1,4-Dihydroxy-2,2,6,6-tetramethylpiperidine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity (bacterial reverse mutation assay) = Negative, OECD 471,

Genetic Toxicity (in vitro mammalian chromosome aberration test) = Positive; OECD 473 (read-across)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test (with and without S9 mix): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 1 and Experiment 2 (with and without S9 mix): 50, 150, 500 and 1500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of bacterial strains

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

The positive controls 2AA and BP were used in the series of plates with S9-mix.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of bacterial strains

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

The positive controls ENNG, 9AA and 4NQO were used in the series of plates without S9-mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar, direct plate incorporation for Experiment 1 and Experiment 2.

DURATION
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
-All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks
-All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
-All tester strain cultures should be in the range of 1 to 9.9 x 10E9 bacteria per ml.
-Each mean positive control value should be at least two times the respective control value for each strain, thus demonstrating both the intrinsic sensitivity ofthe tester strains to mutagenic exposure and the integrity of the S9-mix.
-There should be a minimum of four non-toxic test item dose levels.
-There should be no evidence of excessive contamination.

Evaluation Criteria:
The test material should have induced a reproducible, dose related and statistically significant increase in revertant count in at least one strain of bacteria..

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

Remarks:

Tested up to the maximum recommended dose level of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to the maximum recommended dose level of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST:
The test material was non-toxic to the strains of bacteria (TA100 and WPuvrA-). The test material formulation and the S9-mix used in this experiment were both shown to be sterile.

The number of revertant colonies for the toxicity are shown in the attached background information.

MUTATION TEST:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in the attached background information.and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in the attached background information.).

A history profile of vehicle and positive control values is presented in the attached background information..

No toxicity was exhibited to any of the strains of bacteria used. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B14 of Commission Directive 92/69/EECand the USA, EPA (TSCA) OPPTS harmonized guidelines.

Methods

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia colistrainWP2uvrA were treated with the test item using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and ranged between 50 and 5000µg/plate, in the first experiment. The experiment was repeated on a separate day using the same dose range as in experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.

 

Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused no visible reduction in the growth of the bacterial lawn at any dose level. The test material was therefore tested upto the maximum recommended dose of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial starins, with any dose of the test material, either with or without metabolic activation.

Conclusion

The test item was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Refer to Section 13.2 for the read-across justification.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

(clone No 11)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on a read-across study, the test item induced structural chromosomal aberrations dose dependently within the dose range 150 - 600 μg/ml in the 24 hour treatment and within the dose range 200 - 400 μg/ml in the 48 hour treatment by the direct method and within the dose range 400 - 800 μg/ml by the metabolic activation method. The conclusion is considered valid for this substance based on a one-to-one read-across from an analogue substance.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

other: Notification on Partial Revision of Testing Methods Relating to New Chemical Substances (Notification No 700 of Kanpogyo, No 1039 of Yakuhatsu and No 1014 of 61 Kikyoku, December 5 1986

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

other: Chinese Hamster Lung Fibroblasts (Clone No 11)

Details on mammalian cell type (if applicable):

Chinese Hamster Lung (chl) cells were supplied by National Insitute of Hygienic Sciences on September 28 19988. The modal number of chromosomes is 25 per cell. The time required for doubling the cell number is about 15 hours. CHL cells preserved by freezing were defrosted and cultured. Three days after incubation, a subculture was made, and the cells in logarithmic growth phase were used for the test. The passage number of cells used in the chromosomal aberration test was 26 for 24 and 48-h treatment by the direct method, and 26 by the metabolic activation method.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and 5, 6 benzoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test (Cell Growth Inhibition Test, cell division inhibition):
The dose range of test item used was 0, 100, 200, 400, 600, 800, 1000, 1200, 1400 and 1600 µg/ml.

