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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic in bacteria, as determined in an OECD TG 471 study.

The test substance is not mutagenic in mammalian cells, as determined in an OECD TG 490 study.

The test substance is not cytogenic, as determined in an OECD TG 487 study.

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:
14 Jul 2017 to 20 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
GLP compliance:
yes (incl. QA statement)
Remarks:
Testing laboratory: Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name as mentioned in test report: Nickel dibutyldithiocarbamate
- Batch No. of test material: 70500102
- Expiration date of the lot/batch: 25 May 2019
- Purity test date: set to 100%
- Appearance: dark green powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature (15-25 ⁰C)
Target gene:
- S. typhimurium: his
- E. coli: trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 induced rats livers
Test concentrations with justification for top dose:
MAIN EXPERIMENT
- Strains TA98, TA100, TA1535, TA1537 and WP2 uvrA, with and without S9-mix: 62, 185, 556, 1667, 5000 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: the test substance appeared to be poorly soluble in the aqueous solvent phosphate buffered saline
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See “Any other information on materials and methods inc. tables”
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 hours at ca. 37 ⁰C

NUMBER OF REPLICATIONS:
3 plates/dose
Evaluation criteria:
see section "Any other information on materials and methods incl. tables"
Statistics:
No statistical analysis was performed.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated 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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A dose related precipitation was observed after adding the test substance to the molten top agar and at the plates at all concentrations tested for all strains. At and above 556 μg/plate the precipitation could be observed by macroscopically. This precipitation was considered not to affect counting of the revertants. At and above 1667 μg/plate a slightly more dense background lawn of bacterial growth compared to the concomitant control plates was observed at all strains.

VALIDATION CRITERIA
The mean numbers of his+ and trp+ revertant colonies of the negative controls used were within the acceptable range in all strains and the positive controls gave the expected increase in the mean numbers of revertant colonies. Therefore, the test was considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed in any strain, this was evidenced by an absence of a clearing of the background lawn of bacterial growth compared to the negative controls, no decrease in the mean number of revertants was observed and pinpoint colonies did not occur.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Nov 2017 to 7 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
GLP compliance:
yes (incl. QA statement)
Remarks:
Testing laboratory: Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of the substance as cited in report: Nickel dibutyldithiocarbamate
- Batch No. of test material: 70900202
- Expiration date of the batch: 11 September 2019
- Purity test date: >98%
- Appearance: dark green powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature (15-25 ⁰C)
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
(L5178Y Tk +/- 3.7.2C line)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: MRC Cell Mutation Unit, University of Sussex, United Kingdom
- Methods for maintenance in cell culture: L5178Y cells were grown in growth medium (10% v/v for growing in flasks, and 20% for growing in microtiter plates). The cells were cultured in a humidified incubator at ca 37 °C in air containing ca 5% CO2. Five days prior to treatment, the cells were generated from a frozen stock culture by seeding them in sterile, screw-capped tissue culture flasks (about 10,000,000 cells per flask: growth area ± 75 cm²) containing 50 mL growth medium (with 10% horse serum). Near-confluent cells (i.e. below maximum recommended cell density) were harvested from a number of culture flasks and suspended in growth medium (with 10% horse serum), and the number of cells will be counted. For the gene mutation assay portions of ca. 6,000,000 cells were used per culture in the absence and presence of metabolic activation.
- Modal number of chromosomes: 40 (stable aneuploid karyotype, 2n = 40)

MEDIA USED
- Type and identity of media: Growth medium; RPMI 1640 medium (with HEPES and L-Glutamine) supplemented with heat-inactivated horse serum (10% v/v for growing in flasks, and 20% for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 induced rats livers
Test concentrations with justification for top dose:
JUSTIFICATION FOR TOP DOSE
he test concentrations were selected based on the MN test (Triskelion B.V., V21017/02)

TEST CONCENTRATIONS
Experiment 1
3 hour treatment (without and with S9): 3.9, 7.8, 15.7, 31.3, 62.5, 125, 250 and 500 μg/mL
Experiment 2
24 hour treatment (without S9): 7.8, 15.7, 31.3, 62.5, 125, 180, 250, 320, 400 and 500 μg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: Dimethyl sulfoxide (DMSO) was the preferred solvent for the test substance based on precipitation of the test substance
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 6,000,000 cells 5 mL growth medium

