Registration Dossier

Diss Factsheets

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 December 1986 to 09 February 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report date:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: mouse micronucleus

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl 4-(dimethylamino)benzoate
EC Number:
244-289-3
EC Name:
2-ethylhexyl 4-(dimethylamino)benzoate
Cas Number:
21245-02-3
Molecular formula:
C17H27NO2
IUPAC Name:
2-ethylhexyl 4-(dimethylamino)benzoate
Test material form:
liquid
Details on test material:
- Appearance: Oily liquid
- Storage conditions of test material: Room temperature in the container received from the sponsor (amber glass bottle)

Test animals

Species:
mouse
Strain:
CD-1
Details on species / strain selection:
Mice have historically been used in the Micronucleus test and have been shown to exhibit micronuclei indicative of chromosome breakage or lagging chromosomes. Intraperitoneal administration was chosen to maximise bioavailability of the test material.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 11 weeks old
- Weight at study initiation: Males: 35 to 44 g; Females: 30 to 35 g.
- Assigned to test groups randomly: Yes using a random number table
- Housing: 5 per cage according to sex and dose group in stainless steel wire mesh cages
- Water: Tap water ad libitum
- Acclimation period: 33 days

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Lot No: 55F-0601 (Sigma Chemical Company)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
-The test material was weighed and diluted in corn oil. A good solution was obtained and maintained at all dose levels. Dosing solutions were prepared fresh and dosed within two hours of preparation. There was no apparent change in the physical state of the test material during the assay.
- Dose volume: 10 mL/kg
Duration of treatment / exposure:
The dosing solutions were administered in a single intraperitoneal dose.
Frequency of treatment:
Once
Post exposure period:
Groups were sacrificed 30, 48 and 72 hours after exposure.
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals per sex per group
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: triethylenemelamine (TEM)
- Preparation: Dissolved in 0.9 % saline
- Route of administration: Intraperitoneal
- Doses / concentrations: 0.5 mg/kg/bw (dose volume 10 mL/kg)

Examinations

Tissues and cell types examined:
Slides were prepared from the bone marrow of the femora and stained. The numbers of micronucleated polychromatic erythrocytes and normochromatic erythrocytes were recorded.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose selection was determined as the result of the range finding test conducted at doses of 500, 1500, 3000, 4000 and 5000 mg/kg of body weight.

TREATMENT AND SAMPLING TIMES: A single dose was administered with sacrifice after 30, 48 or 72 hours.

DETAILS OF SLIDE PREPARATION: The femora were opened carefully at the proximal end with scissors until a small opening to the marrow canal became visible. A 1 mL tuberculin syringe filled with approximately 0.2 mL foetal bovine serum was inserted into the bone and the bone marrow was gently flushed (to assure maximum dispersion) into 1.0 mL of foetal bovine serum in a 3 mL conical centrifuge tube. The femora were flushed with foetal bovine serum until all the marrow was out and the bone appeared almost transparent. If necessary the distal ends were opened and flushed. The suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed leaving a small amount of foetal bovine serum with the remaining cell button. The button was mixed with a Pasteur pipette to assure a homogenous mixture. A small drop of the mixture was immediately placed near the frosted end of a glass microscope slide previously cleaned in absolute ethanol and pulled behind a clean slide at a 45 ° angle. The slides were quick dried on a slide warmer at approximately 56 °C. Following preparation of the smears, they were dipped in absolute methanol and allowed to air dry.

STAINING: The slides were stained according to the following procedure: Fix in absolute methanol for 5 minutes and air dry. Remove metallic film from surface of Giemsa working solution (5 % Giemsa in pH 6.8 phosphate buffer solution) using a paper towel. Stain for 20 minutes in Giemsa working solution. Rinse twice. The first rinse is in deionised water adjusted to pH 4.0 to 4.5 and the second rinse in deionised water adjusted to pH 7.0. Clean back of slide with absolute methanol. Dry on 56 °C slide warmer. Clear in xylene. Mount in Permount with cover glass.

METHOD OF ANALYSIS: The slides were screened for good preparation, i.e. well spread, undamaged, perfectly stained. One thousand (1000) PCE per animal were counted for the presence of micronuclei. The data were expressed as the number of micronucleated PCE versus total normal PCE in 1000 total PCE per animal. A total of 1000 polychromatic and normochromatic erythrocytes (NCE) was also counted per animal. These data were expressed as the ratio of polychromatic erythrocytes to normochromatic erythrocytes.
Evaluation criteria:
Micronuclei are uniform, darkly stained, typically round bodies in the cytoplasm of PCE. Occasionally, micronuclei appear almond, or tear drop shaped. Inclusions in PCEs which are reflective, improperly shaped or stained, or which are not in the focal plane of the cell are judged to be artefacts and are not scored as micronuclei. Cells containing more than one micronucleus are only scored as micronucleated PCE.
Assessment of a test material as positive is based upon its ability to produce a statistically significant increase in the number of micronucleated polychromatic erythrocytes (PCE) as compared to the vehicle control.