24-hour tretament without metablic activation: 0, 150, 300 and 600 µg/ml
48-hour treatment withou metabolic activation: 0, 100, 200 and 400 µg/ml

With metabolic activation: 0, 200, 400 and 800 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: pure water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Used in the absence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Used in the presence of S9

Details on test system and experimental conditions:

Cell culture
5ml of 10% Newborn Calf Serum/Minimum Essential Medium (NCS/MEM) containing 15000 or 5000 cells/ml ws plated in Petri dish and cultured for 2 or 3 days. Monolayer cells in logarithmic growh phase were exposed to the test item at 2 or 3 days after culturing under the following conditions
Temperature: 37 ± 0.5°C
Humidity : almost 100%
Atmosphere: air containing 5% CO2

Test substance and positive control solutions
Test solutions: The test substance was dissolved in pure water. The solution was diluted with pure water to make the required concentrations and added to the medium at the rate of 1%. Test solutions were prepared immediately before the start of each test and used within 2 hours.

Positive control solutions: Positive control substances were dissolved in pure water and diluted with pure water to the required concentration. Positive control solutions were prepared immediately before the start of each test and used witin 2 hours.


Cell Growth Inhibition Test : Cell cultures were exposed to the test material for 24 or 48 hours. Test with metabolic activation were treated for 6 hours. At the end of the treatment period S9 Mix / MEM and test material was removed and the dishes rinsed. 5ml 10% NCS/MEM was then added to the dishes and the cell in cubated for 18 hours. At the end of incubation, the medium was removed and the cells detached from each dish with 2ml of trypsin and counted using a Microcell Counter. Two dishes were used and counted for each concentration.


Cell Division inhibition test: Cell culture and treatment were as for the cell growth inhibition test. Two hours prior to the end of incubation, colcemid was added to the medium. Chromosome preparations were made and observed microscopically for the incidence of mitotic cells.


CHROMOSOMAL ABERRATION TEST :
Test substance - the test was carried out at 3 dose levels prepared by diluting the maximum test concentration. The treatment regimes were-
24 hour treatment by the direct method at 150, 300 and 600 μg/l.
48 hour treatment by the direct method at 100, 200 and 400 μg/l.
6 hour treatment by the meatbolic activation method at 200, 400 and 800 μg/l.

Positive control substances - The following test methods and concentrations were used
Mitomycin by the direct method 0.05μg/l.
Cyclophosphamide by the metabolic activation method 10μg/l.

Direct method
Cell cultures were exposed to the test material for 24 or 48 hours. Two hours prior to the end of incubation colcemid was added to the medium after which chromosome preparations were made
A vehicle (pure water) control and Mitomycin positive control groups were maintained under identical conditions but not exposed to the test item.

Metabolic activation method
Cell cultres treated with S9 Mix/MEM were exposed to the test material for 6 hours after whch the S9 Mix/MEM including the test material was removed and the dishes rinsed. 5ml of 10% NCS/MEM was then added to the dishes and the cells were incubated for 18 hours. Two hours prior to the end of incubation colcemid was added. Chromosome preparations were made at the end of incubation.

A vehicle control (pure water) and Cyclophosphamide positive control groups were maintained under identical conditions but not exposed to the test item.
A culture without S9 Mix was treated with test substance at the same concentrations and treatment times to clarify the effect of metabolic activation.

NUMBER OF REPLICATIONS: Two


NUMBER OF CELLS EVALUATED:
100/culture, 200/ test concentration

OBSERVATION OF CHROMOSOMAL ABERRATIONS:
The number of cells with numerical aberrations (polyploid, endoreduplication) and with chromatid-type or chromosome-type structural aberrations ( gap, break, exchange etc) were checked by observing 100 well-spread metaphases from each culture. The incidence of cells with numerical and / or structural aberrations was obtained by observing 200 metaphases for each dose. The structural aberration incidence was calculated with and without gaps.
A gap was only scored when a clear discontinuity (larger than a chromatid width) was evident and when the distal part of the chromatid or chronosome showed no dislocation. Microscope slides were coded and scored blind.

