Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 11th, - March 19th, 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
The micronucleus assay was conducted using established and validated procedures (Heddle, 1973; Hayashi et al., 1994; Mavournin et al ., 1990).

- Heddle, J.A. 1973. A rapid in vivo test for chromosomal damage. Mutation Res. 18:187-190
- Hayashi, M., et al., 1994. In vivo rodent erythrocyte micronucleus assay. Mutation Res. 312: 293-304
- Mavournin, K .H., et al., 1990. The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res. 239: 29-80.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): p-Butyl-alpha-methylhydrocinnamic aldehyde
- Analytical purity: not reported
- Physical state and appearance: liquid, colourless and clear
- Lot No.: 9000349505
- Storage: in the dark, at room temperature

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Sprague Dawley, Inc., Frederick, MD
- Age at study initiation: 6-8 weeks
- Body weight range at study initiation: MN Study; 29.7 - 33.6g (male), 25.7 - 30.7 g (female), Toxicity Study: 25.0 - 28.4 g (male), 25.7 - 28.6 g (female)
- Housing: 5/sex/cage
- Diet: ad libitum (Harlan TEKLAD certified Rodent 7012C). There were no relevant contaminants in the feed
- Water: ad libitum (Washington Suburban Sanitary Commission, Potomac Plant). Met USEPA drinking water standards
- Acclimation: at least 5 days (animals were healthy at begin of study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.9
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle: corn oil
- Rationale for choice: Based on information provided by the Sponsor and because the vehicle was compatible with the test system animals.
- Application volume: 2 ml/100 g bw
Duration of treatment / exposure:
24 h and 48 h
Frequency of treatment:
single administration
Doses / concentrationsopen allclose all
Dose / conc.:
150 mg/kg bw (total dose)
Dose / conc.:
300 mg/kg bw (total dose)
Dose / conc.:
600 mg/kg bw (total dose)
No. of animals per sex per dose:
See table 1 below
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide (CP)
- Vehicle: sterile water
- Concentration tested: 2.5 mg/ml
- Route of application: ip

Examinations

Tissues and cell types examined:
Bone marrow cells were examined for micronucleated polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- Based on the findings of a preliminary toxicity assay, the highest dose in MN assay was set at approximately 75% of the LD50 of 800 mg/kg bw for male and females.

TREATMENT AND SAMPLING TIMES:
- Administration volume: 2 ml/100g bw
- Sampling time: 24 h (all doses), 48 h (control and highest dose)

DETAILS OF SLIDE PREPARATION:
- Flushing of femur: with fetal calf serum
- Centrifugation of bone marrow cells: 100 x g, 5 min, in fetal calf serum
- Fixation agent: methanol
- Staining agent: May-Gruenwald-Giemsa

METHOD OF ANALYSIS:
- Using oil immersion, 2000 polychromatic erythrocytes were scored for micronuclei
- The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes
- Micronuclei defined as; round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte
Evaluation criteria:
CRITERIA FOR POSITIVE TEST
The test article was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes is observed and one or more doses were statistically elevated relative to the vehicle control (p<=0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group is significantly elevated at one sacrifice time with no evidence of a dose-response, the assay is considered a suspect or unconfirmed positive and a repeat experiment is needed. The test article is judged negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control values are observed at any sampling time.

CRITERIA FOR VALID TEST
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p<= 0.05, Kastenbaum-Bowman Tables).
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Number of animals: 5 males/5 females
- Dose range: 300, 500, 700, or 1000 mg/kg bw/d
- Clinical signs of toxicity in test animals: lethargy and piloerection in male and female mice at 300, 500, 700 and 1000 mg/kg. Prostration and irregular breathing were observed in males and females at 700 and 1000 mg/kg, crusty eyes in males at 700 and in males and females at 1000 mg/kg and convulsions in males and females at 1000 mg/kg
- Mortality: 5/5 male mice and 5/5 female mice at 1000 mg/kg, occured within 3 days
- Solubility: The test article was soluble in corn oil at 50 mg/ml, the maximum concentration tested in the study.

RESULTS OF DEFINITIVE STUDY
- Mortality: no mortality was noted at any dose
- Clinical signs: see table 2 and 3 of result
- Genotoxicity: Systemic bioavailability was clearly demonstrated by the PCE/total erythrocyte ratio in the top dose group at 24 hours sacrifice interval (-15% or -30% of control [male; female]).

A statistically significant increase of micronucleated cells (MN) in males after 48 hours and a non-significant increase in females after 24 hours in the highest dose group (600 mg/kg bw/d) was found, the only dose showing signs of systemic toxicity in each tested animal. The statistically significant increase of micronucleated cells in males after 48 hours is not considered biologically relevant, since only one animal of this dose group had 3 MN/2000 polychromatic erythrocytes (PCE) which is well within the historical range of solvent control (0-7 MN/1000 PCE). All other animals had no more than 2 MN/2000 PCE (2,1,2 and 1 MN/2000 PCE, respectively). The statistical significance stems from the low individual MN values of the respective study control animals (1,0,0,1 and 0 MN/2000 PCE).
Furthermore, the small increase observed in the female high dose group 24 hours after treatment (mean 1.2 MN /1000 PCE), are well within the HC range of 0-7 MN/1000 PCE. Single animal data (control animals: 1,1,1,1,1 MN per 2000 PCE versus treated animals: 1,2,1,4,4 MN per 2000 PCE), show only 2 treated females with slightly increased MN numbers. The treatment group mean value reached no statistical significance. Thus, all single values obtained were within the inter-animal variability of the mouse strain used in this study type. In line with the above, no statistically significant increase and no dose-responsive increase were observed in any other test article treated group regardless of dose level, sex or bone marrow collection time. Furthermore, no significant or dose-related increase was observed in any other dose group regardless of sex or bone marrow collection time.

The sensitivity of the test system was shown since the positive control substance caused chromosomal aberrations.

Any other information on results incl. tables

Table 2: Summary of results of the MN assay after 24 h

Treatment (mg/kg bw)

Sex

Clinical signs

PCE/Total Erythrocytes Mean ± SD

Micronucleuted PCEs/ PCEs scored

0

M

-

0.48 ± 0.08

3/10000

F

-

0.50 ± 0.04

5/10000

150

M

-

0.47 ± 0.07

5/10000

F

-

0.48 ± 0.06

4/10000

300

M

Lethargy and

Piloerection

3/5

0.50 ± 0.05

4/10000

F

4/5

0.46 ± 0.06

2/10000

600

M

Lethargy and Piloerection

(15/15)

Prostration and irregular breathing (2/15)

0.40 ± 0.08

6/10000

F

0.35 ± 0.04

12/10000

CP (50 mg/kg bw)

M

-

0.44 ± 0.06

174*/10000

F

-

0.47 ± 0.13

200*/10000

* significantly increased compared to control p<= 0.05

Table 3: Summary of results of the MN assay after 48 h

Treatment (mg/kg bw)

Sex

PCE/Total Erythrocytes Mean ± SD

Micronucleuted PCEs

/ PCEs scored

0

M

0.55 ± 0.04

2/10000

F

0.57 ± 0.05

2/10000

600

M

0.50 ± 0.05

9sig/10000

F

0.52 ± 0.06

5/10000

sig:significantly increased compared to control p<= 0.05. This response is not considered biologically relevant, since only one animal had 3 MNPCE which is within the historical range of solvent control (0-7 MN/2000 PCE/animal). All other animals had no more than 2 MNPCE.

Applicant's summary and conclusion