Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was examined for mutagenicity in vitro in bacteria and mammalian cells, for clastogenicity in mammalian cells and for micronuclei formation in mice. The Ames test and the mouse lymphoma assay yielded negative results for mutagenicity. Clastogenicity was seen at concentrations where precipitation and strong cytotoxicity was observed. Single high dose administration to mice did not induce micronuclei formation. The substance is therefore not considered to be genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
No indepedent repeat experiment for incubation with S9 mix. Dose with S9 mix should have been slightly higher. It resulted only in 24% viability instead of the required 10 - 20% viability.
GLP compliance:
yes (incl. certificate)
Remarks:
The report includes GLP certificates from the German, Dutch and Indian Authorities. All certificates are valid for the period of the study.
Type of assay:
mammalian cell gene mutation assay
Target gene:
tk - thymidinkinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Arochlor 1254 induced rat live
Test concentrations with justification for top dose:
17, 34, 68 and 136 µg/ml (3h, with S9)
55, 110, 220, 440 µg/ml (3h and 24h, without S9)
Vehicle / solvent:
DMSO

The results of the solubility test of Study No. 04733 for the same compound indicated that the test item forms a workable suspension with DMSO at the required concentration of 250000 /lg/mL. Also, DMSO is one of the organic solvents compatible with the test system. Hence, DMSO was selected as the vehicle to prepare the stock and dilutions ofthe test item as weil as the positive controls.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h and 24h
- Expression time (cells in growth medium): 48h

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT) at 4 mug/mL

NUMBER OF REPLICATIONS: tests were done in duplicate

DETERMINATION OF CYTOTOXICITY
- Method: cell growth
Evaluation criteria:
There are several criteria for determining a positive result, such as a concentration related, or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response. A test item, for which the results do not meet the above criteria is considered non mutagenic in this system.
Positive results for an in vitro mammalian cell gene mutation test indicate that the test substance induces gene mutations in the cultured mammalian cells
used. A positive concentration response that is reproducible is most meaningful. Negative results indicate that, under the test conditions the test
substance does not induce gene mutations in the cultured mammalian cells used.
Statistics:
The number of negative contraI values are assumed to follow Poisson Distribution. Test of significance of difference between negative control values over different dose groups and the positive control was carried out using a method of analysis suggested by M.R.Thomas (Cole and Arlett, 1984), based on chi2. All analysis and significance tests were evaluated at 5% level of signiftcance (P<0.05).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RTG was 24%
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Moderatetyl cytotoxic: RTG was 29 (24h) and 39 (3h). The next highest concentration was found to be non scorable based on precipitates upon centrifugation in pretests.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the presence of metabolie activation at the end of exposure to treatment, the pH of the test medium ranged between 7.05 to 7.22 with 7.10 in the DMS0 control. Similarly, in the absence of metabolic activation, the pH of the treatment medium ranged between 7.05 and 7.20 Wilh 7.21 in the DMSO control at the end of exposure to treatment.
- Precipitation: from 800 µg/ml onward, in presence and absence of metabolic activation

RANGE-FINDING/SCREENING STUDIES:
Preliminary test results of Study No. 04733 for the same test item indicated heavy precipitation of the test item at and above 2400 µg/ml. This precipitation interfered with the observation of the cells. Hence, the test item was checked fo it precipitation in the treatment medium (TM) up to the maximum concentration of 1600 µg/ml of the medium. At and abave 800 µg/mL, the test item precipitated In the form of a pellet in the centrifuge tubes both in the presence and absence of metabolic activation in the 3-hour as weil as in the 24-hour exposure regime. Hence cell counts could not be determined. Based on these observations, it was decided to test up to 136 µg/mL in the presence of metabolie aetivation for the 3-hour exposure and up to 440 µg/mL for the 3-hour as well as the 24-hour exposure in the absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, solvent and positive control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the preliminary toxicity test, the test item was toxic to the cells at and above 200 µg/mL when exposed for 3 hours in the presence of metabolic activation resulting in dead and disfigured cells. There was evidence of growth inhibition as relative total growth (RTG) at 100 µg/mL. However, this growth inhibition was not significant. In the 3-hour as well as the 24-hour exposure in the absence of metablic activation, there was evidence of growth inhibition as relativ total growth (RTG) at 400 µg/mL. However, this growth inhibition was not significant.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Presence of Metabolic Activation (Experiment 1)

