals showed increased respiratory rate, piloerection, hunched posture and lethargy
phase II: no mortality occured, moderate piloerection was observed in all animals directly after dosing, at 2500 and 3128 mg/kg bw animals showed lethargy, hunched posture, twitching, staggering gait, ptosis and increased respiratory rate
- Other: 3128 mg/kg bw was the maximum tolerated dose and chosen as highest dose in the micronulceus test

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei:
MCPP-p 2EHE did neither cause any substantial increases in micronucleated polychromatic erythrocyte counts (mnp) nor of micronucleated normochromatic erythrocytes (mnn) at any sampling time. The test item failed to cause any significant decreases in the proportion of polychromatic erythrocytes.

OTHER
- Chemical analysis by high performance liquid chromatography using ultraviolet detection proved the stability and homogeneity of MCPP-p 2EHE in the vehicle. In addition, it was confirmed that the dose formulations were accurately prepared and the mean result was within +4% of the nominal concentration.

Any other information on results incl. tables

Table 1 Summary of results - group totals/means for the entire experiment and results of statistical analysis

Sampling time

Treatment

Dose (mg/kg bw)

p/p + n (mean %)C

Incidence mnp (mean %)A

Incidence mnn (total %)

24 hours

Vehicle control

 

 

MCPP-P 2-EHE

 

 

Mitomycin C

-

 

782

1564

3128

 

12

52

 

51 ns

49 ns

49 ns

 

40 **

0.03

 

0.10 ns

0.07 ns

0.08 ns

 

4.66 **

0.00

 

0.04

0.00

0.00

 

0.09

48 hours

Vehicle control

 

 

MCPP-P 2-EHE

-

 

782

1564

3128

 

50

 

51 ns

51 ns

43 ns

0.03

 

0.09 ns

0.10 ns

 0.17 * (j)

0.04

 

0.00

0.04

0.07

48 hours

re-examination

 

 

 

24 hours

re-examination

Vehicle control

 

 

MCPP-P 2-EHE

 

  

 Mitomycin CB

-

 

782

1564

3128

 

12

not examined

0.01

 

0.13 ns

0.14 ns

   0.13 ns (j)

 

4.38

not examined

72 hours

Vehicle control

 

 

MCPP-P 2-EHE

-

 

782

1564

3128

 

54

 

52 ns

54 ns

55 ns

0.07

 

0.06 ns

0.04 ns

0.15 ns

0.06

 

0.00

0.02

0.04

p/(p+n) % Proportion of polychromatic (immature) erythrocytes

mnp       Number of micronucleated cells observed per 100 polychromatic erythrocytes

mnn      Number of mirconucleated cells observed per 100 normochromatic erythrocytes (calculated total = number of mnn observed in the group / number of normochromatic erythrocytes examined X 100%)

A            Results of statistical analysis using Kruskal-Wallis´, Jonckheere´s and Wilcoxon´s test as appropriate:

               ns (P > 0.01), * (P< 0.01), ** P< (0.001) one-sided probabilities; j = result using Jonckheere´s test for trend only

B            Positive control slides from the 24 hour time point were combined with slides from the 48 hour time point for examination by second slide reader

C            Small apparent errors of ± 1% are due to rounding of individual values for presentation in tables

Applicant's summary and conclusion

Conclusions:
MCPP-P 2-EHE did not show any evidence of chromosome-damaging activity in the in vivo micronucleus assay.
Executive summary:

An in vivo micronucleus test according to OECD TG 474 (Version 1982) and GLP was employed in male and female CD-1 mice to investigate a possible clastogenic or aneugenic effect in bone-marrow erythroblasts. Mice were administered by gavage single dosages of 782, 1564 or 3128 mg/kg bw. The negative control group received the vehicle (aqueous 1% methylcellulose) and the positive control group was treated with mitomycin C at 12 mg/kg bw. Bone marrow smears from five males and five females were obtained at three sampling times after dosing (24 h, 48 h and 72 h). One smear of each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. In addition, the proportion of polychromatic immature erythrocytes was assessed and the incidence of micronucleated normochromatic erythrocytes was recorded. Three male and four females died after treatment with the highest dose of MCPP-P 2-EHE in the micronucleus test, with non of these animals showing signs of misdosing.

At 24 h and 72 h, mice treated with MCPP-P 2-EHE did not show any significant increase in the frequency of micronucleated polychromatic erythrocytes. A significant increase was observed after 48 h. This increase was not confirmed in the re-examination of the slides and since all the original individual and mean values fell within the laboratory historical negative control range, it was concluded that the original increase was not treatment-related. The positive control mitomycin C induced a significant increase of micronucleated polychromatic erythrocytes and a decrease in the proportion of polychromatic erythrocytes. The negative control did not induce an increased frequency of micronucleated polychromatic erythrocytes. Therefore, MCPP-P 2-EHE has not shown any evidence of chromosome-damaging activity in the in vivo micronucleus assay.