CRITERIA FOR A VALID TEST
If the spontaneous rate of micronuclei in the polychromatic erythrocytes is less than 0.5 % and the positive control is statistically greater (p≤0.05) than the spontaneous and at least seven animals per group survived the treatment, the results are deemed acceptable.
Statistics:
A one-tailed t-test was used to make pairwise comparisons between each treatment group with the vehicle control for statistically significant increases in the number of micronucleated PCE. The PCE/NCE ratio was also calculated based on 1000 erythrocytes for each animal. The proportion of PCE per 1000 erythrocytes per animal was evaluated by pairwise t-tests after an arcsin transformation was performed. Statistical significance was judged at the P≤0.05 and P≤0.01 levels.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Some animals exhibited decreased body tone and/or abdominal gait and/or body drop
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1500, 3000, 4000 and 5000 mg/kg/bw
- Clinical signs of toxicity in test animals: No signs were observed at any dose level immediately after administration. At 500 mg/kg/bw no signs were observed at 4 hours post-dose and only one male had a decrease in body tone at 24, 48 and 72 hours. Signs were observed at all other dose levels. One or more animals had a decrease in body tone at 4, 24, 48 and 72 hours in all test groups. Additional signs included abnormal gait at 72 hours in one female in each of the 1500, 4000 and 5000 mg/kg group. Body drop was observed at 72 hours in one male in the 3000 mg/kg group, in one male at 4000 mg/kg and in two males in the 5000 mg/kg group. No other signs were observed and no mortality occurred in the dose range study.

RESULTS OF DEFINITIVE STUDY
No statistically significant increases in the incidence of micronucleated PCE were detected in animals administered the test material, at any of the sacrifice times evaluated. No statistically significant differences were observed in the PCE/NCE ratio in any group of animals administered the test material.
Animals treated with the positive control gave a statistically significant increase in the incidence of micronucleated PCE and a statistically significant depression of the PCE/NCE ratio.
The study fulfils the criteria of a valid test.

PHARMACOTOXIC EFFECTS OF TREATMENT IN DEFINITIVE STUDY
Immediately after dose administration, one male dosed with the test material exhibited decreased body tone. At 4, 24, 48 and 72 hours several animals exhibited decreased body tone and/or abdominal gait. Body drop was observed in a few animals at 48 hours and in two animals at 72 hours. No mortality occurred in this study.
No signs were observed in the animals administered the positive or negative controls.

Any other information on results incl. tables

Table 1: Micronucleated PCE per 1000 Polychromatic erythrocytes per animal

Animal number

Sex

Controls

Test material

Corn oil (48 hours)

TEM (30 hours)

5000 mg/kg

10 mL/kg

0.5 mg/kg

30 hours

48 hours

72 hours

1

M

0

48

0

0

0

2

M

0

46

0

0

0

3

M

0

97

1

1

0

4

M

1

52

0

0

2

5

M

0

42

0

0

0

6

F

0

47

0

0

0

7

F

0

65

1

0

0

8

F

0

40

0

0

1

9

F

0

74

0

1

0

10

F

0

45

2

0

0

Mean ± S.D.

0.10 ± 0.32

55.60 ± 18.01

0.40 ± 0.70

0.20 ± 0.42

0.30 ± 0.67

t value

-

9.744**

1.236

0.600

0.848

**Denotes statistical significance at P0.01

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the test material is not considered to be a clastogenic agent.
Executive summary:

An assay was conducted to evaluate the potential of the test material to induce micronuclei in mice following intraperitoneal administration using methodology equivalent to the standardised guideline OECD 474 under GLP conditions.

In a preliminary dose range finding test the test material was administered intraperitoneally at dose levels of 500, 1500, 3000, 4000 and 5000 mg/kg of bodyweight. Although some clinical signs were observed (decreased body tone, abnormal gait and body drop) the 5000 mg/kg of bodyweight dose was selected for the definitive study.

In the micronucleus test three groups of ten animals (5 males and 5 females per group) were given single doses of the test material at 5000 mg/kg in corn oil by intraperitoneal injection and sacrificed at 30, 48 or 72 hours. Similar groups (5 males and 5 females) were dosed with positive (triethylenemelamine) or vehicle (corn oil) controls and sacrificed at 30 and 48 hours respectively. Slides were prepared from the bone marrow of the femora and stained. Coded slides were scored for the number of polychromatic erythrocytes (PCE) with micronuclei in 1000 PCE per animal. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal.

No statistically significant increases in the incidence of micronucleated PCE were detected in animals administered the test material, at any of the sacrifice times evaluated. No statistically significant differences were observed in the PCE/NCE ratio in any group of animals administered the test material.

Animals treated with the positive control gave a statistically significant increase in the incidence of micronucleated PCE and a statistically significant depression of the PCE/NCE ratio.The study fulfils the criteria of a valid test.

The test material was negative in the micronucleus test at 5000 mg/kg of bodyweight. These findings are based upon the inability of the test material to produce a significant increase in the incidence of micronuclei per 1000 polychromatic erythrocytes per animal in the treated groups versus the vehicle control group. 

Under the conditions of the study, the test material is not considered to be a clastogenic agent.

Categories Display