DURATION
- Exposure duration: 4 hours in the presence and absence of S9-mix (first experiment) or 24 hours in the absence of S9-mix (second experiment)
- Expression time (cells in growth medium): the cell suspensions were diluted to ca. 300,000 cells per mL and the cultures were incubated for ca. 48 hours. After 20-24 hours the cell suspensions were diluted, if required, to ca. 300,000 cells per mL and further incubated
- Selection time: 10-12 days

SELECTION AGENT: 4 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: test substance and positive controls, single cultures; negative controls, duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (RSG) and the relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: The RSG is a measure for the cumulative growth rate of the cells 24 hours and 48 hours after treatment compared with untreated control cultures and the RTG is the product of the relative initial cell yield after treatment, the RSG and the relative colony-forming ability (‘cloning efficiency’) of the cells 48 hours after treatment compared with untreated control cultures.

ADDITIONAL DETAILS ON METHODS
- On the day of treatment, the growth rate and viability (>90 %; by trypan blue exclusion) of the cells were checked.
- At the start of the treatment all cell cultures were checked visually for precipitation of the test substance, discoloring of the medium due to pH change and/or any other aberrancy.
- At the end of the treatment period, the flasks were checked for visibly aberrant effects
(e.g. precipitation of the test substance, presence of lysed cells), and viability was checked. The medium containing the test substance, negative control or positive control was removed and the cells were washed twice with growth medium (with 10% horse serum). Finally, the cells were resuspended in growth medium (with 10% horse serum) and the number of cells was counted. Subsequently, the cell suspensions were diluted to ca. 300,000 cells per mL and the cultures were incubated for ca. 48 hours (after 20-24 hours the cell suspensions were diluted, if required, to ca. 300,000) to allow near-optimal phenotypic expression of induced mutations.
- For determining the cloning efficiency, after ca. 48 hours after treatment a portion of the cells was diluted to a density of 10 cells per mL in growth medium (with 20% serum). The remaining cells were used for determining the frequency of TFT-resistant mutants. Portions (200 μL) of each dilution at 10 cells per mL were transferred to each well of two 96-well microtiter plates, and the plates were incubated for 10-12 days.
- At the end of the 10-12 days incubation, the number of wells without growth of cells were
counted and the cloning efficiency was determined using the zero term of the Poisson distribution (Cole et al., 1983) as follows: Cloning efficiency (CE) = (-ln [number of empty wells/total number of wells])/plated number of cells per well
- The ratio of the cloning efficiency of cells treated with the test substance or the positive control compared to that of the vehicle control yields the relative cloning efficiency (RCE). The suspension growth (SG) was calculated as follows: Suspension growth (SG) = (cell count at 24 h/300,000) x (cell count at 48 h/300,000). A previous day’s cell count was used if lower than 300,000
- The ratio of the SG of treated cells to that of the vehicle control yields the RSG. The relative total growth (RTG) is adjusted for growth during treatment to obtain a measure for cytotoxicity that occurs in all phases of the assay. The RTG was calculated as follows: Relative total growth (RTG) = (cell count after treatment/cell count vehicle conrol) x RSG x RCE. Reduction of the cell count after treatment, or of the RSG and of the RTG is a measure for the cytotoxicity of the test substance.
- A decision for the concentration levels to be tested for mutation induction was made after data on viability and growth rate were available. At least four cultures were selected for gene
mutation analysis.
- The frequency of TFT-resistant mutants was determined after ca. 48 hours after treatment. The cell suspensions were diluted to a density of ca. 10,000 cells per mL in growth medium (with 20% horse serum) containing 4 μg TFT per mL. Portions (200 μL) of each dilution were transferred to each well of two 96-wells microtiter plates. The plates were incubated for 10-12 days.
- The mutant frequency (MF) per 1,000,000 clonable cells was finally calculated as follows: Mutant frequency (MF) = (Mutant Cloning efficiency / Cloning efficiency) * 1,000,000
- For the negative (solvent) and positive control cultures, the mutant colonies were scored using the criteria of small (covers <25% of the well area) and large (covers >25% of the well area) colonies.
Evaluation criteria:
see "Any other information on materials and methods incl. tables"
Statistics:
No statistical analysis was performed.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects reported
- Precipitation: No precipitation in the test is reported.
- Other confounding effects: The stock solutions of 50 mg/mL and 6.25 mg/mL in DMSO were dark green suspension containing test substance particles and a dark green solution (which contained precipitating test substance particles in the first experiment only), respectively.

