Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

REACH_negative | TA 98, TA 100, TA 1535, TA 1537 and Ecoli WP2 uvr A | OECD 471 | with and without | #key study#

REACH_positive │V79 cells│OECD 473 | with and without | #key study#

REACH_negative | V79 cells | OECD 476 | with and without | #key study#

Sa 190 did not induce gene mutations in Salmonella typhimurium and Escherichia coli strains applying pre-incubation or plate incorporation up to 5000 µg/plate in a GLP compliant study according to OECD Technical Guidelines 471 and is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster according GLP compliant study following the OECD Technical Guidelines 476.

But Sa 190 induced structural chromosomal aberrations in the V79 Chinese hamster cell line in presence of metabolic activation with S9 homogenate, when performing a GLP compliant study according to OECD Technical Guidelines 473. The aberration rates found at the concentrations of 0.4 mM (6.8%) and 8.0 mM (5.0%) were clearly increased above the historical negative control data of the testing facility. Thus, the numher of aberrant cells found at these concentrations showed a biologically relevant increase compared to the corresponding solvent control without dose-response relationship.


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
, but deviation was related to source of S9 mix and did not influence the quality or integrity of the study
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
induction of chromosome breakage
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix (phenobarbital and β-naphthoflavone induced)
Test concentrations with justification for top dose:
Pre-Experiment: 0.020; 0.039; 0.078; 0.16; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0 mM
Experiment I: without metabolic activation: 0.1; 5.0; 10.0 mM
Experiment I: with metabolic activation: 0.316; 1.0; 5.0; 10.0 mM
Experiment II: without metabolic activation: 0.1; 5.0; 10.0 mM
Experiment II: with metabolic activation: 0.4; 8.0; 10.0 mM
Vehicle / solvent:
The test item was dissolved in ethanol at a concentration of 1000 mM and was diluted in cell culture medium (MEM)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
V79 cells in vitro were widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells were chosen because of their relatively small number of chromosomes (diploid number, 2n = 22), their high proliferation rate (doubling time of the BSL BIOSERVICE V79 done in stock cultures: 12 - 14 h) and the high plating efficiency of untreated cells (normally more than 50%). These facts were necessary for the appropriate performance of the study.
The V79 (ATCC, CCL-93) cells are stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking for mycoplasma infections was carried out before freezing.
For the experiment, thawed cultures were set up in 75 cm2 cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 10E5 cells per flask were seeded in 15 mL o f MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made every 3 - 4 days.
Evaluation criteria:
ACCEPTABILITY
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
- the number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.0% - 4.0% (with and
without metabolic activation),
- the positive control substance should produce biologically relevant increases in the number of cells with structural chromosome aberrations.

EVALUATION
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells
(with and without metabolic activation)).

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results and a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data, both biological and thoroughly evaluated statistical significance should be considered together.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid

In experiment I and II with and without metabolic activation, no biologically relevant decrease of the relative mitotic index as indication of cell toxicity was noted at all concentrations evaluated. Furthermore, the cell density was not decreased below 70%.

In experiment I without metabolic activation the aberration rates of the negative control (l.5%)‚ solvent control (4.0%) and all dose groups treated with the test item [2.3% (0.1 mM)‚ 3.0% (5.0 mM) and 2.0% (10.0 mM)] were within the historical control data of the testing facility (0.0% - 4.0%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding solvent control. With metabolic activation, the aberration rates of the negative control (2.0%), solvent control (2.0%) and the higher dose groups treated with the test item [3.8% (1.0 mM), 2.0% (5.0 mM) and 3.0% (10.0 mM)] were within the historical control data of the testing facility (0.0% - 4.0%). An increase of the aberration rate above the historical negative control data of the testing facility was observed at a concentration of 0.316 mM (4.5%). A dose-response relationship was not found.

