Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative induction of mutation frequence.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The reliability of the original study was established to be R1: Justification for read-across is detailed at section 13.
Justification for type of information:
Justification for read-across is detailed at section 13.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Cytotest Cell Research GmbH & Co. KG
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: HAM's F12 (Seromed, D-1000 Berlin, FRG) supplemented with 10 % fetal calf serum (FCS; Seromed)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 microsomal fraction
Test concentrations with justification for top dose:
Experiment 1
30, 100, 200, 300, 400, 800 µg/ml (without S9 mix)
1, 5, 20, 100, 1000, 2000, 2700, 3420 µg/ml (with S9 mix)

Experiment 2
80, 300, 600, 800, 1000, 1200 µg/ml (without S9 mix)
342, 1000, 1692, 2000, 2700, 3420 µg/ml (with S9 mix)

Experiment 3
1, 5, 10, 20, 30, 50 µg/ml (with S9 mix)
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: substance is not soluble in water.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 or 16 days; colony staining with 10 % methylene blue in 0.01 % KOH solution.

NUMBER OF REPLICATIONS: in duplicate per experimental point

NUMBER OF CELLS EVALUATED: 10^6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency.
Evaluation criteria:
Test article is classified as positive if it induces either a significant concentration-related increase in the mutant frequency or a reproducible and significant positive response for at least one of the test points.
A test article producing neither a significant concentration related increase in the mutant frequency nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
Statistics:
Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduced plating efficiency observed in some plates with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRETEST
In a pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test article was observed and compared to the controls. Toxicity of the test article was evidenced by a reduction in plating efficiency (PE). The plating efficiency of the CHO cells was reduced after treatment with 1000 µg/ml (without metabolic activation). With metabolic activation toxicity was observed after treatment with 2000 µg/ml and between 40 µg/ml and 300 µg/ml. Therefore, the first experiment was performed with six (without metabolic activation) and eight concentrations (with metabolic activation) ranging from 1 to 3420 µg/ml.
Remarks on result:
other: all strains/cell types tested

Results:

Experiment µg/ml S9 mix mean number cells/flask factor mean number mutant colonies/flask mean number mutant colonies/10^6 cells
seeded found
1 negative control - 542 280,5 0,52 2.8 ± 0.8 13,8
600, EMS - 530 186 0,35 61.0 ± 6.6 542,9
30 - nc
100 - 618 298,5 0,48 1.4 ± 1.1 7
200 - 608 362 0,6 2.4 ± 2.1 9,4
300 - nc
400 - 612 459 0,75 5.2 ± 2.9 17,8
800 - 610 423,5 0,69 3.8 ± 1.3 13,4
negative control + 498 289,5 0,58 2.6 ± 1.1 12,1
solvent control + 498 260,5 0,52 0.2 ± 0.4 0,8
3850, DMBA + 519 123 0,24 109.4 ± 14.2 1215,6
1 + nc
5 + nc
20 + nc
100 + 508 221 0,44 4.6 ± 2.9 28,6
1000 + 525 233,5 0,44 1.2 ± 1.1 7,6
2000 + nc
2700 + 454 273 0,6 0.8 ± 0.8 3,1
3420 + 492 264 0,54 2.6 ± 1.7 13,5
2 negative control - 519 282,5 0,54 3.2 ± 2.9 13,8
600, EMS - 506 148,5 0,29 142.6 ± 3.5 1343,5
80 - 529 253,5 0,48 1.4 ± 0.9 6,7
300 - 504 232 0,46 1.4 ± 1.7 7,5
600 - nc
800 - 522 294 0,56 0.2 ± 0.4 0,8
1000 - 507 282 0,56 0.4 ± 0.5 1,7
1200 - nc
negative control + 506 267 0,53 2.0 ± 0.7 8,4
solvent control + 514 258 0,5 1.8 ± 1.1 8
3850, DMBA + 500 159 0,32 153.0 ± 14.8 1048,5
342 + 511 236,5 0,46 1.2 ± 1.3 5,8
1000 + nc
1692 + 525 211 0,4 0.2 ± 0.4 1,3
2000 + nc
2700 + 515 224,5 0,44 0.2 ± 0.4 1
3420 + 503 251,5 0,5 2.0 ± 2.0 8,8
3 negative control + 530 341 0,64 1.2 ± 0.8 4,5
solvent control + 506 314 0,62 0.4 ± 0.5 1,7
3850, DMBA + 504 298 0,59 77.2 ± 2.9 320,7
1000 + 531 290,5 0,55 0.4 ± 0.5 2,1
5000 + nc
10000 + 525 320 0,61 1.6 ± 1.5 6,2
20000 + 504 260,5 0,52 0.2 ± 0.4 0,9
30000 + 532 313 0,59 0.6 ± 0.5 2,4
50000 + 519 264 0,51 0.4 ± 0.5 1,9