Evaluation criteria:

The results were assessed as follows. The incidence of cells (mean value for two flasks) with aberrations including gaps was
less than 5% - Negative (-)
5% or more but less tha 10% - Suspect Positive (±)
10% or more but less than 20% - Positive (+)
20% or more but less than 50% - Positive (++)
50% or more - Positive (+++)

The test substance is judged to be positive for induction of chromosomla aberration when the incidence of 10% or more was dose related or reproducible. Reexamination was performed whenever the result was suspect positive, or positive at only one dose level.

Species / strain:

other: Chinese Hamster Lung Fibroblast (clone No 11)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The values for the two dishes were not markedly different, the incidence of cells with aberrration did ot exceed 5% in the negative (vehicle) control groups, the incidence of cells with structural aberrations excluding gap was 10% or more in the positive control groups and there was no contamination with microorganisms in the cultures. The test resuts were therefore considered to be valid.

Inhibition test on cell growth and cell division
Cell growth inhibition see Table 1 and Fig 1 in attached background information
The growth rate in the vehicle control was considered to be 100%. 50% growth inhibition concentrations of the test substance were about 400 μg/ml for 24 hour treatment and about 120 μg/ml for 48 hour treatment by the direct method, and about 1000μg/ml by the metabolic activation method.
The cell division inhibition test was carried out at several concentrations close to the LC50 to determine the possible dose level in the chromosomal aberration test. Mitotic metaphases of chromosomes sufficient to assess chromosomal aberration were observed at doses of 400 μg/ml or less for 24 hour treatment, 800 μg/ml or less for 48 hour treatment by the metabolic activation method and 600 μg/ml or less by the metabolic activation method. Therefore 600 μg/ml for 24 hour treatment and 400μg/ml for 48 hour treatment by the direct method, and 800 μg/ml by the metabolic activation method were used as the maximum concentrations in the chromosomal aberration test. Additionally, two lower concentrations with geometric progression of 2 were used in each treatment.

Chromosomal aberration test
Direct method (see table 2 and Fig 2 in attached background information.)
-24 hour treatment, The incidence of cells with structural chromosomal aberrations including gaps were
150μg/ml = 5.0% , suspect positive
300μg/ml = 13.0% , positive
600μg/ml = 39.5%, positive
The incidence of structural aberration was dose related
The incicence in the vehicle control was 1.5% and within normal range,
The incidence in the positive control group treated with Mitomycin was 55%, indicating induction of chromosomal aberration.
The incidence of polyploid cells was negative in each treatment group.
-48 hour treatment, The incidence of cells with structural chromosomal aberrations including gaps were
100μg/ml = 2.50% , negative
200μg/ml = 7.5% , suspect positive
400μg/ml = 59.5%, positive
The incicence in the vehicle control was 1.0% and within normal range,
The incidence in the positive control group treated with Mitomycin was 90.5%, indicating induction of chromosomal aberration.
The incidence of polyploid cells was negative in each treatment group.

Metabolic activation method (Table 3 and Fig 3)
With S9 Mix -The incidence of cells with structural chromosomal aberrations including gaps were
200μg/ml = 4.5% , negative
400μg/ml = 26.0% ,positive
800μg/ml = 94.0%, positive
The incidence of structural aberration was dose related
The incicence in the vehicle control was 3.0% and within normal range,
The incidence in the positive control group treated with Cyclophophamide was 67.5%, indicating induction of chromosomal aberration.
The incidence of polyploid cells was negative in each treatment group.
Without S9 Mix -The incidence of cells with structural chromosomal aberrations including gaps were
200μg/ml = 4.5% , negative
400μg/ml = 2.0% , negative
800μg/ml = 1.5.0%, negative
The incidence of structural aberration was dose related
The incicence in the vehicle control was 2.5% and within normal range,
The incidence in the positive control group treated with Cyclophophamide was 3.0% and within normal range.
The incidence of polyploid cells was negative in each treatment group.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The test item induced structural chromosomal aberrations dose dependently within the dose range 150 - 600 μg/ml in the 24 hour treatment and within the dose range 200 - 400 μg/ml in the 48 hour treatment by the direct method and within the dose range 400 - 800 μg/ml by the metabolic activation method.

Executive summary:

Introduction. 

