Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in Salmonella typhimurium strains TA ( TA97a, TA98, TA100, TA102 and TA1535) (according to OECD TG 471)

Mutagenicity in mammalian cells: negative in L5178Y mouse lymphoma cells (according to OECD TG 471)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July - August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium: Histidine gene
Species / strain / cell type:
other: TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Metabolic activation system:
S-9
Vehicle / solvent:
0.9% NaCl (saline) and PEG 400
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoflourene and 2-aminoanthracene
Details on test system and experimental conditions:
TEST ARTICLE PREPARATION: The amount of material tested was based on ISO extraction ratios. An aliquot of the solvents used were included and incubated in parallel with the test articles to serve as solvent controls (negative controls).
Extract solvent 1: 0.9% NaCl (saline) – Extraction ratio : 6cm2/mL – Test Article/Extraction Solvent amount : 42 cm2/7 mL – Extraction parameters : Temp = 37 ± 1ºC, time: 72 ± 2 hours (with agitation).
Extract solvent 2: PEG 400 – Extraction ratio : 6cm2/mL – Test Article/Extraction Solvent amount : 42 cm2/7 mL – Extraction parameters : Temp = 37 ± 1ºC, time: 72 ± 2 hours (with agitation).

PROCEDURE:
BROTH CULTURE PREPARATION: Commercial culture discs were used to inoculate nutrient broth for testing. The cultures were incubated at 37 ± 2ºC for 10-14 hours on an orbital shaker until when measured spectrophotometrically at 660nm, an absorbance reading of approximately 1.0 to 2.0 was obtained. Validation data of the cultures showed absorbance readings in the range resulted in concentrations of approximately 10E9 CFU/mL.

STRAIN GENOTYPE VERIFICATION: The culture disc lot number is checked for presence of appropriate strain genotype characteristics. These tests included verification of the following:
- Presence of uvrB mutation
- Presence or absence of R-factor plasmid
- Presence of rfa mutation
- Requirement for histidine
The uvrB mutation was verified by demonstrating UV sensitivity (lack of repair system). The R-factor was checked by determining sensitivity or resistance to ampicillin (0.08% in 0.02 NaOH). The presence if the rfa mutation was verified by demonstrating sensitivity to crystal violet (0.1% in water) on nutrient agar plates. The histidine requirement was assured by plating onto minimal glucose agar plates spread with 0.1 mL of 0.5 mM biotin and both with and without 0.1 mL of 0.1 M histidine.

METABOLIC ACTIVATION SYSTEM: The S-9 activation system was used to screen for the presence of mutagens from byproducts of the test article. Rat liver S-9 homogenate was obtained from Molecular Toxicology, Inc. The homogenate was kept frozen at ≤ -60ºC upon receipt. Plates requiring activation contained approximately 20 µL rat liver S-9 per plate. When working with soft agar plates did not exceed 47ºC.

TOP AGAR PREPARATION: Aliquots of top agar were melted and maintained at 45 ± 2ºC. Each 100 mL aliquot of top agar was fortified with 5-10mL of 0.5 mM histidine prior to use.

PLATE INCORPORATION TESTS: Each test article extract and solvent control was tested both with and without S-9 metabolic activation. The S-9 specific chemical controls (2-aminofluorene and 2-aminoanthracene) were also tested both with and without S-9 metabolic activation. Strain specific non-metabolic chemical controls were also included (Sodium Azide, Mitomycin-C and 4-nitro-0-phenylene-diamine. The non-metabolic chemical controls were tested without S-9 activation only.
Sterile 13 x 100 mm test tubes were transferred to a waterbath held at 45 ± 2ºC. Two mL aliquots of top agar were transferred to each test tube. Three replicates for each test article or control were prepared.
1) Solvent controls: 2mL top agar, 100 µL test organism and 100 µL of each solvent control.
2) Test article Extracts: 2mL top agar, 100 µL test organism and 100 µL of each test article extract.
3) Chemicals controls: 2mL top agar, 100 µL test organism and 10 µL of the specified chemical.
Each replicate requiring S-9 metabolic activation has 0.5 mL of the prepared S-9 mix added.
The replicates were vortexed, poured onto MGPA plates, swirled to form an even layer and allowed to solidify. The plates were incubated for growth 37± 2ºC for 48-72 hours.