Group Test item concn. (µg/mL) Non-selective medium     Selective medium    
P(0) individual plates
M.F x 10-6 M.F/survivor x 10-6 Induced M.F x 10-6 RTG
P(0) C.E. RCE R1 R2 Total
G1 DMSO (200 µI) 16/96 90 100 78/96 83/96 161/192 88 0.98 - 100
G2 17 18/96 84 93 79/96 84/96 163/192 82 0.98 - 69
G3 34 16/96 90 100 86/96 81/96 167/192 70 0.78 - 60
G4 68 20/96 78 88 89/96 80/96 169/192 64 0.81 - 43
G5 136 22/96 74 82 79/96 87/96 166/192 73 0.99 - 24
G6 CPA 12 23/96 71 80 23/96 36/96 59/192 590+ 8.26 7.28 43

Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Absence of Metabolic Activation (Experiment 2)

Group Test item concn. (µg/mL) Non-selective medium     Selective medium    
P(0) individual plates
M.F x 10-6 M.F/survivor x 10-6 Induced M.F x 10-6 RTG
P(0) C.E. RCE R1 R2 Total
G1 DMSO (200 µI) 14/96 96 100 78/96 84/96 162/192 85 0.88 - 100
G2 55 16/96 90 93 81/96 85/96 166/192 73 0.81 - 80
G3 110 16/96 90 93 83/96 80/96 163/192 82 0.91 0.03 62
G4 220 18/96 84 87 78/96 79/96 157/192 101 1.20 0.32 51
G5 440 18/96 84 87 76/96 78/96 154/192 110 1.32 0.44 39
G6 MMS 20 21/96 76 79 42/96 33/96 75/192 470+ 6.18 5.30 50

Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Absence of Metabolic Activation (Experiment 3)

Group Test item concn. (µg/mL) Non-selective medium     Selective medium    
P(0) individual plates
M.F x 10-6 M.F/survivor x 10-6 Induced M.F x 10-6 RTG
P(0) C.E. RCE R1 R2 Total
G1 DMSO (200 µJ) 15/96 93 100 80/96 83/96 163/192 82 0.88 - 100
G2 55 15/96 93 100 79/96 81/96 160/192 91 0.98 0.10 82
G3 110 18/96 84 90 86/96 76/96 162/192 85 1.01 0.13 74
G4 220 19/96 81 87 77/96 80/96 157/192 101 1.24 0.36 44
G5 440 20/96 78 85 81/96 84/96 165/192 76 0.97 0.08 27
G6 MMS20 20/96 78 85 36/96 32/96 68/192 519+ 6.62 5.74 29

M.F = Mutant Frequency

C.E = Cloning Efficiency

RCE = Relative Cloning Efficiency

RTG = Relative Total Growth

+ = Significantly higher than control (p ≤ 0.05)

Conclusions:
The study indicated that the test item does not have the potential to cause gene mutation at the tk locus at the concentrations tested and under the conditions of testing.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Information on purity and expiry date not given. Substance identified by Trade name, and the product specification for this is the substance (no formulation).
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Arochlor-induced rats
Test concentrations with justification for top dose:
31.3, 62.5, 125, 250, 500 and 1000 µg/mL for the non-activaled 4 hour exposure group
250, 500, 1000, 1250, 1500 and 2000 µg/mL repeat experiment for the non-activaled 4 hour exposure group
31.3, 62.5, 125, 250, 500, 1000, 1500 and 3000 µg/mL for the S9 activated 4 hour and the non-activated 20 hour exposure groups

Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 16 - 24h
- Exposure duration: 4h or 20h
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 500 (mitotic index), 200 metaphase cells

DETERMINATION OF CYTOTOXICITY
- Method: Trypane blue exclusion

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
The frequency of cells with structural chromosome aberrations in the solvent control must be witln the range of the historical solvent control. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p < 0.05, Fisher's exact test) relative to the solvent control.
Statistics:
Statistical analysis of the percent aberrant cells was perormed using the Fisher`s exact test.
Fisher`s exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher`s exact test at any test article dose level, the Cochrane Aritage test was used to measure dose-reponsiveness.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:not affected by the test item
- Effects of osmolality:not affected by the test item
- Precipitation: Visible precipitate was obsered in treatment medium at dose levels ≥ 62.5 µg/mL al the begining of the treatment period.

RANGE-FINDING/SCREENING STUDIES:
In the prelimar toxicity assay, the maxum dose tested was 5000 µg/mL. The test article formed a workable suspension in DMSO at concentrations ≥ 1500 µg/mL and was soluble in DMSO at concentrtions ≤ 500 µg/mL. Visible precipitate was obsered in treatment medium at dose levels ≥ 50 µg/mL at the beginning of the treatment period. Dose levels ≤ 15 µg/mL were soluble in treatment medium at the begining of the treatment period. Selection of dose levels for the chromosome aberration assay was based on cell grwth inhibition relative to the solvent control. Substantial toxicity (i.e., at least 50% cell growth inhibition, relative to the solvent control) was observed at dose levels ≥ 500 in the non-activated 4 hour exposure group and at dose levels ≥ 15 µg/mL in S9 activated 4 hour exposure grup. Due to excessive toxicity, no viable cells were observed at 5000 µg/mL in the non-activated 20 hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 31.3 to 1000 µg/mL for the non-activated 4 hour exposure group, and from 31.3 to 3000 µg/mL for the S9 activated 4 hour and the non-activated 20 hour exposure groups.

The reults of the assays are summarzed in the following table:

Treatment Time Recovery Time Harvest Time S9 Toxicity* at highest dose scored (µg/mL) Mitotic Index Reduction
**
LED for Structural Aberrations LED for Numerical Aberrations
4hr 16 hr 20hr - 50% at 2000 52% 2000 (µg/ml) None
20hr 0 hr 20hr - 6% at500  53% None None
4hr 16 hr 20hr + 35% at 250 51% None None

* cell growth inhibition

** relative to solvent control at high dose evaluated for chromosome aberrtions

LED = lowest effective dose

The perentage of cells with structural aberrations in the non-activated 4 hour exposure group was signficantly increased above thaI of the solvent control at 2000 µg/mL ( p< 0.05, Fisher's exact test). The Cochran-Armitage test was also positive for a dose reponse (p <0.05). The percentage of cells with numerical aberrations in the non-activated 4 hour exposur group was not signficantly increaed above that of the solvent control at any dose level (p<0.05, Fisher`s exact test).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1983
Deviations:
yes
Remarks:
other positive controls used as recommended
GLP compliance:
no
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): XR-1476
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9-mix
Test concentrations with justification for top dose:
1, 10, 50, 100, 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: TA1535 and TA100: MNNG; TA1537: 9-AA; TA1538 and TA98: 2-NF
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: TA1535: 2-AA; TA100 and TA98: Aflatoxin B1; TA1537 6-AC; TA1538: 2-AF
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours

