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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A reliable bacterial reverse mutation assay and a Rat Hepatocyte Primary Culture /DNA repair test (UDS) are available.

Both studies indicate a negative results and both are taken into consideration.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 5, 1985 - March 18, 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 5601-49-1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Room temperature in container received
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble and stable
Target gene:
Histidine locus
Species / strain / cell type:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100
Metabolic activation:
with and without
Metabolic activation system:
S-9
Test concentrations with justification for top dose:
50, 166, 500, 1666, and 5000 µg/plate.
Vehicle / solvent:
Distilled deonized water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
Negative controls without activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration:48-72 h at 37 degrees C

NUMBER OF REPLICATIONS: 3

- OTHER: 2 ml aliquots of molten top agar, 0.5 ml biotin and histadine, 0.1 ml tester strain, 0.1 ml test compound concentration were vortexed and poured onto minimal glucose plates in a thin layer. Plates needing activation were suplimented with 0.5 ml S-9 rat liver homogenate. Revertant colonies were counted with an electronic colony counter interfaced with a computer.
Evaluation criteria:
A positive result is a dose-related significant increase in the number of histidine dependent colonies as determined by the program developed by Fenton and Moore. A negative result is the absence of reproducible increase in the number of histidine dependent colonies.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Conclusions:
The test material was not found to increase mutation frequencies in any strain of S. typhimurium, either with or without metabolic activation. The substance is considered to not be mutagenic.
Executive summary:

The test article was received as a clear liquid. The solvent used throughout the assay was distilled/deionized water and 5000 microgram/plate.

Dose levels in a preliminary toxicity screen were 50, 166, 500, 1666 and 5000 microgram/plate. Strains TA1538 and TA100 of Salmonella typhimurium exhibited no inhibition of bacterial lawn growth at any of the dose levels tested.

Therefore , the top dose selected for the plate incorporation mutation assay was 5000 microgram/plate.

The test article was evaluated in strains TA 1535, TA1537, TA 1538 and TA100 of Salmonelle typhimurium both with and without metabolic activation preparation at dose of 50, 166, 500, 1666 and 5000 microgram/plate.

There were 0.10 mL of S-9 supernatant (31.1 mg protein/ml) per 1.0 ml of S9 -mix in the rat liver metabolic activation preparation.

The test article was negative in strains TA 1535, TA 1537, TA 1538, TA98 and TA100 of Salmonella typhimurium with and without metabolic activation preparation at dose of 50, 1661 500, 166 and 5000 microgram/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable limits of mean historical data.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 20, 1985 -April 19, 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.18 (DNA Damage and Repair - Unscheduled DNA Synthesis - Mammalian Cells In Vitro)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor, 5601-49-1
Species / strain / cell type:
hepatocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Freshly harvested rat liver
- Suitability of cells: 88% viability


Additional strain / cell type characteristics:
not applicable
Test concentrations with justification for top dose:
Triplicate cultures were seeded with 1 x 10^5 rat liver cells and treated with 20 µl of test substance at doses of 0.33, 1.0, 3.3 10, 33, 100, 333, 1000, 3333, and 10,000 µg/well.
Vehicle / solvent:
Distilled water dilution to test concentration.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1 x 10^5

DURATION
- Exposure duration: 2 hours to test material, 7 days to photographic emulsion
- Expression time (cells in growth medium):20 hours

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 1 x 10^5 viable cells were inoculated in 12 well cluster dishes containing 15mm plastic Thermanox coverslips in WMES containing 10% calf serum. The cells were allowed to attach for 2 hours at 37 degrees C in CO2 incubator. Cultures were rinsed, and serum-free media containing test material and 10 uC/ml H-thymidine added. 18-20 hours later, the cultures were washed 3 times with 3 ml phosphate buffered saline by aspiration. The cells were swelled in sodium citrate, and fixed in 3 30 min changes of 3:1 ethanol:glacial acetic acid. Slips were air dried, and mounted cell side up on slides with permount. Slides were dipped in NTB-2 photographic emulsion in the dark, dried overnight, and stored at 4 degrees C in lightproof boxes containing Drierite for one week. Then, slides were developed for 4 min in D19, washed, fixed, stained with Harris Alum hematoxylin, rinsed and air dried.

