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Genetic toxicity in vitro

Description of key information

Two reliable in vitro genetic toxicity studies are available. In an Ames test performed according to OECD guideline 471 (BioReliance, 2010) the test substance demonstrated to be negative for mutagenicity with and without metabolic activation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvr A.
In a CHO/HGPRT assay performed according to OECD guideline 476 (BioReliance, 2010), the test substance demonstrated negative for mutagenicity with and without metabolic activation. No excessive toxicity was observed. No in vitro cytogenicity / chromosome aberration study in mammalian cells was performed as adequate in vivo data is available.


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-20 - 2010-11-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
other: ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (1997)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Amine C8
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: temperature: ambient; all articles will be stored in the dark
Target gene:
Histidine locus, tryptophan locus and additional gene mutations
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
5000, 1500, 500, 150, 50, 15, 5.0 and 1.5 µg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene at 10 µg/plate
Remarks:
with metabolic activation, E coli strain WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene at 1.0-2.0 µg/plate
Remarks:
with metabolic activation, Samonella strains
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation, at 1.0 µg/plate (Salmonella strain TA98)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation, at 75 µg/plate (Salmonella strain TA100, TA1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation, at 1000 µg/plate (E coli WP2urvA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applciable (plate incorporation method)
- Exposure duration: at the end of the working day, each inoculated flask will be placed in a shaker/incubator programmed to begin shaking at approximately 125 to 175 rpm and incubating at 37± 2°C for approximately 12 to 14 hours before the anticipated time of harvest. All cultures will be harvested by spectrophotometric monitoring of culture turbidity rather than by duration of incubation since overgrowth of cultures can cause loss of sensitivity to some mutagens. Cultures will be removed from incubation at a density of approximately 1E09 cells/mL.
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): 48 to 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS:duplicate (initial toxicity - mutation assay), triplicate (confirmatory mutagenicity assay)

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: A minimum of three non-toxic dose levels will be required to evaluate assay data. A dose level is considered toxic if it causes a > 50% reduction in the mean number of revertans per plate relative to the mean vehicle control value (this reduction must be accompanied by an abrupt dose-dependent drop in the revertant count) or a reduction in the background lawn. In the event that less than three non-toxic dose levels are achieved, the affected portion of the assay will be repeated with an appropriate change in dose levels.
Evaluation criteria:
For a test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:
- Strains TA 1535 and TA 1537: data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-times the mean vehicle control value
- Strains TA98, TA100 and WP2 uvrA: data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value. An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No precipitation was observed.
Conclusions:
Under the conditions of the test,the test substance tested negative with and without metabolic activation in the bacterial reverse mutation assay. No toxicity nor precipitation was observed.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-20 - 2010-11-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
other: ICH of Technical Requirements for Registration of Pharmaceuticals for Human Use, Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, S2A
GLP compliance:
yes
Type of assay:
other: CHO/HGPRT Assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Amine C8
- Physical state: clear brown liquid
- Analytical purity: 100%, an adjustment for purity or active ingredient was not made
- Lot/batch No.: AD16UT
- Storage condition of test material: storage temperature: ambient; the test article will be stored in the dark
Target gene:
hypoxanthine-guanine phosphoribosyl trasnferase locus
Species / strain / cell type:
other: CHO-K1-BH4 cell line
Details on mammalian cell type (if applicable):
Type and identity of media: F12 medium without hypoxanthine, supplemented with 5% dialyzed fetal bovine serum (F12FBS5-Hx), at a density of 5 x E05 cells/25 cm2 surface area and will be incubated at 37 ± 1°C in a humidified atmosphere of 5±1 % CO2 in air for 18-24 hours
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: Each freeze lot of cells has been tested and found to be free of mycoplasma contamination.
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
0.5; 1.5; 5; 15; 50; 150; 500; 1500; 5000 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation at a concentration of 2 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation at a concentration of 4 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 5 hours
- Expression time (cells in growth medium): For expression of the mutant phenotype, the replicates from each treatment condition will be subcultured independently in F12FBS5-Hx, at a density of no greater than 1E06 cells/100 mm dish. Subculture at 2-4 day intervals will be performed for the 7-9 day expression period. At this time, selection for the mutant phenotype will be performed.
- Selection time (if incubation with a selection agent): For the selection of the TG-resistant phenotype, cells from each treatment condition will be plated into a maximum of five dishes at a density of 2 x 1E05 cells/100 mm dish in F12FBS5-Hx contining 10 µM TG. For cloning efficiency at the time of selection, 100 cells/60 mm dish will be plated in triplicate in medium free of TG.
- Fixation time (start of exposure up to fixation or harvest of cells): After 7-10 days of incubation, the colonies will be fixed, stained and counted for both clining efficiency at selection and mutant selection.