HISTORICAL CONTROL DATA (means and standard deviation, confidence interval (95%), number of assays)
- Positive historical control data: MCA 10 µg/mL 4 h (+S9): 907 ± 299, 467-1631, 102; MMS 100 μM 24 h (-S9): 1292± 466, 640-2045, 99
- Negative (solvent) historical control data: 4 h (-S9): 75 ± 24, 46-122, 25; 4 h (+S9): 82 ± 26, 41-141, 100; 24 h (-S9): 94 ± 37, 44-134, 91

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RTG
- Other observations: In the first experiment, at the end of the treatment, viability of the cells (as determined with trypan blue dye exclusion) was 95% and 99% at the highest test concentration, in the absence and presence of S9-mix, respectively. In the second experiment, at the end of the treatment, viability of the cells was 96% at the highest test concentration in the absence of S9-mix. In both the first and second experiment, the test substance was slightly cytotoxic to the cells resulting in a reduction of the relative total growth (RTG). At the highest concentration tested (500 μg/ml) the RTG was 70% and 76% after 4 hours treatment in the absence and presence of S9-mix, respectively, and 43% after 24 hours treatment in the absence of S9-mix.

ASSAY VALIDATION
- Acceptance criteria for the positive and negative control substance were met.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Oct 2017 to 23 Nov 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
GLP compliance:
yes (incl. QA statement)
Remarks:
Testing laboratory: Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name as cited in report: Nickel dibutyldithiocarbamate
- Batch No.of test material: 70900202
- Expiration date of the batch: 11 September 2019
- Purity test date: >98 %
- Appearance: dark green powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature (15-25 ⁰C)
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
CELLS USED
- Sex, age and number of blood donors: two young healthy, non-smoking donors (38 and 27 years old, for the first and second test, respectively)
- Whether whole blood or separated lymphocytes were used: whole blood
- Methods for maintenance in cell culture: Whole blood (0.5 mL) was added to sterile screw-capped tubes containing 4.5 ml culture medium. The blood cultures were incubated for ca. 48 hours at ca. 37 °C in humidified air containing ca. 5% CO2.

MEDIA USED
- Type and identity of media: RPMI 1640 medium (with HEPES and Glutamax-I), supplemented with heat-inactivated (30 min, 56ºC) fetal calf serum (20% v/v), penicillin (100 U/mL medium), streptomycin (100 μg/mL medium) and phytohemagglutinin (2.4 μg/mL)
Cytokinesis block (if used):
Cytochalasine B (6 μg/mL) for 20 hours (pulse treatment group) or 24 hours (continuous treatment group)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 induced rats livers
Test concentrations with justification for top dose:
JUSTIFICATION OF THE TOP DOSE
According to OECD guideline 487, for poorly soluble test substances that are not cytotoxic at concentrations lower than the lowest insoluble concentration, the highest concentration analyzed should produce turbidity or a precipitate visible by eye or with the aid of an inverted microscope at the end of the treatment with the test substance. The top dose was selected based on solubility, osmolality and pH tests. Stock solutions of 200 mg/mL and 100 mg/mL were prepared. Subsequently, the 100 mg/mL was 2-fold diluted with DMSO to obtain a 50 mg/mL solution. From this 50 mg/mL solution, six dilutions with 2-fold spacing were prepared in DMSO. In addition, a 6.25 mg/mL stock solution was prepared to check if this was a clear solution. The final concentrations of the test substance in culture medium of 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 μg/mL. Shortly after preparation at ambient temperature and after 3 hours incubation in a humified incubator at ca. 37 °C and ca. 5% CO2, changes with respect to the test substance were checked visually. In addition, pH and osmolality measurements were performed.