nc: culture not continued

Conclusions:
No induction of gene mutation at the HGRPT locus in CHO cells.
Executive summary:

The potential of the test substance to induce gene mutations at the HGPRT locus using chinese hamster ovary cells was tested in vitro with and without metabolic activation according to OECD guideline 476 (tested up to 3420 μg/ml). Cytotoxicity was observed in an intermediate concentration range around 20 µg/ml of test substance, presumably induced by inhibition of enzymes of the S9-mix. The test substance showed no mutagenic effects on the HGPRT locus of CHO cells.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The reliability of the original study was established to be R2: Justification for read-across is detailed at section 13.
Justification for type of information:
Justification for read-across is detailed at section 13.
Qualifier:
no guideline available
Principles of method if other than guideline:
The intrasanguineous host-mediated assay (IHMA) employs microbial indicator cells to assay for mutagens using the intact animal as the source of chemical transformation, detoxification, systemic distribution, and excretion. By intrasanguineous injection of the bacteria strains Salmonella typhimurium TA 1535, TA 98 and TA 100, the indicator organisms were brought into close contact with the mammalian metabolic organ(s). Subsequent recovery and plating of the cells for mutations might indicate the ultimate mutagenic metabolites of a test material within the animal body.
GLP compliance:
yes
Target gene:
his
Species / strain / cell type:
other: TA1535, TA98, TA100
Metabolic activation:
with
Metabolic activation system:
mouse liver, in vivo
Vehicle / solvent:
- Vehicle/solvent used: CMC (carboxymethyl cellulose)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
N-dimethylnitrosamine
Evaluation criteria:
The data obtained from the mutagenicity tests are calculated on a test by test basis followed by an examination of the total response pattern using all the data. Mutant numbers as well as the calculated frequencies are compared to the vehicle control data. Dose-related increases in mutants and mutation frequencies are the most convincing data in assessing mutagenic activity of a test material. A test material is considered to have positive mutagenic response if it produces a dose response over three concentrations with the highest increase is equal to twice the solvent control value for the bacterial strain used.
Statistics:
Chi-square test (Campbell, 1974), the level of significance was set at p < 0.05.
Species / strain:
other: TA1535, TA98, TA100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No mutagenic potential on all strains/types tested

Dose Animal No. Revertants/plate
TA 100 TA 98 TA 1535
Control 1 9 17 4
2 28 15 15
3 4 25 226
4 16 22 2
5 - - 3
50 µg/kg DMN 1 182   115
2 247   85
3 266   170
4 234   108
5 374   271
100 mg/kg 2-AAF 1   192  
2   932  
3   222  
4   87  
5   101  
0.4 g/kg 1 12 21 17
2 23 14 3
3 21 151 4
4 25 21 -
5 - 29 -
1.3 g/kg 1 9 17 5
2 21 18 3
3 16 18 2
4 48 16 2
5 36 23 5
4.0 g/kg 1 14 11 7
2 40 19 5
3 17 25 -
4 - 18 -
5 - - -
Conclusions:
Negative mutagenic potential.
Executive summary:

The test material did not exhibit genetic activity in the host mediated assay using the strains TA100, TA98 and TA1535 of Salmonella typhimurium injection into mice and was considered inactive under the test conditions employed, according to the evaluating criteria.