SELECTION AGENT (mutation assays): 6-thioguanine
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 1E06 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: not applicable

OTHER: not applicable
Evaluation criteria:
The mutant frequency (MF) for each treatment condition is calculated by dividing the total number of mutant colonies by the number of cells selected, corrected for the cloning efficiency of cells prior to mutant selection, and is expressed as TG-resistant mutants per 1E06 clonable cells. Mutant frequencies generated from doses giving < 10% relative survival are not considered as valid data points and are not included in the analysis. The wide acceptable range (0 to 25 mutants per 1E06 clonable cells) in spontaneous mutant frequency also suggest the need to set a minimum mutant frequency for a response to be considered positive. In this laboratory, a more conservative approach is used which sets the minimum significant level at > 40 mutants per 1E06 clonable cells. If a single point above 40 mutants per 1E06 clonable cells is observed at the highest dose, the results will be considered equivocal. If no culture exhibits a mutant frequency of > 40 mutants per 1E06 clonable cells, the test article will be considered negative.
Key result
Species / strain:
other: CHO-K1-BH4 cell line
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Under the conditions of the test, the test substance tested negative with and without metabolic activation in the In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT Assay). Cloning efficiency was not effected by the substance: no cytotoxicity was observed.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in vivo micronucleus test on mouse bone marrow erythrocytes (BioReliance, 2010), performed according to OECD guideline 474, the animals were exposed orally (via gavage) with 500, 1000, 2000 mg/kg. This did not induce a statistically positive increase in micronuclei in the hemopoietic cells of the mouse bone marrow at the time intervals evaluated under the experimental condition of this assay. No toxicity was observed. Vehicle and positive controls were valid.

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:
2010-10-21 - 2010-11-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
other: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human use (1994, 1996 and 1997)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
- Name of test material (as cited in study report): Amine C8
- Physical state: dark liquid
- Analytical purity: the test substance is a complex mixture, and the purity will be considered to be 100%
- Lot/batch No.: AD16UT
- Storage condition of test material: at ambient (15 to 30°C), protected from light
Species:
mouse
Strain:
other: Hsd:ICR (CD-1)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation:
- Assigned to test groups randomly: yes, under following basis: Initially for the dose range finding study, animals are assigned to one group of 5 male and 5 female mice, as per study design. For the definitive micronucleus study animals are assigned to 7 groups of 5 males and 5 females each. The animals are assigned using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight variation of animals will not exceed ± 20% of the mean weight. Following randomization, animals are identified by sequentially numbered ear tags assigned to each animal during randomization process. The cage card contains, at least, the animal number, sex, study number, test substance identification, treatment group number, dose level, and route of administration. Cage cards are color coded as a function of treatment group.
- Fasting period before study: no fasting
- Housing: Animals are housed in an AAALAC-accredited facility with a controlled environment. Mice of the same sex are housed up to five per rodent Micro-Barrier cage. Cages are placed on the racks equipped with an automatic watering system and MicroVENT full ventilation, HEPA filtered system. The purpose of this system is to supply uninterrupted positive air to each individual rodent Micro-Barrier cage and to capture the effluent air from each cage and re-filter the air (HEPA) prior to indroducing the air back into the cage. If needed, alternative housing system will be implemented. Heat treated hardwood chips are used for bedding to absorb liquids.
- Diet (e.g. ad libitum): A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) is provided at libitum. The food is analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients.
- Water (e.g. ad libitum): Animals have free access to tap water, which meets U.S. EPA drinking water standards. Drinking water is monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens.
- Acclimation period: 5 days prior to dose administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ± 22°C
- Humidity (%): 50 ± 20% relative humidity
- Air changes (per hr): The animal rooms are supplied with at least 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: purified (deionized water)
- Justification for choice of solvent/vehicle: good workability/solubility of the test substance in the vehicle and compatibility of the vehicle with the test system.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For each study, the test substance formulations are be prepared prior to dose administration. Each concentration is prepared by mixing an appropriate amount of the test substance with the appropriate volume of the vehicle. The mixtures are vortexed, if needed homogenized and stirred in order to achieve workable or soluble formulations.