The effect of the test item on chromosomal aberration induction was investigated using Chines hamster lung fibroblasts (CHL cells). The method was conducted in accordance with the Notification on Partial Revision of Testing Methods Relating to New Chemical Substances (Notification No 700 of Kanpogyo, No 1039 of Yakuhatsu and No 1014 of 61 Kikyoku, December 5 1986, complaint with OECD guidelines.

Methods. 

Chromosomal aberration tests were carried out at doses of 150, 300 and 600 μg/ml for 24 hour treatment, at 100, 200 and 400 μ

g/ml for 48 hour treatment by the direct method (without metabolic activation), and at 200, 400 and 800 μg/ml by the metabolic activation method. Positive control Mitomycin was used at 0.05 μg/ml for 24 and 48 hour treatment by the direct method, and Cyclophophamide was used at 10 μg/ml by the metabolic activation method.

Results.

The test item induced structural chromosomal aberrations dose dependently within the dose range 150 - 600 μg/ml in the 24 hour treatment and within the dose range 200 - 400 μg/ml in the 48 hour treatment by the direct method and within the dose range 400 - 800 μg/ml by the metabolic activation method.

The incidence of polyploid ccells was negative in each treatment group.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the normal range.

The positive control groups induced chromosomal aberrations.

Conclusion.

The test item was considered to be clastogenic to CHL cells in vitro

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Genetic Toxicity (in vivo mammalian erythrocyte micronucleus test) = Negative; OECD 474

Genetic Toxicity (in vivo mammalian erythrocyte micronucleus test) = Negative; ISO 10993-3:2003

Link to relevant study records

Reference

Reference 1

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:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Korea National Institute of Environmental Research Public Notice No 2006-4, Annex 5 Genotoxicity Test and SOPs of KTR.

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species: Mouse, ICR (Crl:CD l BR), male
- Source: Orient Incorporation, 699-13 Mokdong-Ri, Buk-Myeon, Gapyoung-Gun, Kyonggi-do, Korea
- Age at study initiation: 6 weeks at reception
- Weight at study initiation: 21.83 to 26.81g
- Assigned to test groups randomly: Yes, stratified random sampling method with the index of animal bodyweight and excluding animals with clinical signs
- Housing: 6 animals / polycarbonate cage
- Diet (e.g. ad libitum): Pelleted food for experimnetal use, (Purina Korea Incorporation, 85-1 Jangdang-Dong, Pyongtaek-Si, Kyonggi-do, Korea) ad libitum
- Water (e.g. ad libitum): Tap water purified by fitration ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 to 70
- Air changes (per hr): 12 to 15 times / hour
- Photoperiod (hrs dark / hrs light): twelve hours light and twelve hours darkness.

Route of administration:

intraperitoneal

Vehicle:

Water

Details on exposure:

Groups, each of six male mice, were dosed once only via the intraperitoneal route with the test item at 0, 137.5, 275 or 550 mg/kg. A further group of six males was treated with the positive control Cyclophosphamide.The animals were killed by cervical dislocation 24 hours following treatment.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Once only

Post exposure period:

24 Hours

Remarks:

Doses / Concentrations:
137.5, 275 or 550 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

6 males / group

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control item was supplied by Sigma Chemical Co, as follows:

Supplier's identification: Cyclophosphamide
Supplier's lot number: 87H0207
Purity: 98%
Storage conditions: Refrigeration 4ºC

For the purpose of this study the positive control item was dosed at 10 ml/kg at a dose level of 70 mg/kg.

Tissues and cell types examined:

Mammalian erythrocytes.

Details of tissue and slide preparation:

The animlas were sacrificed 24 hours after administration, the femur was removed and both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone marrow was flushed with foetal bovine serum. The cells were collected to the centrifuge tube and centrifuged at 1000rpm for 5 minutes. The supernatant was discarded and the concentrated cells were suspeneded with a little fresh serum. The cell suspension was smeared on a clean slide. The smeared slide was air dried and therafter fixed for 5 minutes with methanol and stained with 40μg/ml acridine orange.