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

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Chromosome Aberration
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
The reliability of the source study was established to be R2: Justification for read-across is detailed at section 13.
Justification for type of information:
Justification for read-across is detailed at section 13.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, WIGA, D-8741 Sulzfeld, FRG
- Weight at study initiation: mean 26.7 g
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon cages, type MI
- Diet: Standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland); ad libitum.
- Water: drinking water from bottles; ad libitum.
- Acclimation period: about one week.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: CMC (carboxymethyl cellulose)
- Concentration of test material in vehicle: 8.5, 17, 34 g/100 ml
- Amount of vehicle: 20 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
All test substance formulations were prepared immediately before administration. The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment.
Duration of treatment / exposure:
16, 24, 48 h for the highest dose of test material; 24 h for all other dose groups and controls
Frequency of treatment:
Single application
Post exposure period:
16, 24, 48 h for the highest dose of test material; 24 h for all other dose groups and controls
Dose / conc.:
1 700 mg/kg bw/day (nominal)
Remarks:
#1
Dose / conc.:
3 400 mg/kg bw/day (nominal)
Remarks:
#2
Dose / conc.:
6 800 mg/kg bw/day (nominal)
Remarks:
#3
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Substance: cyclophosphamide
- Route of administration: oral by gavage
- Doses / concentrations: 40 mg/kg bw
Tissues and cell types examined:
Bone marrow of the two femora
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION
In the determination of the acute oral toxicity all animals survived the high dose of 6800 mg/kg body weight without any clinical signs or symptoms. A volume as high as 20 ml/kg body weight had to be selected in order to be able do administer this amount. Higher doses suspended in an 0.5 % aqueous CMC formulation led to a viscous mass which could no longer be administered.

DETAILS OF SLIDE PREPARATION
Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained. Staining in eosin and methylene blue solution for 5 minutes. Rinsed in aqua dest., then placed in fresh aqua dest. for 2 or 3 minutes. Staining in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Entellan.

METHOD OF ANALYSIS
As a rule, 1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occur, are also scored.
Evaluation criteria:
The increase in the micronucleus rate in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome breaking (clastogenic) effect or of a spindle activity of the substance tested.
Statistics:
Two statistical tests were used to answer the questions of whether there are significant differences between control group and dose group or between the individual dose groups concerning the rate of micronuclei in polychromatic erythrocytes: first, the exact test according to FISHER, which was applied to register significant differences between the relative frequencies of a characteristic of two groups, and, second, the asymptotic U test according to MANN-WHITNEY (rank test modified according to WILCOXON). The relative frequencies of cells with micronuclei per animal were use das a criterion of the rank determination for the U test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY- Clinical signs of toxicity in test animals: The single oral administration in doses of 6800 mg/kg, 3400 mg/kg or 1700 mg/kg body weight was tolerated by all animals without any signs of toxicity.-Necropsy: The gross-pathological examination of the animals sacrificed at the end of the study did not reveal any changes of the internal organs which could the attributed to the test substance administered.

Results:

Substance Dose (mg/kg bw) Interval: 16 hours Interval: 24 hours Interval: 48 hours
Polychromatic erythrocytes investigated Normocytes / 10000 polychromatic erythrocytes Cells with micronuclei Polychromatic erythrocytes investigated Normocytes / 10000 polychromatic erythrocytes Cells with micronuclei Polychromatic erythrocytes investigated Normocytes / 10000 polychromatic erythrocytes Cells with micronuclei
per 1000 polychromatic erythrocytes per 1000 normochromatic erythrocytes per 1000 polychromatic erythrocytes per 1000 normochromatic erythrocytes per 1000 polychromatic erythrocytes per 1000 normochromatic erythrocytes
vehicle control, 0.5% CMC - 10000 3485 1.8 0
Säurebraun 6229 6800 10000 2988 1.4 1.34 10000 4349 1.4 1.61 10000 4359 1.3 1.61
Säurebraun 6229 3400 - 10000 3506 1.4 0.86
Säurebraun 6229 1700 - 10000 4307 1.9 0.46
Cyclophospamide 40 - 10000 5623 23.4 1.78
Conclusions:
No induction of chromosome breaking (clastogenic) effect or a spindle activity in polychromatic erythrocytes of the bone marrow of the femora mice.
Executive summary:

The substance was tested for chromosome aberration potential following OECD 474, by oral administration.