NUMBER OF CELLS EVALUATED: 60 cells per dose

- OTHER:Unscheduled DNA repair was quantified by nuclear and cytoplasmic black silver grain counts using an Artek 880 colony counter
Evaluation criteria:
Test material was considered positive if it produced a mean nuclear grain count of 5 or greater than the control mean nuclear grain count at any concentration.
Key result
Species / strain:
hepatocytes:
Remarks:
Rat liver
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Remarks on result:
other: no quantitative result
Conclusions:
The test material was considered to be negative under the conditions of the assay.
Executive summary:

The test item was supplied as a clear liquid and was disssolved in distilled water at the suggestion of the sponsor. The liver of a amle Fischer 344 rat was perfused, excised and combed yielding 2.11 X 10E6 cells per mL of media with a 88% viability.

Triplicate cultures were seeded with 1X10E5 viable cells and treated with 20 microliter of test item at doses of 0.33 / 1.0 / 3.3 / 10 / 33 / 100 / 333 / 1000 / 3333 / and 10 000 microgram/well.

In addition, a distilled water group, an untreated control (WME only) and 2 -Acetamidofluorene (2AAF) at a final concentration of 1x10E-5M, serving as the positive control, were evaluated concurrently. The highest dose scored in the DNA Repair Test was 1000 microgram/plate in 2 Ml of media due to excessive toxicity at the higher doses.

Unscheduled DNA repair synthesis was quantified by a net nuclear increase of black silver grains for 20 cells/coverslip. This value was determined by subtracting the highest of three adjacent cytoplasmic counts from the nuclear counts. Three coverslips at each dose point were evaluated for a total of 60 cells/dose. The coverslips were evaluated at a mignification of approximately 1500X.

The results for the test item, were negative in the Rat Hepatocyte Primary Culture/DNA Repair test under conditions of the assay. These findings are absed upon the inability of the test item to produce a mean nuclear grain count of five or greater than the vehicle control mean nuclear grain count at any level of concentration.

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

Genetic toxicity in vivo

Description of key information

One additional in vivo study was performed, micronucleus study and was found to be negative (OECD 475).

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:
March 5, 1985 - April 10, 1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Micronucleus induction
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor; 5601-49-1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in receiving container
- Stability under test conditions: There was no apparent change in the physical state of the test and control articles during the assay.
- Solubility and stability of the test substance in the solvent/vehicle: soluble and stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Diluted to dose concentration with distilled water
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Age at study initiation: 7.5 weeks
- Weight at study initiation: Males: 26-32 grams; Females: 23-28 grams
- Assigned to test groups randomly: yes
- Fasting period before study: No data
- Housing: By dose group, by sex, 5 per cage, standard stainless steel wire mesh cages.
- Diet (e.g. ad libitum): ad libitum, Purina Certified Lab Blox
- Water (e.g. ad libitum): ad libitum, fresh tap water
- Acclimation period: No data

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Solubility
- Concentration of test material in vehicle: 125 mg/kg
- Lot/batch no. (if required): 6-246
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test article was weighed and diluted with distilled H2O. Good solutions were obtained and maintained at all levels evaluated. The positive control was dissolved in distilled water and administered at 10mlkg at a dose of 0.05 mg/kg of body weight. The test article solutions and positive control solutions were prepared fresh and dosed within two hours of preparation.
Duration of treatment / exposure:
Single dose, intraperitoneal
Frequency of treatment:
Once
Post exposure period:
Sampled at 30, 48 and 72 hours
Dose / conc.:
125 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males and 5 females per group (3 groups)
Control animals:
yes
Positive control(s):
triethylenemelamine
- Justification for choice of positive control(s): Not specified
- Route of administration: Intraperitoneal
- Doses / concentrations: 0.05 mg/kg
Tissues and cell types examined:
Stained micronuclei of femur bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Range-finding study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 30, 48, and 72 h after dosing