Evaluation criteria:

Stained bone marrow smears were coded and examined blind using fluorescent microscope at magnification over 400X.
Criteria for judgement
Polychromatic erythrocytes (PCE) are determined by appearing orange fluorescent light without nuclei

Nonchromatic erythrocytes (NCE) are determined by appearing only their black shadows without fluorescent light

Criteria for Micronuclei
-Size : From as small as distinguishable to as large as a half the diameter of erythrocytes
-Shape: mainly round and included the form of a donut shape, half moon shape
-Colour: THe same colour with near cell nucleus: green fluorescent in acridine orange

Observation of Specimen : Micronucleated polychromatic erythrocytes (MNPCE) were counted in 2000 polychromatic erythrocytes per animal. Additionally Polychromatic erythrocyte frequency in 100 whole erythrocytes (PCE+NCE) was calculated

Statistics:

Statistical analyses were determined on the base of P value 0.05 between the vehicle control's micronucleated polychromatic erythrocyes and the test item groups. The determination of dose dependence was by trend test according to the statistical results.

Sex:

male

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The frequencies of MNPCE were
Negative control = 0.13% ± 0.05%
137.5 mg/kg = 0.14 ± 0.05%
275 mg/kg = 0.13% ± 0.04%
550 mg/kg = 0.16 ± 0.05%
Positive control = 6.76 0.65%
There was no statistically significant difference at any dose level of the test item.
The positive control showed a statistically significant increase (p<0.01)

The ratio of PCE's to total erythrocytes as an index of bone marrow toxicity, were
137.5 mg/kg = 50.81
275 mg/kg = 51.28
550 mg/kg = 50.48
Positive control = 49.38
There was no significant difference in the ratio between any test item treated group and negative control


There were no deaths. However in some test item groups there was a statistically related decrease in bodweights compared to the vehicle control.

See attached background information

Conclusions:

Interpretation of results (migrated information): negative
The test item was considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction. 

The study was conducted to determine if the test material would cause genotoxic changes in chromosomes or the miotic apparatus of murine polychromatic erytjhrocytes The study was conducted in accordance with toxicity test guideline of Korea National institute of Environmnetal Research Public notice No 2006 -4, Annex 5 Genotoxicity test and the method was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No.474 "Mammalian Erythrocyte Micronucleus Test".

Methods. 

The test item was administered once intraperitoneally to groups of 6 male rats at a dose of either 0, 137.5, 275 or 550. Animals were killed 24 hours later, the bone marrow extracted, and smear preparations made and stained. The number of Micronucleated Polychromatic Erythrocytes was counted in 2000 Polychromatic Erythrocytes.

Results. 

There was no statistically significant increase in the frequencies of Micronucleated Polychromatic Erythrocytes at any dose level tested. and no significant difference was observed in the ratio of Polychromatic Erythrocytes to total erythrocytes between the test item groups and the vehicle control.

A statistically significant increase was observed in the positive control group hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusion. 

The test item was considered to be non-mutagenic under the conditions of the test.

Reference 2

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Non GLP study conducted in compliance with agreed protocols.

Qualifier:

according to guideline

Guideline:

other: ISO 10993-3:2003

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

not specified

Sex:

male/female

Route of administration:

intraperitoneal

Vehicle:

0.9% sodium chloride

Details on exposure:

Definitive Mutagenicity Study
Groups, of ten animals of each sex, were dosed once only via the intraperitoneal route at a volume of 20ml/kg with the test item at 0, 55.3, 174.8, or 552.3 mg/kg in males and 0, 29.8, 94.4, or 298.3 mg/kg in females. Positive control groups consisting of five animals of each sex were treated with cyclophosphamide at either 15 or 75 mg/kg. Half of the animals treated with the test item or negative control were sacrificed after 24 hours following treatment / induction. All of the positive control groups were sacrificed twenty four hours after treatment. The remaining mice in the test item groups and the negative control were sacrificed ater a forty eight hour induction..