The test substance, under the experimental conditions, did not induce chromosome breaking (clastogenic) effect or a spindle activity in polychromatic erythrocytes of the bone marrow of the femora mice.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The reliability of the read-across study was established to be R2: comparable to guideline study with acceptable restrictions.
Justification for type of information:
Justification for read-across is detailed at section 13.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
yes
Remarks:
limited given data
GLP compliance:
yes
Remarks:
HAZLETON LABORATORIES AMERICA, INC.
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: adult- Weight at study initiation: 150 - 300 g
- Assigned to test groups randomly: yes
- Diet: Purina Certified Rodent Chow (Formula 5002); ad libitum.
- Acclimation period: minimum of 5 days prior to use.
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: water
- Amount of vehicle (if gavage or dermal): 9 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
Fresh preparations of test article in vehicle were used for any testing purpose.
Duration of treatment / exposure:
4 h
Frequency of treatment:
single application
Post exposure period:
4 h
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
#1
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
#2
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
#3
Dose / conc.:
4 000 mg/kg bw/day (nominal)
Remarks:
#4
No. of animals per sex per dose:
3 animals/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Dimethylnitrosamine (DMN)
- Justification for choice of positive control: known to induce UDS in vivo in rat hepatocytes.
- Route of administration: intraperitoneal.
- Doses / concentrations: ca. 10 mg/kg bw
Tissues and cell types examined:
hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION
In the preliminary study to determine dose and perfusion time, an attempt was made to gavage two animals with 5000 mg/kg, but the test material formed a sludge in CMC that could not be forced through a syringe. The highest dose that could be administered was between approximately 3200 mg/kg and 3400 mg/kg although one animal died due to gavage back up. The amount administered was not exact because the sludge was difficult to measure. One rat was sacrificed (liver perfusion) approximately 4.5 hours later and the other approximately 15 hours later and slides were prepared for UDS. Microscopic examination of the slides prepared at the two time points indicated that they were similar. It was decided that the UDS assay would be performed approximately 4 hours after administration of a single dose of the test material.

DETAILS OF SLIDE PREPARATION
UDS based on the procedures in rats described by Williams (1980) and Mirsalis, Tyson and Butterworth (1982): Briefly, isolated hepatocytes were cultured for 1.5 to 2 hours at 37 °C in a humidified atmosphere containing 5 % C02. Three of the replicate cultures from each animal were used for the UDS assay, thus labeld and fixed with acetic acid : ethanol (1:3) and dried for at least 24 hours.

METHOD OF ANALYSIS
The cells were examined microscopically at approximately 1500x magnification under oil immersion and the field was displayed on the video screen of an automatic counter. UDS was measured by counting nuclear grains and subtracting the average number of grains in three nuclear-sized areas adjacent to each nucleus (background count). This value is referred to as the net nuclear grain count. The coverslips were coded to prevent bias in grain counting. The net nuclear grain count was determined for 50 randomly selected cells on each coverslip. Only nuclei with normal morphologies were scored, and any occasional nuclei blackened by grains too numerous to count were excluded as cells in which replicative DNA synthesis occurred rather than repair synthesis. The mean net nuclear grain count was determined from the triplicate coverslips (150 total nuclei) for each treatment condition. Occasionally, a coverslip is recounted at a later date or by a different technician. Since a different cell population will generally be scored, the average count for 50 cells was used in the calculation of the mean for the triplicate treatment.
Evaluation criteria:
The test material is considered active in the UDS assay at applied concentrations that cause:
- an increase in the mean net nuclear grain count to at least six grains per nucleus after subtraction of the concurrent negative control value, and/or
- an increase in the percent of nuclei having 6 or more net grains to at least 10 % of the analyzed population after subtraction of the concurrent negative control value, and/or
- the percent of nuclei with 20 or more grains to reach or exceed 2 % of the analyzed population.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 3200 - 5000 mg/kg bw
- Solubility: material formed a sludge in CMC that could not be forced through a syringe at 5000 mg/kg bw.