In experiment II without metabolic activation the aherration rates of the negative control (2.5%), solvent control (0.0%) and all dose goups treated with the test item [2.5% (0.1 mM), 3.0% (50 mM) and 0.5% (10.0 mM)] were within the historical control data of the testing facility (0.0% - 4.0%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding solvent control. With metabolic activation the aberration rates of the negative control (3.0%), solvent control (2.0%) and the highest dose group treated with the test item (l.5% (10.0 mM)) were within the historical control data of the testing facility (0.0% - 4.0%). The aberration rates found at the concentrations of 0.4 mM (6.8%) and 8.0 mM (5.0%) were clearly increased above the historical negative control data of the testing facility. Thus, the numher of aberrant cells found at these concentrations showed a biologically relevant increase compared to the corresponding solvent control without dose-response relationship.

Conclusions:
In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item Sa 190 induced structural chromosomal aberrations in the V79 Chinese hamster cell line in presence of metabolic activation with S9 homogenate.
Therefore, the test item Sa 190 is considered to be clastogenic in this chromosome aberration test.
Executive summary:

To investigate the potential of Sa 190 to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out. The chromosomes were prepared 20 h after start of treatment with the test item, The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 0.1, 5.0 and 10.0 mM

with metabolic activation: 0.316, 1.0, 5.0 and 10.0 mM

Experiment II:

without metabolic activation: 0.1, 5.0 and 10.0 mM

with metabolic activation: 0.4, 8,0 and 10.0 mM

Precipitation of the test item was observed at the end of the treatment by the unaided eye in the experiment I with and without metabolic activation at concentrations of 5.0 mM and higher. In experiment II, precipitation of the test item was seen by the unaided eye without metabolic activation at concentrations of 5.0 mM and higher, with metabolic activation at concentrations of 8.0 mM and higher.

No toxic effects of the test item were noted with and without metabolic activation at all concentrations evaluated in experiment I and II In both experiments without metabolic activation no biologically relevant increase of the aberration rates was noted after treatment with the test item. With metabolic activation an increase of aberrant cells was found in experiment I at a concentration of 0.316 mM and in experiment II at concentrations of 0.4 mM and 8.0 mM

In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls. EMS ( 400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according 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
Target gene:
TA 1537: his C 3076; rfa-; uvrB-:, frame shift mutations
TA 98: his D 3052; rfa-; uvrB-; R-factor, frame shift mutations
TA 1535: his G 46; rfa-; uvrB-:, base-pair substitutions
TA 100: his G 46; rfa-; uvrB-; R-factor, base-pair substitutions
WP2 uvrA: trp-; uvrA-:, base-pair substitutions and others
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver S 9 mix (Phenobarbital/beta-Naphthoflavone induced rat liver S9)
Test concentrations with justification for top dose:
Pre-Experiment / Experiment I: 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
DMF (Dimethylformamid)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD)
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research GmbH according to B. Ames et al. and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH. The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

According to the direct plate incorporation or the pre-incubation method the bacteria are exposed to the test item with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies are counted.
To establish a dose response effect at least 6 dose levels with adequately spaced intervals were tested. The maximum dose level was 5000 μg/plate.
To validate the test, reference mutagens were tested in parallel to the test item.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- the positive control substances should produce a significant increase in mutant colony frequencies:

EVALUATION
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
No statisitcal analysis performed
Key result
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
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Sa 190 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of Sa 190 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Experiment II: 33, 100, 333, 1000, 2500, and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Sa 190 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - June 2012
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
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT-gene for hypoxanthin-guanin-phosphoribosyl-transferase
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix (phenobarbital and β-naphthoflavone induced)
Test concentrations with justification for top dose:
Experiment I: with and without metabolic activation
0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5.0, 7.5, 10 mM
Experiment II:
without metabolic activation
0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, , 2.5, 5.0, 7.5, 10 mM
with metabolic activation
0.025, 0.05, 0.10, 0.25, 0.50, 1.0, 2.0, 4.0, 7.0, 10 mM
Vehicle / solvent:
The test item was dissolved in EtOH, processed by ultrasound for 5 min, diluted and added to the cell culture medium prior to treatement.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are
characterized by their high proliferation rate (12 - 14 h doubling time of the BSL BIOSERVICE stock cultures) and their high cloning efficiency of untreated cells, usually more than 50%. Thesefactsare necessary for the appropriate performance of the study.
The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections. Thawed stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM).
For purifying the cell population of pre-existing HPRT mutants cells were exposed to HAT medium containing 100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine and 10.0 µM glycine for several cell doublings (2-3 days).

Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h lang time exposure assay.
Evaluation criteria:
ACCEPTABILITY
A mutation assay is considered acceptable if it meets the following criteria:
- Negative and/or solvent controls fall within the performing Iaborateries historical control data range: 1-39 mutants/10E6 cells;
- The absolute cloning efficiency: ([number of positive cultures x 100) / total number of seeded cultures) of the negative and/or solvent controls is > 50%
- The positive controls (EMS and DMBA) induce significant increases (at least 3-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range ofnegative controls) in the mutant frequencies.

EVALUATION
A test is considered to be negative if there is no biological relevant increase in the number of mutants.
There are several criteria for determining a positive result:
- a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
- a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed;
- if there is by chance a low spontaneaus mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid

Precipitation of the test item was noted in the experiments at concentrations of 1.25 mM and higher, in experiment II (with metabolic activation) at 1.0 mM and higher.

No biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment I and II with and without metabolic activation.

In experiment I without metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data. No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies of the negative control were found to be 20.95 and 27.78, of the solvent control 45.76 and 38.43 mutants/106 cells and in the range of 16.74 to 38.63 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 0.92 was found at a concentration of 0.0390 mM with a relative growth of 98.1%. At the concentration of 10 mM (without metabolic activation) the number of cells per flask and the cloning efficiency was not detectable because of a pipetting error. Due to the fact that at this concentration neither a cytotoxic effect has been observed nor very differing cloning efficiencies or number of mutant colonies per 106 cells have been detected all over the concentration range tested (including negative and solvent controls), the cloning efficiency should also be in the range between 53% and 73%. Assuming the "worst" detected cloning efficiency of 53%, the calculation would result in 25.71 mutants per 106 cells and a mutation factor of 0.61. Furthermore, in experiment II without metabolic activation also no reduction in cloning efficiency was observed at the highest concentration tested (10 mM). Due to these facts, the experiment is still considered as valid and the results are reliable. With metabolic activation all mutant values of the negative, the solvent controls and most mutant values of the test item concentrations found were within the historical control data (about 2-39 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls. Mutation frequencies of the negative control were found to be 29.60 and 28.92. of the solvent control 34.42 and 36.51 mutants/106 cells and in the range of 19.56 to 62.71 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.77 was found at a concentration of 5.0 mM with a relative growth of 82.1%.

In experiment II without metabolic activation all mutant values of the negative, the solvent controls and most mutant values of the test item concentrations found were within the historical control data (about 1-39 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies of the negative control were found to be 38.84 and 22.64, of the solvent control 27.59 and 15.05 mutants/106 cells and in the range of 11.23 to 48.36 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 2.27 was found at a concentration of 10.0 mM with a relative growth of 88.6%.

In experiment II with metabolic activation most mutant values found were within the historical control data of the test facility (about 2-39 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies of the negative control were found to be 25.49 and 30.08, of the solvent control 42.35 and 18.62 mutants/106 cells and in the range of 12.85 to 48.44 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.59 was found at a concentration of 2.0 mM with a relative growth of 84.5%. DMBA (1.0 and 1.5 pg/mL) and EMS (300 pg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Conclusions:
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Sa 190 is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

The test item Sa 190 was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster. The selection of the concentrations was based on data from the pre-experiments. In experiment I and II 10 mM (with and without metabolic activation) was selected as the highest concentration. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h lang time exposure assay.