Duration of treatment / exposure:

24 hours & 48 hours

Frequency of treatment:

Once only

Remarks:

Doses / Concentrations:
55.3, 174.8 or 552.3 mg/kg males
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
29.8, 94.4 or 298.3 mg/kg females
Basis:
nominal conc.

No. of animals per sex per dose:

10 males and 10 females for test item and negative control groups
4 males and 5 females for positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control item was Cyclophosphamide

For the purpose of this study the positive control item was dosed at a dose level of 15 0r 75 mg/kg.

Tissues and cell types examined:

Mammalian erythrocytes.

Details of tissue and slide preparation:

Once the mice were sacrified one femur was removed from each animal and the bone marrow was flushed with foetal bovine serum. Clean glass slides were labelled with a blind code. Immediately after harvest three smears were prepared from the marrow obtained from each mouse. The slides were then fixed for five minutes in methanol and air dried. Slides were stained with 0.24mM Acridine Orange fluorescent stain and visualised under 40x power on a fluorescent microscope.

Evaluation criteria:

2000 PCE from each animal was scored for the presence of micronuclei.
The proportion of PCE to 500 mature erythroctes was also determined on each slide scored as an estimation of toxicity

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose Range Finding studies
Initial study
Severe signs of toxicity were noted immediatley after treatment at 707mg/kg for both males and females. Several animals died within minutes of dosing. Remaining mice from the 707 mg/kg dose group were terminated.
Second study
Dose levels of 223.7, 298.3, 447.5 and 552.3 were evaluated. Females died within 15 minutes of treatment

Definitive Mutagenic Study
Based on the results of the dose range finding studies to determine the maximum tolerated dose, dose levls of 55.3, 174.8 and 552.3 mg/kg in males and 29.8, 94.4 and 298.3 mg/kg in females was chosen for mutagenic analysis.
Within 15 minutes of intraperitoneal injection 3 males from the top dose level of 552.3 mg/kg had died. All other animals appeared healthy.

The erythropoietic ratios in animals treated with the test item were not significantly different from those in the negative controls

There was no increses in mPCE production in the test item treated groups compared to the concurrent negative controls.

Treatment with the high dose of Cyclophosphamide led to a significant increase in micronuclei induction in both males and females. Accodingly, this assay met the criterial for a valid assy.

See attached background information

Conclusions:

The test item was considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction. 

The study was conducted to evaluate the potential of the test material to induce in vivo clastogenic events or damage to the mitotic spindle in polychromatic erythrocytes obtained from mouse bone marrow. (Schmid et al 1976).

Methods. 

Ten mice of each sex were treated with the test item at 0, 55.3, 174.8 or 552 mg/kg in males and 29.8, 94.4 or 298.3 mg/kg in females. Positive control groups consisted of five animals / sex. Half of the animals in the test item or negative control group were sacrifcied at either 24 or 48 hours after treatment. All mice in the positive control groups were sacrificed 24 hours after treatment. Once the mice were sacrificed, the bone marrow was extracted from one femur and smear preparations made and stained. The number of Micronucleated Polychromatic Erythrocytes was counted in 2000 Polychromatic Erythrocytes and the proportion of PCE to 500 mature erythrocytes was also determined on each slide as an estimation of toxicity.

Results. 

There was no statistically significant increase in the frequencies of Micronucleated Polychromatic Erythrocytes at any dose level tested. and no significant difference was observed in the ratio of Polychromatic Erythrocytes to total erythrocytes between the test item groups and the vehicle control.

A significant dose response was induced by the positive control Cyclophosphamide, mice treated with 70 mg/kg showed mPCE rates that were significantly higher than those in the negative controls.

The negative control group showed spontaneous production of micronuclei within or slightly below the acceptable range.

Conclusion. 

The test item was considered to be non-mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Not applicable, the substance is not considered mutagenic.

Additional information

In Vitro

Ames Test

A "Bacterial Reverse Mutation Test", was performed using Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia colistrainWP2uvrA. The test was performed using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose ranged between 50 and 5000µg/plate, in the first experiment. The experiment was repeated on a separate day using the same dose range as in experiment 1.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.The test item caused no visible reduction in the growth of the bacterial lawn at any dose level. The test material was therefore tested upto the maximum recommended dose of 5000μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial starins, with any dose of the test material, either with or without metabolic activation.

