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:
January-April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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)
Qualifier:
according to
Guideline:
other: ISO 10993-3, 2003, Biological Evaluation of Medical Devices - Part 3: Tests for Genotoxicity, Carcinogenicity and Reproductive Toxicity
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Bond.Ease™ Topical Skin Adhesive (polymerized)
- Batch no.: 10312101
- Physical state: Liquid
- Purity: >96%

Test animals

Species:
mouse
Strain:
Swiss
Remarks:
Albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, Indianapolis, IN.
- Age at study initiation: 6 to 12 weeks old
- Weight at study initiation: 23.0-29.4 grams
- Health status: healthy, not previously used in other experimental procedures
- Animal identification: ear punch
- Housing: group housed (5 per cage of same sex)
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days, under same conditions as for the actual test
- Animal selection: selected from larger pool and examined to ensure lack of adverse clinical signs

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 ± 5ºF
- Humidity (%): 30-70%
- Air changes (per hr): 10 to 15
- Photoperiod: 12-hour light/dark cycle, full spectrum fluorescent lights

Administration / exposure

Route of administration:
other: intravenous and intraperitoneal
Vehicle:
- Vehicles/solvents used: 0.9% Sodium Chloride (NaCl) and Cottonseed Oil (CSO).
- Route of administration: Intravenous (NaCl) and intraperitoneal (CSO)
- Extration ratio: The test article (165 cm2) was combined with 27.5 mL of vehicle at a ratio of 6 cm2 per 1 mL per ISO 10993-12 guidelines.
- Extraction conditions: 37ºC for 72 hours
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
Test article administered only once.
Doses / concentrations
Dose / conc.:
20 other: mL/kg bw
No. of animals per sex per dose:
5 male and 5 female
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide (CP)
- Route of administration: intraperitoneal
- Doses / concentrations: 25 µg/g bw.

Examinations

Tissues and cell types examined:
Bone−marrow cells
Details of tissue and slide preparation:
Post-Dose Procedure:
- Clinical observations were conducted daily.
- The animals were weighed and then euthanized by CO2 inhalation.
- Animals from the test, negative, and positive control groups were sacrificed 24 hours after treatment. Additional animals from the test and negative control groups were sacrificed 48 hours after treatment. Immediately after sacrifice, one or both femurs were exposed by appropriate surgical techniques. The bone marrow was collected and placed on a clean, pre−labeled microscope slide. Using a thick plastic cover glass, a fine feather edge smear was prepared and allowed to air dry. The slides were fixed in methanol and stained with Giemsa.
- Scoring:
The treatment and control slides were coded prior to scoring to reduce any possible bias. A total of at least 2000 polychromatic erythrocytes (PCEs) per animal were scored for the presence of micronuclei. The scored elements were the number of micronucleated cells, and not the number of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was also determined for each animal by counting corresponding number of normochromatic erythrocytes per 2000 polychromatic cells. Data were collected and presented separately for male and female animals.
Evaluation criteria:
The number of micronucleated PCEs for the positive and negative controls should be in the range of expected historical control values. The frequency of micronucleated PCEs in the positive control group should be statistically significant compared to the negative control group.
If these conditions are not met the test should be repeated.
Statistics:
Data are analyzed separately for male and female animals. The test results are analyzed using a statistical method such as the “t−test” using Graph Pad Prism Software by Analytical Software, Inc. (Analyzing Data with Graph Pad Prism®, Harvey Motulsky). This statistical method determines if there is a significant (p ≤ 0.05) increase in the incidence of micronucleated cells in the test article group as compared to the negative control group. A dose related response is determined, if appropriate, by a linear regression analysis. Biological and statistical significance is considered in the evaluation.

Positive Dose Response:
The test article is considered to have caused a positive response in the assay if a statistically significant increase in micronucleated PCEs over its concurrent negative control article is observed.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Animal weights (prior to dosing and prior to sacrifice), dosing data, and clinical observations were recorded for all the animals during the course of the study.
A total of 23 animals in the negative control control group, 7 animals in the positive control group and 26 animals in the test article group lost an insignificant amount of weight (less than 8%). All other animals gained weight.
No clinical signs of toxicity were observed for any of the test or control animals.

Applicant's summary and conclusion

Conclusions:
The USP 0.9% Sodium Chloride for Injection (NaCl) and Cottonseed Oil (CSO) extracts of the test article were tested at the neat concentration for their ability to induce a statistically significant increase in the number of micronucleated erythrocytes in rodent bone marrow. The results showed that neither of the test article extract induced a statistically significant increase in micronucleated erythrocytes as compared to the respective negative controls 24 and 28 hours after dosing. The positive control, Cyclophosphamide (CP), caused a statistically significant increase in micronucleated erythrocytes as compared to the negative controls, validating the functioning of the assay.
Based on the criteria of the study protocol, the test article is considered non-clastogenic, under experimental conditions utilized.
Executive summary:

The purpose of the study was to evaluate the ability of the test article and/or its metabolites to induce micronuclei in maturing erythrocytes of mice. This procedure is designed to detect damage to the chromosomes or mitotic apparatus caused by the test article.

The study was conducted based upon:

- ISO 10993 -3, 2003, Biological Evaluation of Medical Devices - Part 3: Tests for Genotoxicity, Carcinogenicity and Reproductive Toxicity.

- OECD 474, Organization for Economic Co-Operation and Development (OECD), Guidelines for the Testing of Chemicals, "Mammalian Erythrocyte Micronucleus Test", adopted 21 July 1997.

The study was conformed to the current FDA 21 CFR, Part 58 - Good Lbaboratory Practice for Non-Clinical Laboratory Studies.

The USP 0.9% Sodium Chloride for Injection (NaCl) and Cottonseed Oil (CSO) extracts of the test article were tested at the neat concentration for their ability to induce a statistically significant increase in the number of micronucleated erythrocytes in rodent bone marrow. The results showed that neither of the test article extract induced a statistically significant increase in micronucleated erythrocytes as compared to the respective negative controls 24 and 28 hours after dosing. The positive control, Cyclophosphamide (CP), caused a statistically significant increase in micronucleated erythrocytes as compared to the negative controls, validating the functioning of the assay.

Based on the criteria of the study protocol, the test article is considered non-clastogenic, under experimental conditions utilized.