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:
From April 18 to July 19, 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Basic data given. Positive control group not included, number of micronucleated immature erythrocytes for each animal not reported.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2010

Materials and methods

Principles of method if other than guideline:
According to methodology of MacGregor et al. (1990).
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Yellow liquid
- Stability under test conditions: Stable
- Storage condition of test material: Stored at or below - 20° C, protected from light, in 1-L Teflon® bottles.

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc., Germantown, NY
- Age at study initiation: 6-7 weeks
- Assigned to test groups randomly: Yes; animals were distributed randomly into groups of approximately equal initial mean body weights.
- Housing: Male animals were housed individually and females were housed 5/cage in polycarbonate cages.
- Diet: NTP-2000 irradiated wafer or pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water: Tap water (Columbus, OH, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), ad libitum
- Acclimation period: Females: 13 days; males: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 °F
- Humidity: 50 ± 15 %
- Air changes: ≥ 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES:
From: April 18, 2001 To: July 19, 2001

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amounts of test material and corn oil were placed in a glass mixing container, capped, and thoroughly mixed with a paint shaker for approximately 5 minutes. Dose formulations were prepared approximately monthly during the study.

ANALYSIS OF FORMULATION:
Test material formulations were analysed three times during the study period. Homogeneity studies of 0.2 and 120 mg/mL formulations and stability studies of the 0.2 mg/mL formulation were performed by the analytical chemistry laboratory using GC. Homogeneity was confirmed, and the 120 mg/mL dose formulation was found to be suitable for gavage. Stability was confirmed for up to 35 days for dose formulations stored in amber glass bottles with Teflon® -lined lids at – 20 °C, 5 °C, and room temperature, as well as for 3 h under simulated animal room conditions. All the formulations analysed were within 10 % of the target concentrations.

DOSE VOLUME: 10 mL/kg bw/day
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
5 days/week
Post exposure period:
No data
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
37.5 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (except vehicle control in females where 8 animals used)
Control animals:
yes, concurrent vehicle
Positive control(s):
None

Examinations

Tissues and cell types examined:
- Frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each animal per treatment group was determined.
- Percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined as a measure of bone marrow toxicity.
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES:
- At the end of the 3-month toxicity study, peripheral blood samples were obtained from male and female animals.

DETAILS OF SLIDE PREPARATION:
- Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded.

METHOD OF ANALYSIS:
- Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each animal per treatment group.
- Percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined as a measure of bone marrow toxicity.
Evaluation criteria:
- In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups.
- A final call of positive for micronucleus induction is preferably based on reproducibly positive trials.
- Statistical as well as biological factors are considered.
Statistics:
- The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean.
- The frequency of micronucleated cells among NCEs was analysed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the control group.
- In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.
- Significance was considered at p≤ 0.025.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
positive
Remarks:
female mice
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The frequencies of micronucleated erythrocytes were not increased in peripheral blood of male animals exposed to 37.5 to 600 mg/kg bw/day of test material by gavage for 3 months; in contrast, a 3.2-fold increase of micronucleated erythrocytes in females at 600 mg/kg bw/day were observed.

Any other information on results incl. tables

Table 7.6.2/1. Micronucleus data

Dose

(mg/kg bw/day)

No. of animals with erythrocytes scored

Micronucleated

NCEs/1000 NCEs

(mean ± SE)

P value*

PCEs (%)

0 (corn oil)

5M

0.90 ± 0.37

 

2.3

37.5

5M

1.60 ± 0.46

0.0806

2.8

75

5M

0.70 ± 0.25

0.6915

3.1

150

5M

0.90 ± 0.24

0.5000

2.8

300

5M

0.30 ± 0.12

0.9584

2.5

600

5M

0.90 ± 0.19

0.5000

2.9

 

P = 0.841#

 

0 (corn oil)

8F

0.50 ± 0.16

 

2.8

37.5

5F

1.10 ± 0.19

0.0408

3.5

75

5F

0.20 ± 0.12

0.8850

3.1

150

5F

0.70 ± 0.30

0.2568

2.7

300

5F

1.00 ± 0.35

0.0680

3.4

600

5F

1.60 ± 0.40

0.0022

2.4

 

P = 0.001#

 

Keys:

NCE = Normochromatic erythrocyte

PCE = Polychromatic erythrocyte

* Pairwise comparison with the vehicle control; dosed group values are significant at P ≤ 0.005.

# Significance of micronucleated NCEs/1000 NCEs tested by the one-tailed trend test; significant at P≤ 0.025

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the test material showed a statistically significant increase in the frequency of micronucleated erythrocytes in female mice. This was concluded by the authors to be evidence of a positive response. However, the female vehicle control group had a particularly low mean and standard deviation for the frequency of micronucleated erythrocytes and there was no clear dose response relationship, therefore the response was considered to an artefact. The test material was not considered to be clastogenic or aneugenic in male or female mice.
Executive summary:

In an in vivo bone marrow micronucleus test, groups of B6C3F1 mice (5/sex/dose) were exposed to test material in corn oil at doses of 37.5, 75, 150, 300 and 600 mg/kg bw/day, 5 days/week for 14 weeks, by gavage. At the end of the study period, peripheral blood samples were obtained from treated animals and smears were prepared immediately. Slides were fixed, stained and coded for analysis. Frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each animal per treatment group was determined. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined as a measure of bone marrow toxicity.

 

No significant changes in the percentage of PCEs were observed over the dose range tested in either males or females, indicating an absence of treatment-related toxicity to the bone marrow. The frequencies of micronucleated erythrocytes were not increased in peripheral blood of male mice at any dose level. In contrast, female mice had a 3.2-fold increase of micronucleated erythrocytes at 600 mg/kg bw/day.

 

Under the test conditions, the test material showed a statistically significant increase in the frequency of micronucleated erythrocytes in female mice. This was concluded by the authors to be evidence of a positive response. However, the female vehicle control group had a particularly low mean and standard deviation for the frequency of micronucleated erythrocytes and there was no clear dose response relationship, therefore the response was considered to an artefact. The test material was not considered to be clastogenic or aneugenic in male or female mice.