DETAILS OF SLIDE PREPARATION: Bone marrow was flushed with 0.2 ml bovine serum, into 1 ml additional serum, and centrifuged at 1000 rpm for 5 min. The supernatant was drawn off, and a drop of the stirred cell button was pulled across a slide at a 45 degree angle. The slides were quick-dried at approximately 56 degrees C, dipped in absolute methanol, and allowed to air-dry overnight. The slides were stained for 29 minutes in 5% Giemsa working solution, and rinsed twice. The first rinse was distilled water adjusted to pH 4-4.5, and the second was distilled water adjusted to pH 7.0. Slides were then dried on a slide warmer at 56 degrees C, cleared in xylene for 5 minutes, and mounted in Permount with a cover glass.

METHOD OF ANALYSIS: 1000 polychromatic eurythrocytes per animal were counted for the presence of micronuclei. The data were expressed as a ratio of micronucleated polychromatic eurythrocytes to bormal polychromatic eurythrocytes.
Evaluation criteria:
Assessment of the test substance as positive is based upon its ability to induce a statistically significant increase in micronucleated polychromatic eurythrocytes as compared to negative control.
Statistics:
The test substance is considered as positive based upon its ability to induce a statistically significant increase in micronucleated polychromatic eurythrocytes as compared to negative control, via one tailed t test. Significance was judged at p
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 166-5000 mg/kg body weight
- Clinical signs of toxicity in test animals: Mortality observed at all doses by 72 h

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): none
- Ratio of PCE/NCE (for Micronucleus assay): Mean 1.66
Conclusions:
The test substance was found to be negative in the micronucleus test at a dose level of 125 mg/kg in a single intraperitoneal dose, with sacrifice times of 30, 48, and 72 hours. Therefore, the test substacne is not to be classified as mutagenic, according to the CLP Regulation
Executive summary:

In a preliminary dose-rage-finding, the test article ws administred intraperitoneally to 6 groups (2 males and 2 females per group) of CD-1 mice at dose levels of 166 / 300 / 500 / 1666 / 3000 and 5000 mg/kg of body weight..

Total mortality occured at the four highest dose levels. In the lowest two dose groups one male per group died and signs were observed at all levels.

Due to the high mortality and severity of signs in the study, 125 mg/kg was seleceted as the dose for the micronucleus test as an estimate of the maximum tolerated dose.

In the micronucleus test, three groups of ten animals (5 males and 5 females/group) were given single doses by intraperitoneal injection at 125 mg/kg and sacrified at 30 / 48 and 72 hours.

Similar groups, dosed with the posiitve control, triethylenemelanine (TEM) and negatice control, distilled water, were evaluated concurrently.

Slides were prepared from the bone marrow of the femur and stained. Coded slides were scored for the number of polychromatic erythrocytes per 1000 erythrocytes per animal was determined.

The results for the test item were negative in the micronucleus test at a dose level of 125 at all of the time intervals evaluated. These findings are based upon the inability of the test article to produce a statistically significant increase in the number of micronuclei in 1000 polychromatic erythrocytes per animal in the treated groups versus the negactive control group.

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

Additional information

Several mutagenicity studies have been conducted in in-vitro test systems where the Ames test turned out to be negative and in the Rat Hepatocyte Primary Culture /DNA repair test (UDS), the result was found to be also negative, indicating clearly a non mutagenic potential in-vitro.

One in-vivo mutagenicity assays was performed: OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test) and the test item has no significant mutagenic potential in-vivo.

Altogether, it was judged that the test substance is neither mutagenic in vitro and nor mutagenic in in-vivo experiment.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).