Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Read-Across - OECD 471 (in vitro gene mutation study in bacteria) - Ames NR-deficient strains - K1 (reliable without restrictions) - KS (key study) - positive with and without S9;


- Read-Across - OECD 476 (In vitro gene mutation study in mammalian cells) - K1 (reliable without restrictions) - Supporting Study - negative.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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(s)/solvent(s) 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
Remarks:
Without metabolic activation: 600 µg/ml = 4.8 mM dissolved in medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation: 3.85 μg/ml = 15.0 μM dissolved in DMSO
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 S-9 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:
The analogue substance was tested for gene mutation in mammalian cells following OECD 476. The tested substance under the reported experimental conditions did not induce gene mutation at the HGRPT locus in CHO cells.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: expert assessment
Adequacy of study:
weight of evidence
Study period:
2022
Reliability:
1 (reliable without restriction)
Qualifier:
no guideline required
Principles of method if other than guideline:
Expert statement
GLP compliance:
no
Type of assay:
other: expert statement
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: TA 98NR, TA 100NR
Metabolic activation:
with and without
Metabolic activation system:
Uninduced rat hamster liver S9 Mix
Test concentrations with justification for top dose:
Test concentrations: 5, 16, 50, 160, 500, 1600 and 5000 µg/plate (final concentrations)
Vehicle / solvent:
Purified water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Species / strain:
other: refer to the attached expert statement
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Based on the expert statement, the test substance is not considered as genotoxic in gene mutation studies on bacteria.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- Read-Across - OECD 474 (in vivo mammalian somatic cell study: cytogenicity/erythrocyte micronucleus) - K2 (reliable with restrictions) - KS (key study) - negative;


- Read-Across - OECD 486 (In vivo mammalian cell study: DNA damage and/or repair) - K2 (reliable with restrictions) - SS (supporting study) - negative.


- OECD 489 (Comet Assay): Read-Across in progress.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic and germ cell study: gene mutation
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
not yet defined
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: analogue substance 04


CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
This part refers to the available studies on the target substance Acid Brown 165:1 (EC: 943-697-5):
- Available GLP studies: not available.
- Available non-GLP studies: not available.
- Historical human data: not available.
- (Q)SAR: not available.
- Weight of evidence: not available.
- Grouping and read-across: this part refers to the available studies on the analogue substance 03, that were used in read across to cover the endpoint of genotoxicity of Acid Brown 165:1 (EC: 943-697-5).
✓ In vitro gene mutation study in bacteria (OECD 471) – NR-deficient strains.
✓ In vivo mammalian cell study: DNA damage and/or repair (OECD 486).
✓ In vitro gene mutation study in bacteria (OECD 471) – only TA100 strain.
✓ In vivo mammalian somatic cell study: cytogenicity/erythrocyte micronucleus (OECD 474).
✓ In vitro gene mutation study in bacteria (OECD 471) – IHMA.
✓ In vitro gene mutation study in mammalian cells (OECD 476).


CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Under Annex VIII Section 8.4., column 2 of REACH, further mutagenicity studies must be considered in case of a positive result in an in vitro gene mutation study in bacteria.
Guidance on information requirements R7a, section 7.7.6 (2017), states that regarding Annex VIII, when both the mammalian cell tests are negative but there was a positive result in the bacterial test, it will be necessary to decide whether any further testing is needed on a case-by-case basis. For example, suspicion that a unique positive response observed in the bacterial test was due to a specific bacterial metabolism of the test substance could be explored further by investigation in vitro. Alternatively, an in vivo test may be required.
The submitted dossier contains results for the in vitro gene mutation study in bacteria, following the OECD 471 with nitro-reductase deficient strains, which raises the concern for in vivo gene mutation, since the influence of the nitro-reductase is demonstrated but needs further evidence. The reported study was conducted on the analogue substance 03.
In particular, annex VIII, Column 2 requires the registrant to consider appropriate mutagenicity in vivo studies already at the Annex VIII tonnage level, which involves studies mentioned in Annex IX (among OECD 474 Mammalian Erythrocyte micronucleus test, OECD 488 Transgenic Rodent Mutation Assay, OECD 489 In vivo mammalian Alkaline Comet Assay and OECD 486 Unscheduled DNA Synthesis).

