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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

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 somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
other: read across from analogue substance
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):
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 analogue substance
Adequacy of study:
key 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

refer to the attached document

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.