The test item was investigated at the following concentrations:

Experiment I with and without metabolic activation: 0.0390, 0.0780, 0.1560, 0.3125, 0.625, 1.25, 2.5, 5.0, 7.5, 10 mM

Experiment II without metabolic activation: 0.0390, 0.0780, 0.1560, 0.3125, 0.625, 1.25, 2.5, 5.0, 7.5, 10 mM and

with metabolic activation: 0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 2.0, 4.0, 7.0, 10 mM

Precipitation of the test item was noted in the experiments at concentrations of 1.25 mM and higher, in experiment II (with metabolic activation) at 1.0 mM and higher. No biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 105.7% for the highest concentration (10 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 10 mM with a relative growth of 86.4%. In experiment II without metabolic activation the relative growth was 88.6% for the highest concentration (10 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a relative growth of 80.6%.

In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

REACH_negative | mouse (male/female) | OECD 474 | #key study#

In order to clarify the genotoxic potential, an in vivo combined micronucleus assay was conducted according to OECD TG 474. In the respective micronucleus study the test substance was not clastogenic or aneugenic in the bone marrow of male rats up to a dose of 2000 mg/kg under the experimental conditions.

In conclusion Sa 190 is not considered to have a genotoxic potential in vivo.

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:
April - June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
ANIMALS

- Species: mouse (mus musculus)
- Strain: NMRI, young healthy adult
- Source: Charles River, 97633 Sulzfeld, Germany
- Number of animals: 5 of each sex per dose group
- Initial age at start of acclimatisation: 6 - 12 weeks
- Age at start of treatment: minimum 7 weeks

The animals were derived from a controlled full barrier maintained breeding system (spf). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes and undergo an adequate acclimatisation period after arrival. The animals were randomly distributed into test groups and individually marked for identification by tail and ear drawing.

HUSBANDRY

The animals were barrier maintained (semi-barrier) in an air conditioned room. The experiment was conducted under standard laboratory conditions.
- Housing: 5 animals of identical sex per cage
- Cage type: IVC cage (Polysulphone), Type II L
- Bedding: Altromin saw fiber bedding (Batch: 02102150227)
- Feed: Free access to Altromin 1324 (Batch: 1239) maintenance diet for rats and mice
- Air change: at least 10 x per hour
- Water: Free access to tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
- Environment: temperature 22 ± 3 °C; relative humidity 55 ± 10%; artificial light 6:00 - 18:00
Route of administration:
intraperitoneal
Vehicle:
Cottonseed oil
Details on exposure:
PREPARATION OF THE TEST ITEM
The test item was grinded thoroughly in a mortar and suspended in Cottonseed oil (Sigma, Batch MKBQ5465V, MKBS9702V) within 1 h before treatment. All animals received a single volume ip of 10 mL/kg bw. The solvent was chosen according to its relative non-toxicity for the animals.
Duration of treatment / exposure:
single administration
Frequency of treatment:
single administration
Post exposure period:
44 and 68 h
Dose / conc.:
400 other: mg/kg bw (nominal)
Dose / conc.:
1 000 other: mg/kg bw (nominal)
Dose / conc.:
2 000 other: mg/kg bw (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Name: CPA (cyclophosphamide)
- CAS No.: 50-18-0
- Supplier: Sigma
- Catalogue No.: C0768
- Batch No.: SLBG4216G
- Dissolved in: physiological saline
- Dosing: 40 mg/kg bw
- Route and frequency of administration: ip, single
- Volume administered: 10 mL/kg bw

The solution was aliquoted and stored at <=-15°C. On day of administration the solution was freshly thawed. The stability of CPA at room temperature is quite good (3.5% is hydrolysed per day in aqueous solution). It is acceptable that the positive control can be administered by a route different from the test agent and sampled at only a single time. The sampling time for the positive control is 44 h after treatment.
Tissues and cell types examined:
Blood cells - erythrocytes
Evaluation criteria:
There are several criteria for determining a positive result:
- dose-related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups.
According to the OECO guideline, the biological relevance as well as the statistical significance of the results are the criteria for the interpretation.
A test item is considered to be negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level.
Statistics:
For the statistics the nonparametric Mann-Whitney test wass used. However, both biological relevance and statistical significance (at a level of p< 0.05) were considered together.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
other: vehicle served as negative control
Positive controls validity:
valid

PRE-EXPERIMENT

In the pre-experiment a concentration of 200 mg/mL of the test item was evaluated. Three male and three female mice received a single dose of 2000 mg/kg bw ip and showed mild toxicity such as reduction of spontaneous activity, constricted abdomen, piloerection, bradykinesia and half eyelid closure. Thus, it could be concluded that the test substance was systemically available. Due to the results obtained in the pre-experiment 2000 mg/kg bw was chosen as maximum tolerable dose (1 MTD) in the main experiment.

