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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reliable GLP and guideline compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
Please refer to confidential details on test material.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: 7 weeks old
- Weight at study initiation: 200.4 – 220.4 g for males and 168.4 – 182.1 g for females
- Housing: Rats of the same sex were housed up to five per rodent Micro-Barrier cage
- Diet: certified laboratory rodent chow, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 50 ± 20%
- Air changes: At least 10 changes of fresh HEPA-filtered air every hour
- Photoperiod: 12 hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Polyethylene glycol 300 (PEG 300)
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance dose formulations were prepared fresh on each day prior to dose administration. An appropriate amount of the test substance was weighed separately for each concentration and was combined with an appropriate reduced volume of the vehicle. Each formulation was vortexed (2 minutes), homogenized (4 minutes) and stirred for 10 minutes. Each formulation was adjusted to the final required volume and was stirred for 1 hour, prior to use. All formulations appeared as dark yellow suspensions.
Duration of treatment / exposure:
Two oral gavage treatments, approximately 24 hours apart (treatment and control groups)
Exposure was terminated approximately 24 hours before animals were sacrificed.
Frequency of treatment:
test and vehicle control substance: twice, approximately 24 hours appart
positive control substance: once
Post exposure period:
Animals were sacrificed for bone marrow collection approximately 24 hours after the last dose.
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw/day (dose volume: 10 mL/kg bw)
Basis:

No. of animals per sex per dose:
5 rats
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate at a dose of 40 mg/kg bw and a dose volume of 10 mL/kg bw.

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
BONE MARROW COLLECTION AND SLIDE PREPARATION
Approximately 24 hours after the last dose, animals were euthanized by CO2 asphyxiation as verified by toe pinch reflex. Immediately following, the bone marrow was aspirated into a syringe containing fetal bovine serum. Bone marrow cells were transferred to a labeled centrifuge tube containing approximately 3 mL cold fetal bovine serum and pelleted by centrifugation (~100 x g for 5 minutes). Cells were re-suspended, and a small drop of bone marrow suspension was spread onto clean glass slides (two/animal). All slides were air dried and fixed in methanol, and one set of slides was stained with acridine orange for microscopic evaluation. The other set served as back up slides that could be used if necessary.

SCORING FOR MICRONUCLEI (BONE MARROW EVALUATION)
To control for bias, bone marrow slides were coded using a random number table by an individual not involved with the scoring process. Using a fluorescent microscope and medium magnification an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (1000X), the following cell population and cellular components were evaluated and enumerated:

Polychromatic erythrocytes (PCEs)
At least 2000 PCEs/animal (10.000PCE/group) were scored for the presence of micronuclei (mnPCEs) whenever possible.

Normochromatic erythrocytes (NCEs)
The number of NCEs and micronucleated NCEs (mnNCEs) in the field of 1000 total erythrocytes (PCEs + NCEs) were determined for each animal.

The proportion of polychromatic erythrocytes to total erythrocytes was determined per total of 1000 erythrocytes (PCEs + NCEs) for each animal as an indication of bone marrow cytotoxicity. Statistical significance was determined in each treatment group and sex.
Evaluation criteria:
EVALUATION OF TEST RESULTS
The frequency of mnPCEs and the proportion of PCEs to total erythrocytes was determined for each animal and treatment group. Once the criteria for a valid assay were met, the results were evaluated as follows:
- A test substance is considered to be positive if it induces a significant increase in mnPCE frequency at any dose level or sampling time (p ≤ 0.05).
- A test substance will be considered to be negative if no significant increase in mnPCE frequency is observed (p > 0.05).
- Other criteria also may be used in reaching a conclusion about the study results (e.g., magnitude of any increase, dose-dependency, comparison to historical control values, biological relevance, etc.). In accordance with OECD Guideline 474 biological relevance of the results should be considered first. In such cases, the Study Director uses sound scientific judgment and clearly report and describe any such considerations.

CRITERIA FOR A VALID TEST
All criteria for a valid test were met because:
- The mnPCE frequency of the vehicle controls was within the historical vehicle control range; and the positive control induced a significant increase in mnPCE frequency (p ≤ 0.05).
- 5 animals/sex/group were available for analysis.
Statistics:
Statistical significance (p ≤ 0.05) was determined using Dunnett comparisons (One-way ANOVA or T-test) or the binomial distribution (Kastenbaum and Bowman, 1970).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
There were signs of systemic toxicity in high dose males.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Dose Formulation Analysis
Based on the analytical results, all test substance formulations were prepared accurately and were homogeneous.

In Life Evaluation
No mortality was observed in any of the treatment groups. Diarrhea was noted in two of the male rats in the vehicle control group on Day 2. Piloerection was noted after the second 2000 mg/kg bw dose in all animals and it persisted in the males until euthanasia. All other animals appeared normal during the study period. No appreciable changes in group mean body weights were observed in most groups, although a slight weight loss was observed in the high dose males between Days 1 and 2.

Bone Marrow Evaluation
No statistically significant decreases in the proportion of PCEs to total erythrocytes were observed at any dose level, indicating the test item did not inhibit erythropoiesis. However, individual high dose males exhibited decreased PCE proportions as compared to the concurrent controls. Collectively, the clinical observations, the loss in body weight between Days 1 and 2, and individually low PCE proportions may be indicative of systemic exposure to the test substance in males, and systemic toxicity, at the highest dose evaluated.
No statistically significant increases in mnPCE frequencies were observed at any dose of the test item as compared to the concurrent vehicle control (p > 0.05). In contrast, the CP positive control induced a statistically significant increase in mnPCE frequencies (p ≤ 0.05). All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

Any other information on results incl. tables

Table 1: Frequency of micronucleated polychromatic erythrocytes (mnPCE) in vivo after treatment with the test item

Group

Dose level

[mg/kg bw/day]

mnPCE/1000 PCE

(mean number ± SD)

PCE/Total Erythrocytes

(mean number ± SD)

Male rats

 

NC

     0

0.2 ± 0.27

0.537 ± 0.02

test item

 500

0.4 ± 0.42

0.548 ± 0.02

1000

0.2 ± 0.45

0.532 ± 0.05

2000

0.3 ± 0.27

0.490 ± 0.09

PC

   40

17.1 ± 5.59

0.383 ± 0.05**

Female rats

 

NC

     0

0.4 ± 0.42

0.553 ± 0.05

test item

 500

0.7 ± 0.57

0.566 ± 0.04

1000

0.4 ± 0.42

0.560 ± 0.03

2000

0.3 ± 0.27

0.537 ± 0.04

PC

   40

13.1 ± 5.28

0.412 ± 0.04**

mnPCE:: micronucleated polychromatic erythrocytes

NC: negative control,

PC: positive control,

SD: standard deviation

**: statistically significant p ≤ 0.01 (two-sample t-test)

 

Applicant's summary and conclusion