NUMBER OF REPLICATIONS: three

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Before an agent is reported to be either active or inactive in the Salmonella/microsomal assay, the following criteria must be met:
All sterility controls must be negative for bacterial growth. The spontaneous revertants for each strain must be within acceptable limits. All 3 control plates must have approximately equal numbers of revertant colonies. The solvent controls must have approximately the same number of colonies as spontaneous reversion controls. Positive mutagens must give at least 4x the number of colonies as the controls for spontaneous reversion. Any test with a strain which does not meet these criteria must be repeated on a separate day.
All criteria noted above must be met before results with an unknown agent can be evaluated. To be considered positive, an unknown agent should exhibit a dose response effect. That is, there should be an increasing number of mutants with increased amounts of test agent. For strains TA1535, TA1537, TA1538 and TA98, the first positive dose must produce 3x the number of colonies as the negative control. For strain TA100, at least one dose tested must produce 3.5x the number of mutants as the spontaneous reversion control.
Statistics:
Linear Regression Analysis
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:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
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

Results without metabolic activation

Strain TA 1535 TA 1537 TA 1538 TA 100 TA 98
Dose (µg/plate)
Bacteria alone 30 5.7 3.7 154 11.7
DMSO 36.3 3.7 1.3 143.7 9.6
MNNG 1017 NT NT 1818.7 NT
9-AA NT 435.7 NT NT NT
2-NP NT NT 347.7 NT 372
1 35 3.7 4.7 136.3 13
10 42.7 3.3 4.3 135.3 12
50 34.7 2.7 4.7 119.7 9
100 38.3 2.3 3.7 143.3 7
1000 36 5 4.7 138.3 10

NT = not tested

Results with metabolic activation

Strain TA 1535 TA 1537 TA 1538 TA 100 TA 98
Dose (µg/plate) 30 5.7 3.7 154 11.7
Bacteria alone 43.3 10.7 18.3 179.9 37.7
DMSO 49 11.7 26.7 161.7 31
2-AA 323.3 NT NT NT NT
6-AC NT 325.7 NT NT NT
2-AF NT NT 524.3 NT NT
Aflatoxin NT NT NT 827 586.3
1 52.3 9 21.7 176.7 29
10 32.7 9 23.7 190 21
50 35.3 9.3 51 186.7 32.3
100 37.3 9 42 159.7 18
1000 54.3 9.7 34.7 175.3 26

NT = not tested

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Single high dose administration to mice did not induce micronuclei formation.

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:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD (USA)
- Age at study initiation: approximately 6 to 8 weeks old
- Weight at study initiation: male 28.5 - 32.5 g, 20.5 - 25.7 g
- Assigned to test groups randomly: random
- Fasting period before study:
- Housing: Mice of the same sex were housed up to five per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 °F
- Humidity (%): 50 - 20%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2003-07-28 To:2009-09-01
Route of administration:
intraperitoneal
Vehicle:
Corn oil
Details on exposure:
The test arcle-vehicle mixture, the vehicle alone, or the positive control was administered by a single intrperitoneal injection at a dose volume of 20 mUkg. Intraperitoneal injection was selected to maximize delivery of the test arcle to the test system. All mice in the experimental and control groups were weighed immediately before dose administration, and the dose volume was based on individual body weight. Mice were observed afer dose adnistration for clinical signs of toxicity.
Duration of treatment / exposure:
24h (all dose groups)
48h (high dose group)
Frequency of treatment:
single
Post exposure period:
24h (all dose groups)
48h (high dose group)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide was dissolved in sterile distiled water at a concentrtion of 2.5 mg/mL for use as the positive control.
Tissues and cell types examined:
Bone marow cells (polychromatic eryocytes, PCEs)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Range-finder study.