Signs of toxicity (2000 mg/kg bw)

 Signs Time post-application / sex (3 male and 3 female mice)
   30 min  1 h  2 h  3 h  4 h  24 h  48 h  72 h
   m  f  m  f  m  f  m  f  m  f  m  f  m  f  m  f
 Red. spont. activity  0  0  0  0  0  0  0  0  3  1  2  1  0  0  0  0
 Constricted abdomen  0  0  0  0  0  0  0  0  1  1  0  0  0  0  0  0
 Piloerection  0  0  0  0  0  0  0  0  2  2  0  1  0  0  0  0
 Half eyelid closure  1  0  1  0  0  0  0  0  3  3  2  0  0  0  0  0
 Bradykinesia  0  0  0  0  0  0  0  0  1  1  0  0  0  0  0  0

MAIN EXPERIMENT

Toxicity

2000 mg/kg bw was tested as the maximum tolerable dose (1 MTD) in the main experiment. The volume administered ip was 10 mL/kg bw.

All animals treated with the highest dose (1 MTD) showed mild toxic effects. After 24 h no toxic symptoms were observed anymore in male as well as female animals. The signs of toxicity noted were reduction of spontaneous activity, bradykinesia and half eyelid closure. Animals treated

with 0.5 MTD and 0.2 MTD showed no signs of toxicity.

Signs of toxicity (2000 mg/kg bw)

 Signs Time post-application / sex (5 male and 5 female mice)
   30 min  1 h  2 h  3 h  4 h  24 h  44 h  68 h
   m  f  m  f  m  f  m  f  m  f  m  f  m  f  m  f
 Red. spont. activity  5  5  5  5  5  5  5  0  5  0  0  0  0  0  0  0
 Half eyelid closure  5  5  5  5  0  0  0  0  0  0  0  0  0  0  0  0
 Bradykinesia  5  0  5  5  0  0  0  0  0  0  0  0  0  0  0  0

Relative PCE

The relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes) was determined for each animal. The relative PCE is the supportive endpoint to assess cytotoxicity, which helps to demonstrate a target cell exposure with the test item.

The negative controls (44 h, 68 h) were within the range of the historical negative control data control (0.88% - 4.30%). The mean values noted for the 44 h negative control were 1.47% (male mice) and 1.43% (female mice). The mean values detected for the 68 h negative control were 2.17% (male mice) and 1.47% (female mice).

The animal group treated with 0.2 MTD showed mean values of the relative PCE of 1.93% (male and female mice). The mean values observed in both groups were increased as compared to the corresponding negative control. However, these increases were not statistically significant.

The dose groups which were treated with 0.5 MTD showed mean values of the relative PCE of 1.97% (male mice) and 1.07% (female mice). The value observed in the male group was increased but this increase was not statistically significant. The value observed in the female group was decreased as compared to the corresponding negative control but this decrease was not statistically significant.

The animals who received 1 MTD (44 h treatment) showed mean values of 1.69% (male mice) and 1.63% (female mice). The values observed in both groups were increased as compared to the corresponding negative control. However, these increases were not statistically significant.

The animal group which was treated with 1 MTD (68 h treatment) showed mean values of the relative PCE of 2.90% (male mice) and 2.61% (female mice). The values observed in both groups were increased as compared to the corresponding negative control. However, these

increases were not statistically significant.

The decrease and/or increase of PCE values in treated animals compared to control animals is a hint of a target cell exposure of the test item.

Micronucleated polychromatic erythrocytes

For all dose groups, including positive and negative controls, at least 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes.