Results:

Test condition Dose Level Animal UDS grains/nucleus Average* % nuclei with
>= 6 grains >= 20 grains
water - 1 0.45 ± 0.18 0.7 0.0
2 -0.43 ± 0.10 0.7 0.0
3 -0.163 ± 1.59 0.7 0.0
DMN 10 mg/kg bw 1 25.22 ± 8.79 94.7 64.7
2 15.47 ± 2.65 83.3 41.3
3 17.2 ± 7.8 84.0 41.3
Saeurebraun 6229 4000 mg/kg bw 1 0.36 ± 0.52 2.0 0.0
2 -0.08 ± 0.73 0.0 0.0
3 0.08 ± 0.66 0.7 0.0
2000 mg/kg bw 1 -1.32 ± 0.50 0.7 0.0
2 -1.00 ± 0.76 0.0 0.0
3 -1.03 ± 1.02 0.7 0.0
1000 mg/kg bw 1 -1.49 ± 0.57 0.0 0.0
2 -1.11 ± 0.88 5.3 0.0
3 -0.57 ± 0.60 0.0 0.0
5000 mg/kg bw 1 -0.20 ± 0.20 1.3 0.0
2 -1.18 ± 0.39 0.0 0.0
3 -0.92 ± 0.75 1.3 0.0

UDS: Average of net nuclear grain counts on triplicate coverslips (150 total cells), ± standard deviation between coverslips

*: Average values for triplicate coverslips

Conclusions:
Inactive in the in-vitro/in-vivo Rat Epatocyte UDS Assay over a dosing range of about 500 to 4000 mg/kg.
Executive summary:

The substance was tested for genotoxicity following OECD 486.

The tets substance was inactive in the in-vitro/in-vivo Rat Epatocyte UDS Assay over a dosing range of about 500 to 4000 mg/kg.

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

Additional information

Experimental data in read across with Similar Substance 01 were available to assess the genetic toxicity in vitro and in vivo.

 

Gene mutation in bacteria

 

Gene mutation in mammalian cells

The potential of the test substance to induce gene mutations at the HGPRT locus using chinese hamster ovary cells was tested in vitro with and without metabolic activation according to OECD guideline 476 (tested up to 3420 μg/ml). Cytotoxicity was observed in an intermediate concentration range around 20 µg/ml of test substance, presumably induced by inhibition of enzymes of the S9-mix. The test substance showed no mutagenic effects on the HGPRT locus of CHO cells.

 

 

Cytogenicity in vivo

The potential of the test substance to induce chromosome damage or spindle poisoning was investigated in an OECD guideline 474 conform standard micronucleus test study in male and female NMRI mice. Test substance was given orally in CMC-vehicle in concentrations of 1700,3400 and 6800 mg/kg bw (>limit dose tested in acute oral toxicity study) and bone marrow was isolated after 16,24 and 48 h. No signs of toxicity and no pathological changes of the organs were observed in any dose groups. Microscopic evaluation of bone-marrow derived erythrocytes showed that the test substance did not impair erythropoeisis, did not induce any chromosome-damaging (clastogenic) effects and did not impair chromosome distribution in the course of mitosis.

 

A second in vivo rat hepatocyte UDS indicator test was done according to OECD guideline 486. The test material was administered in water orally to rats (4000,2000,1000 and 500 mg/kg bw) and 4 h later hepatocytes were isolated, labelled with 3H-thymidine, processed and net nuclear grains were determined via microscopic evaluation. The test substance did not induce nuclear staining differently from vehicle control levels and no dose-related trend was observed, thus, the test material was inactive in the UDS-test in the dose range tested.

Justification for classification or non-classification

The test substance showed genotoxic reactions only in in vitro experiments using bacteria (Ames test). In all other experiments using bacteria in vivo (host mediated assay), mammalian cells in vitro (CHO, hamster; HGPRT test), mammalian cells in a combined in vitro/in vivo study (UDS test) or whole animals (in vivo micronucleus test) the test substance did not exhibit any genetic activity.

Therefore, based on the information currently available, there is no need for classification of the test substance for mutagenic effects under the regulation EC 1272/2008 (CLP).