CONSIDERATIONS ON THE IN VIVO STUDIES INSERTED IN THE DOSSIER AND EXPERT ASSESSMENT ON TESTING PROPOSAL:
In the submitted dossier an OECD 474 (Mammalian Erythrocyte micronucleus test) in vivo study performed on the analogue substance 03 is available and showed negative results. This study is adequate to cover the chromosomal aberration potential of the two substances and to waive the performance of an in vitro cytogenicity in mammalian cells, as laid down in Column II of Annex VIII of the REACH Regulation.
Moreover, a reliable OECD 486 (in vivo UDS assay) is also present on the analogue substance 03 which resulted negative and can be used as supporting information for the in vivo gene mutation properties assessment, since the cells analyzed in the UDS assay involve only those of the liver.

Therefore, in order to further and completely assess its gene mutation properties in different tissues of the animal, a Comet Assay, OECD 489, on Analogue substance 04 was presented as testing proposal and it will be also used in read across for assessing the in vivo potential gene mutation properties of the target substance Acid Brown 165:1 (EC: 700-899-6).
Analogue substance 04 is, in fact, considered as representative of the mutagenic behavior of Acid Brown 165:1 (EC: 700-899-6) as specified in the read-across section.
OECD 489 allows to measure DNA strand breaks, that may result from direct interactions with DNA, alkali labile sites or as a consequence of incomplete excision repair. Therefore, the alkaline comet assay recognizes primary DNA damage that would lead to gene mutations and/or chromosome aberrations, but will also detect DNA damage that may be effectively repaired or lead to cell death. The comet assay can be applied to almost every tissue of an animal from which single cell or nuclei suspensions can be made, including specific site of contact tissues.
OECD 488 is not considered as the first choice for assessing the gene mutation in vivo for this substance, since preliminary data for gene mutation in vivo (OECD 486) already indicates negativity in the somatic cells of the liver. A confirmation by the Comet assay performed over other tissues (and for azo dyes the intestinal tract is the site of major metabolism and dye/metabolites absorptioni) would be sufficient to assess the genotoxic potential of the substance.
Finally, as reported in literature, from the analysis of 91 chemicals with published data from Comet Assay and Transgenic rodent mutation assay (TGR), the comet assay appears to yield similar results to the TGR assay in liver and gastrointestinal tract (predominantly stomach and colon data) and, hence, can be confidently performed to confirm in vivo gene mutation activity in terms of genotoxicity in general.
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes
Type of assay:
mammalian comet assay
Sex:
not specified
Genotoxicity:
other: to be performed
Remarks on result:
other: the test is in read-across from a submitted testing proposal still under evaluation
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
other: read-across from supporting substance (stuctural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system 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 M I
- Diet (e.g. ad libitum): Standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland); ad libitum
- Water (e.g. ad libitum): drinking water from bottles; ad libitum
- Acclimation period: about one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Concentration of test material in vehicle: 8.5, 17, 34 g/100 ml
- Amount of vehicle (if gavage or dermal): 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
Remarks:
Doses / Concentrations:
1700, 3400, 6800 mg/kg bw
Basis:
nominal conc.
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 6810 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 d
as 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:
The analogue substance was tested for chromosome aberration potential following OECD 476, by oral administration. The tested sample 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:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
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 or test system 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 (e.g. ad libitum): Purina Certified Rodent Chow (Formula 5002); ad libitum
- Water (e.g. ad libitum): water; ad libitum
- Acclimation period: minimum of 5 days prior to use
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) 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
Remarks:
Doses / Concentrations:
500, 1000, 2000, 4000 mg/kg bw
Basis:
nominal in water
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(s): 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:
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

For further details refer to the attached document on genotoxicity assessment.

Justification for classification or non-classification

Classification for mutagenicity is warranted for substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans


The classification in Category 2 is based on:


— Positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:


— Somatic cell mutagenicity tests in vivo, in mammals; or


— Other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.


Based on the results of the in vivo and in vitro tests no classification for mutagenicity is applied following Regulation 1272/2008.


A new evaluation of the genotoxic potential will be performed once the result of the "in vivo" COMET Assay on the Analogue substance 04 will be available.