The negative controls (44 h and 68 h) evaluated were within the range of the historical negative control data (0.10 – 0.33%). The mean values of micronuclei observed for the negative control (44 h) were 0.19% (male mice) and 0.15% (female mice). The mean values of the 68 h negative control were 0.23% (male mice) and 0.15% (female mice).

The mean values of micronuclei observed after treatment with 0.2 MTD were 0.22% (male mice) and 0.18% (female mice). The values observed in both groups were within the range of the corresponding negative as well as historical negative control data.

The mean values noted for the 0.5 MTD dose group were 0.22% (male mice) and 0.18% (female mice). The values observed in both groups were within the range of the corresponding negative as well as historical negative control data.

The dose group treated with 1 MTD (44 h treatment) showed mean values of 0.28% (male mice) and 0.18% (female mice). The value observed in the male group was increased compared to the corresponding negative control, but this increase was not statistically significant. Additionally, the value was within the range of the historical negative control data. The value observed in the female group was within the range of the corresponding negative as well as historical negative control data.

The mean values observed for the 1 MTD (68 h treatment) were 0.25% (male mice) and 0.20% (female mice). The value observed in the male group was within the range of the corresponding negative as well as historical negative control data. The value observed in the female group was increased as compared to the corresponding negative control but this increase was not statistically significant. Moreover, the value was within the range of the historical laboratory control data.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p< 0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated.

Cyclophosphamide (40 mg/kg bw) administered ip was used as positive control which induced a statistically significant increase in the micronucleus frequency (mean percentage of cells with micronuclei was 1.95% for male and 1.36% for female mice. This demonstrates the validity of the assay.

Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Sa 190 did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. Therefore, Sa 190 is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.
Executive summary:

This study was performed to investigate the potential of Sa 190 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse, which is the endpoint of this test to assess genotoxicity.

The test item was grinded thoroughly in a mortar and suspended in Cottonseed oil. The volume administered ip was 10 mL/kg bw. Peripheral blood samples were collected for micronuclei analysis 44 h and 68 h after a single application of the test item.

A pre-experiment was performed as dose range finding study based on the OECD guideline 474 and other relevant documents (OECD 420, OECD 423). Based on the outcome of the dose range finding study, a dose of 2000 mg/kg bw was selected as maximum tolerable dose (MTD).

In the main experiment three dose levels were used covering a range from the maximum tolerable dose to little or no toxicity. The following dose groups were selected based on the toxicity observed in the pre-experiment:

 Doses  Concentration [mg/kg bw]
 1 MTD  2000
 0.5 MTD  1000
 0.2 MTD  400

Animals treated with doses of 0.2 MTD and 0.5 MTD showed no signs of systemic toxicity. Animals treated with a dose of 1 MTD showed mild signs of systemic toxicity such as reduction of spontaneous activity, bradykinesia and half eyelid closure.

The observation of systemic toxicity in the pre- as well as in the main-experiment demonstrates that Sa 190 was systemically available after intraperitoneal application and thus was able to reach the target organ.

For all dose groups, including positive and negative controls, 10000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The negative controls (44 h, 68 h) were within the range of the historical negative control data. The mean values noted for the dose groups which were treated with the test item (44 h, 68 h) were within the corresponding and historical negative control data range, except the value observed for the 1 MTD male group (44 h) and 1 MTD female group (68 h). These values were increased as compared to the corresponding negative control. However, these increases were not statistically significant and both values were within the range of the historical negative control data. Therefore they were regarded as not biologically relevant.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p< 0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated.

Cyclophosphamide (40 mg/kg bw) administered ip was used as positive control, which induced a statistically significant increase in the micronucleus frequency. This demonstrates the validity of the assay.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Sa 190 did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.

Therefore, Sa 190 is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.

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

Additional information

Justification for classification or non-classification

The substance was clearly negative in the in vivo micronucleus assay conducted according to OECD TG 474 and in the in vitro gene mutation assays conducted according to OECD TG 471 and 476, thus the classification criteria are not met.