The test item was considered to be non-mutagenic under the conditions of this test.

Chromosome aberration test (Read Across)

The effect of the test item on chromosomal aberration induction was investigated using Chines hamster lung fibroblasts (CHL cells).

Chromosomal aberration tests were carried out at doses of 150, 300 and 600μg/ml for 24 hour treatment, at 100, 200 and 400 μ

g/ml for 48 hour treatment by the direct method (without metabolic activation), and at 200, 400 and 800μg/ml by the metabolic activation method. Positive control Mitomycin was used at 0.05μg/ml for 24 and 48 hour treatment by the direct method, and Cyclophophamide was used at 10 μg/ml by the metabolic activation method.

The test item induced structural chromosomal aberrations dose dependently within the dose range 150 - 600 μg/ml in the 24 hour treatment and within the dose range 200 - 400 μg/ml in the 48 hour treatment by the direct method and within the dose range 400 - 800 μg/ml by the metabolic activation method. The incidence of polyploid cells was negative in each treatment group.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the normal range.The positive control groups induced chromosomal aberrations.

The test item was considered to be clastogenic to CHL cells in vitro.

In Vivo

Mammalian Erythrocyte Micronucleus Test (1)

The study was conducted to determine if the test material would cause genotoxic changes in chromosomes or the miotic apparatus of murine polychromatic erytjhrocytes.The test item was administered once intraperitoneally to groups of 6 male rats at a dose of either 0, 137.5, 275 or 550. Animals were killed 24 hours later, the bone marrow extracted, and smear preparations made and stained. The number of Micronucleated Polychromatic Erythrocytes was counted in 2000 Polychromatic Erythrocytes.

There was no statistically significant increase in the frequencies of Micronucleated Polychromatic Erythrocytes at any dose level tested. and no significant difference was observed in the ratio of Polychromatic Erythrocytes to total erythrocytes between the test item groups and the vehicle control. A statistically significant increase was observed in the positive control group hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. 

The test item was considered to be non-mutagenic under the conditions of the test.

Mammalian Erythrocyte Micronucleus Test (2)

Ten mice of each sex were treated with the test item at 0, 55.3, 174.8 or 552 mg/kg in males and 29.8, 94.4 or 298.3 mg/kg in females. Positive control groups consisted of five animals / sex. Half of the animals in the test item or negative control group were sacrifcied at either 24 or 48 hours after treatment. All mice in the positive control groups were sacrificed 24 hours after treatment. Once the mice were sacrificed, the bone marrow was extracted from one femur and smear preparations made and stained. The number of Micronucleated Polychromatic Erythrocytes was counted in 2000 Polychromatic Erythrocytes and the proportion of PCE to 500 mature erythrocytes was also determined on each slide as an estimation of toxicity.

There was no statistically significant increase in the frequencies of Micronucleated Polychromatic Erythrocytes at any dose level tested. and no significant difference was observed in the ratio of Polychromatic Erythrocytes to total erythrocytes between the test item groups and the vehicle control. A significant dose response was induced by the positive control Cyclophosphamide, mice treated with 70 mg/kg showed mPCE rates that were significantly higher than those in the negative controls.The negative control group showed spontaneous production of micronuclei within or slightly below the acceptable range.

The test item was considered to be non-mutagenic under the conditions of the test.



Justification for selection of genetic toxicity endpoint 


In vivo and in vitro studies are available. The in vivo micronucleus test for substance is selected as it is the most sensitive endpoint tested and performed according to OECD and/or EC guidelines and to GLP principles

Justification for classification or non-classification

The substance produced negative results in the Ames test but a positive result in the read across in vitro chromosome aberration test. This result is mitigated by the negative result in the in vivo micronucleus test tests investigating the same endpoint. Therefore, the substance is not considered to exhibit a potential for genetic toxicity.

The test substance is not classified for genetic toxicity according Regulation (EC) No. 1272/2008.