DETAILS OF SLIDE PREPARATION
At the scheduled sacrifice times, five mice per sex per tratment were sacrificed by CO2 asphyxation. Imediately following sacrifce, the femurs were distaly exposed, cut just above the knee, and the bone marow was aspirated into a syringe contaig fetal bovine serum. The bone marow cells were trasferrd to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. Two slides were prepard from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS
To contrl for bias, slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification (10 x 40), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (10 x 100), 2000 polychromatic eryrocytes per animal were scored for the presence of micronuclei. The number of micronucleated nonnochromatic erythrocytes in the field of 2000 polychromatic eryrocytes was enumerated for each animaL. The proportion of polychromatic eryocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Evaluation criteria:
All conclusions were based on sound scientific judgement; however. as a guide to interpretation of the data, the test arcle was considered to induce a positive response if a dose-reponsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p < 0.05, Kastenbaum-Bowman Tables) at any sampling time.
Statistics:
Kastenbaum-Bowman tables
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
piloerection was seen in male and female mice at 1000 and 2000 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the pilot toxicity study, two male mice each were exposed to the test article at a dose of 1, 10, 100, or 1000 mg/kg body weight while five male and five female mice were exposed to 2000 mg/kg. No mortity was observed during the course of the study and only piloerection was seen in male mice at 2000 mg/kg. In the absence of mortality in the pilot study, the high dose for the micronucleus test was set at 2000 mg/kg.

RESULTS OF DEFINITIVE STUDY
The definitive micronucleus study consisted of seven groups, each containing 5 male and 5 female ICR mice. Animals in five of these groups were treated either with the controls (negative or positive) or with the test item at a dose of 500, 1000, or 2000 mg/kg and were euthanized 24 hour after treatment. Animals in other two groups were treated either with the negative control or the test item at a dose of 2000 mg/kg and were euthanized 48 hours afer treatment. No mortality was observed in any male or female mice in the micronucleus study while piloerection was seen in male and female mice at 1000 and 2000 mg/kg. Bone marow cells (polychromatic eryocytes, PCEs), collected 24 and 48 hours after tratment, were examined microscopically for the presence of micronuclei (MPCEs/2000 PCEs/animal). No appreciable reduction in the ratio of polychromatic eryhrocytes to total eryhrocytes was
observed in the test article-treated groups relative to the vehicle control groups suggesting that the test arcle did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control groups was observed in maIe or female mice at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman Tables).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro assays

Mutagenicity of the test item in vitro was examined in an Ames test (prior-GLP, euivalent to OECD guideline 471). The material, dissolved in DMSO, was tested in 5 Salmonella strains in presence and absence of a metabolic activation system at concentrations up to 1000 µg/plate. All experiments were performed in triplicates. Mutagenicity was not detected; the material was also not cytotoxic but tested up to the limit concentrations.

Clastogenicity in mammalian cells was examined in a chromosome aberration assay (according GLP and OECD guideline 473). The material was tested in CHO cells at concentrations up to 3000 µg/ml in presence and absence of a metabolic activation system. Precipitation of the test substance was observed from 50 µg/ml onward; cytotoxicity was recorded at concentrations from 15 µg/ml onward. The test item was found to induce chromosomal aberrations at 2000 µg/ml in absence of S9 mix. Mutagenicity in mammalian cells was investigated in a GLP and OECD 476 guideline conform in vitro assay. The material, dissolved in DMSO, was applied to mouse lymphoma cells up to concentrations of 4400 µg/ml in presence and absence of a metabolic activation system. Precipitation was observed at concentrations of 800 µg/ml and above. The test item was of moderate cytotoxicity and was not found to be mutagenic in mammalian cells.

In vivo study

The test substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in corn oil, was administered once orally to male and female mice at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration. 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. An increase in the number of polychromatic erythrocytes containing micronuclei was not detected when compared to control. Piloerection in male and female animals at 1000 and 2000 mg/kg bw was observed.

Discussion

The test substance was found to be negative for mutagenicity in bacteria and in mammalian cells as well as for genotoxicity in a micronucleus assay in mice. Formation of chromosomal aberrations was observed in mammalian cells at concentrations were precipitation and high cytotoxicity was recorded. It is therefore difficult to decide whether the aberrations originate from interactions of the substance with DNA or from cytotoxicity. However, the result of the in vivo micronucleus assay clearly showed that single substance application of high doses did not result in micronucleus formation. The test item is therefore not considered to be genotoxic.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.