SPOT TESTS: The test article extracts were also analysed using the spot method on plates with and without the S-9 activation system. Two mL aliquots of the top agar mixture and 0.1 mL of the appropriate test organism was added to minimal glucose agar plates. The plates were allowed to harden then 10 µL of the test article extract was added as a spot on the surface of the plate. The plates were incubated for growth of the organisms at 37± 2ºC ºC for 48-72 hours.

CHEMICAL CONTROL MATERIALS: The following chemical controls were used. The concentrations listed are the amount added per plate: 1.5 µg Sodium Azide, 2.5 µg Mitomycin-C, 20 µg 4-nitro-0-phenylene-diamine (NPD), 20 µg 2-aminofluorene (2-AF) and 7 µg 2-aminoanthracene (2-AA). The chemical controls were tested using the plate incorporation method only.
Evaluation criteria:
The criteria for acceptance of the test and criteria for determination of a mutagen are listed below.
1) Tested strains for genotype verification and achieved the appropriate responses.
2) All chemical controls included in the test gave the appropriate responses.
3) The reversion rates for each tester strains were within the historical ranges as outlined in the standard protocol.
Criteria for a Mutagen: A greater than two-fold increase in the number of revertants in strains TA97a, TA100 and TA102 or a greater than three-fold increase in strains TA98 and TA1535 when compared to the solvent control (percent of control >200% or >300%).
Criteria for a Non-Mutagen: A less than two-fold increase in the strains TA97a, TA100 and TA102 or a less than three-fold increase in strains TA98 and TA1535 when compared to the solvent control (percent of control <200% or <300%).
Key result
Species / strain:
other: TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test article extracts did not produce a two-fold or a three-fold increase in the number of revertants in any of the 5 tester strains. The spot tests showed no zone of increased reversion or of toxicity. In summary, the extracts tested against the five strains did not meet the criteria for a potential mutagen.
Executive summary:

The Salmonella typhimurium reverse mutation assay (Ames test) is used to determine the potential mutagenic activity of a solid test article extract by exposing a large number of the test organisms to the extract fluid in agar plates. The agar plates are monitored for growth of revertants (organisms mutating to the wild type) which are counted and used to estimate the mutagenic potential of the test article.

The Ames test employs several histidine dependent (His+) strains of S. typhimurium which require the amino acid histidine for growth. The test detects mutations which cause the bacterial strains to revert to histidine independent (His-) bacteria which are capable of synthesizing histidine and can grow in the absence of histid ine. The assay uses tester strains TA97a, TA98, TA100, TA102 and TA1535 which were selected to detect various types of mutagens. The test is performed both with and without metabolic

activation using an S-9 activation system. The S-9 activation system is designed to simulate mammalian liver enzyme systems and is used to detect substances which undergo metabolic activation from nonmutagenic forms.

All test method acceptance criteria were met.

Results: The results are calculated using a validated computer program. Manual calculations may differ slightly due to rounding. All results greater than 300 colony forming units (CFU) are considered estimates.

The test article extracts did not produce a two-fold or a three-fold increase in the number of revertants in any of the 5 tester strains. The spot tests showed no zone of increased reversion or of toxicity. In summary, the extracts tested against the five strains did not meet the criteria for a potential mutagen.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 December 2011 to 3 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation 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:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source of cells: American Type Culture Collection (ATCC) (Manassas, Virginia), Cell Line # CRL 9518.
- Cell Culture Preparation:
The L5178Y (TK+/−) cells will be grown in Fischer’s or other suitable medium containing 10% serum. Mouse lymphoma cells are periodically cleansed of spontaneously occurring TK−/− cells by growing the cells in complete medium containing a mixture of thymidine, hypoxanthine, methotrexate, and glycine (THMG). The THMG is removed, and the cells are grown for an additional 24 hours in complete medium containing thymidine, hypoxanthine, and glycine (THG). Cultures of recently cleansed cells will be used in the Mouse Lymphoma assay. Cultures containing 4−8 × 106 cells will be prepared in complete medium prior to the addition of the dosing solutions.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S−9 induced by a combination of cofactors and culture medium
Vehicle / solvent:
NaCl and PEG, used as extraction vehicles, served as negative controls.
MethylmethaneSulfonate (MMS) served as the positive control article for the non-activated assay.
Dimethylbenzanthracene (DMBA) served as the positive control article for the activated assay.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 hours
- Expression time (cells in growth medium): 20 and 44 hours
- Selection time (if incubation with a selection agent): 10-12 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 1 x 10E6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency (RCE)