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): not applicable (gavage)
- Storage temperature of food: no data
Duration of treatment / exposure:
The test substance dose formulations, the vehicle alone or the positive control (cyclophosphamide monohydrate) are given as a single administration.
Frequency of treatment:
The test substance dose formulations, the vehicle alone or the positive control (cyclophosphamide monohydrate) are given as a single administration. The volume of administration will be 20 mL/kg body weight.
Post exposure period:
In the definitive test, approximately 24 and 48 hours after dose administration, animals are euthanized by carbon dioxide inhalation followed by incision of the diaphragm. Animals in the positive control group are euthanized 24 hours after dose administration.
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested gavage
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested gavage
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested gavage
No. of animals per sex per dose:
Dose range finding study: 30 animals/sex
Definitive Micronucleus Study: 35 animals
Control animals:
yes, concurrent vehicle
Positive control(s):
- Route of administration: oral: gavage
- Doses / concentrations: animals will receive cyclophosphamide at a dose of 50 mg/kg body weight
Tissues and cell types examined:
Immediately following euthanasia, the femurs are exposed, cut just above the knee and the bone marrow will be aspirated into a syringe containing fetal bovine serum.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): In the definitive test, approximately 24 and 48 hours after dose administration, animals are euthanized by carbon dioxide inhalation followed by incision of the diaphragm. Animals in the positive control group are euthanized 24 hours after dose administration.

DETAILS OF SLIDE PREPARATION: The bone marrow cells are transferred to a labelled centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells are pelleted by centrifugation and the supernatant is drawn off, leaving a small amount of fetal bovine serum with the remaining cell pellet. The cells are respudended and a small drop of the bone marrow suspension is spread onto a clean glass slide. Each slide will be identified by the experiment and animal number. At least two slides are prepared from each animal, air dried and fixed by dipping in methanol. One set of slides is stained with May-Gruenwald-Giemsa, permanently mounted and used for microscopic evaluation. The other set of slides (not stained) is kept as a backup set.

METHOD OF ANALYSIS: Using a light microscope and a medium magnification (400 X), an area of acceptable quality will be selected such that the cells are well spread and stained. The following cell populations and cell components are recorded using oil immersion (1000X):
-2000 polychromatic erythrocytes (PCEs) are scored per animal for the presence of micronuclei resulting in evaluation of 10000 PCEs per group.
- The number of micronucleated normochromatic erythrocytes (MNCEs) in the field of 2000 polychromatic erythrocytes are also enumerated, but are not used to evaluate the response of the test substance
- The proportion of polychromatic erythrocytes to total erythrocytes is also determined per total of 1000 erythrocytes (PCEs/ECs ratio) for each animal.


Evaluation criteria:
The test substance will be considered to induce a positive response if the incidence of micronucleated polychromatic erythrocytes at one or more doses is statistically elevated relative to the vehicle control (p< or = 0.05, Kastenbaum-Bowman Tables).
The substance will be judged negative if no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance groups relative to the concurrent negative (vehicle) groups is observed.
However, the results of statistical analysis may not be the only criteria in determination of the test substance potential to induce micronuclei. The following criteria may be taken into consideration: values that are statistically significant but do not exceed the range of historical negative or vechicle controls may be judged as not significant and relevant; a dose-dependent increase will also be taken in consideration in determination of the test substance positive response; if criteria for either a positive or negative genotoxic response are not met, the results are judged as equivocal or inconclusive.
Statistics:
Statistical analysis of data is performed using the Kastenbaum-Bowman tables which are based on the binomial distribution.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
In an in vivo micronucleus test on mouse bone marrow erythrocytes, performed according to OECD 474, the animals were exposed orally (via gavage) with 500, 1000, 2000 mg/kg. This did not induce a statistically positive increase in micronuclei in the hemopoietic cells of the mouse bone marrow at the time intervals evaluated under the experimental condition of this assay. No toxicity was observed. Vehicle and positive controls were valid.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:


In a bacterial reverse mutation assay performed according to OECD guideline 471 (Ames; BioReliance, 2011; Klimisch 1), the mutagenic potential of the test substance was investigated in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and E coli WP2 uvrA. Under the test conditions, the test substance demonstrated to be not mutagenic with and without metabolic activation. Toxicity or precipitation was not observed.


The test substance was tested in an in vitro mammalian cell gene mutation test (CHO/HGPRT test), performed according to OECD guideline 476 (Bioreliance, 2011; Klimisch 1). Under the conditions of the test, the test substance demonstrated to be negative with and without metabolic activation in the assay. Cloning efficiency was not affected by the substance as no excessive cytotoxicity was observed.


According to the REACH Regulation, no in vitro cytogenicity study in mammalian cells or in vitro micronucleus study needs to be conducted if adequate data from an in vivo cytogenicity test are available (Annex VIII, Column 2 adaptation).


Genetic toxicity in vivo:


In an in vivo micronucleus test on mouse bone marrow erythrocytes, performed according to the OECD guideline 474 (Bioreliance, 2011; Klimisch 2), the animals were exposed orally (via gavage) with 500, 1000 or 2000 mg/kg test item. This did not induce a statistically positive increase in micronuclei in the hemopoietic cells of the mouse bone marrow at the time intervals evaluated under the experimental condition of this assay. No toxicity was observed. Vehicle and positive controls were valid.

Justification for classification or non-classification

Based on the available data and according to the CLP criteria, the test substance should not be classified as mutagenic or clastogenic.