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:
Evaluation criteria:
The results of the Mouse Lymphoma Mutagenesis Assay are evaluated on the basis of the number of TFT-resistant mutants per 1x10E6 surviving cells.
Statistical Analysis (see below).
Statistics:
Statistical Analysis:
The test results will be analyzed using a statistical methods 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 mutation frequency of the test article compared to the negative control group.

The test article is considered to have caused a positive response in the assay if a statistically significant increase in the number of mutants is observed over its concurrent negative control article.

Confirmatory Assay:
The confirmatory assay will be conducted to validate the reproducibility of the mutagenesis assay, only if specified by the Sponsor, and at an additional cost.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The 0.9% Sodium Chloride for Injection (NaCI) and Polyethylene Glycol 400 (PEG) extracts of the test article, BondEaseTM Topical Skin Adhesive (polymerized), were tested for their potential to induce a statistically significant increase in the number of homozygous thymidine kinase mutants (TK-/-) over the background rate in L5178Y mouse lymphoma cell line, heterozygous at the thymidine kinase locus (TK+/-), in the presence and absence of a metabolic activation system.
The results showed no significant increase in the frequency of homozygous mutants in cells exposed to NaCI or PEG extracts of the test article as compared to the respective controls. The positive controls induced a significant increase in the frequency of homozygous mutants in both non-activated and activated conditions, validating the functioning of the assay. Based on the criteria of the study protocol, the test miicle is considered non-mutagenic, under the experimental conditions utilized.
Executive summary:

Purpose:

The purpose of the study was to evaluate the ability of the test article (MM212) to induce an increase in the formation of homozygous thymidine kinase mutants (TK-/-) over the background rate in a mouse lymphoma cell line, in the presence and absence of a metabolic activation system. The mutant cell line, L5178Y, carries the mutation in the thymidine kinase locus (TK+/-).

References:

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 476, Organization for Economic Co-Operation and Development (OECD) Guidelines for the Testing of Chemicals, "In vitro Mammalian Cell Gene Mutation Test", adopted 21 July 1997.

Compliance:

The study was conducted in compliance with U.S. Food and Drug Administration regulations set forth in 21 CFR, Part 58 under Good Laboratory Practices.

Results:

The results indicated that the test article did not induce a statistically significant increase (p > 0.05) in the number of trifluorothymidine (TFT) resistant mutants per 1 x 10^6 cells as compared to negative control cultures in both the non-activated and activated assays. The positive controls produced a statistically significant increase in the incidence of mutants (p < 0.01) per 1 x 10E6 cells as compared to the negative control cultures.

Conclusion:

The 0.9% Sodium Chloride for Injection (NaCl) and Polyethylene Glycol 400 (PEG) extracts of the test article, BondEaseTM Topical Skin Adhesive (polymerized), were tested for their potential to induce a statistically significant increase in the number of homozygous thymidine kinase mutants (TK-/-) over the background rate in L5178Y mouse lymphoma cell line, heterozygous at the thymidine kinase locus (TK+/-), in the presence and absence of a metabolic activation system.

The results showed no significant increase in the frecuency of homozygous mutants in cells exposed to NaCl or PEG extracts of the test article as compared to the respective controls. The positive controls induced a significant increase in the frecuency of homozygous mutants in both non-activated and activated conditions, validating the functioning of the assay. Based on the criteria of the study protocol, the test article is considered non-mutagenic, under the experimental conditions utilized.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mouse bone marrow micronucleus test: negative (according to OECD TG 474)

Link to relevant study records
Reference
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
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
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
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.
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.
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.
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.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

No adverse effects observed on the available in vitro and in vivo genotoxicity studies, therefore, MM212 is not classified for mutagenicity according to Regulation (EC) No 1272/2008 (CLP).