DOSES (1st and 2nd experiment) 500*, 250, 125*, 62.5, 31.3*, 15.6, 7.8*, 3.9, 2 and 1 μg/mL.
* selected for micronuclei analysis
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: Culture medium was considered not suitable as solvent based on the bacterial reverse mutation test with the test substance (Triskelion study 21005/02). Therefore, DMSO was selected as solvent.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Vinblastine Sulphate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

EXPERIMENT 1, PULSE TREATMENT GROUPS WITH AND WITHOUT S9-MIX
- Preincubation period: 48 hour
- Exposure duration: 4 hours
- Methods exposure: After ca. 48 hours incubation of the blood cultures at ca. 37 °C in humidified air containing ca. 5% CO2, the cells were harvested by low speed centrifugation, suspended in freshly prepared tissue culture medium without foetal calf serum and exposed to 50 μL test substance solutions or negative (solvent) control, or 50 μL of the positive control solution (Cyclophosphamide). To all cultures of the pulse treatment group in the presence of the S9-mix, 0.5 ml of S9-mix was added. The total volume in each culture was 5 mL. The final concentration of DMSO was 1%. After a 4-hours treatment period at ca. 37 ⁰C in humidified air containing ca. 5% CO2, the cultures were checked for visible aberrant effects (e.g. haemolysis of the erythrocytes).
- Recovery time: 20 hours
- Methods recovery: The cells were washed twice with phosphate-buffered saline (pH 7.4) and subsequently supplied with 5 mL freshly prepared culture medium enriched with fetal calf serum (20%), PHA-L and cytochalasin B (final concentration 6 μg/mL, stock solution 600 μg/ml in DMSO). The cells were incubated for an additional ca. 20 hours at ca. 37°C in humidified air containing ca. 5% CO2 and harvested ca. 72 hours after initiation of the cultures (second cell-cycle), followed by the preparation of slides.

EXPERIMENT 2, CONTINUOUS TREATMENT GROUP WITHOUT S9-MIX
- Preincubation period: 48 hour
- Exposure duration: 24 hours with cytochalasin B
- Methods exposure After 48 ± 2 hours incubation of the blood cultures at ca. 37 °C in humidified air containing ca. 5% CO2, the cells were harvested by low speed centrifugation and suspended in freshly prepared tissue culture medium enriched with 20% foetal calf serum, followed by exposure to 50 μL of the test substance solutions or negative (solvent) control, or 50 μL of the positive control solution (Vinblastine Sulphate). The total volume in each culture was 5 ml. The cells were incubated for 24 hours at ca. 37°C in humidified air containing ca. 5% CO2 in the presence of cytochalasin B (final concentration 6 μg/mL, stock solution 600 μg/mL in DMSO) and harvested 72 hours after initiation of the cultures (second cell-cycle), followed by preparation of slides. Since both the test substance and cytochalalsin B were dissolved in DMSO, the final DMSO concentration during treatment was 2%. An additional solvent control (1% DMSO) was included in the experiment to demonstrate that the relatively high percentage of organic solvent did not have an adverse effect.

NUMBER OF REPLICATIONS:
Two cultures per dose

SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Each culture was harvested and processed separately. The cells were harvested by low speed centrifugation, treated with a hypotonic solution (0.075 M potassium chloride), fixed three times with a freshly prepared mixture of methanol and acetic acid, spread on clean slides and air dried. All procedures were performed at ambient temperature. Three slides will be prepared from the negative (solvent) controls, positive controls and from each selected culture treated with the test substance.

STAIN
acridin-orange

NUMBER OF CELLS EVALUATED: Number of cells evaluated: 500 cells per slide (in total 1000 cells per dose level)

CRITERIA FOR SCORING CYTOKINESIS-BLOCKED (BINUCLEATED) CELLS:
The cytokinesis-blocked cells that may be scored for micronuclei frequency should have the following characteristics:
1. The cells should be binucleated.
2. The two nuclei in a binucleated cell should have intact nuclear membranes and be situated within the same cytoplasmic boundary.
3. The two nuclei in a binucleated cell should be approximately equal size. staining pattern and staining intensity.
4. The two nuclei within a binucleated cell may be attached by a fine nucleoplasmic bridge which is no wider than one-fourth of the largest nuclear diameter
5. The two main nuclei in a binucleated cell may touch but ideally should not overlap each other. A cell with two overlapping nuclei can be scored only if the nuclear boundaries of each nucleus are distinguishable.
6. The cytoplasmic boundary or membrane of a binucleated cell should be intact and clearly distinguishable from the cytoplasmic boundary of adjacent cells.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Micronuclei are morphologically identical to but smaller than nuclei. They have the following characteristics:
1. The diameter of micronuclei usually varies between 1/16 and 1/3 of the diameter of the main nuclei.
2. Micronuclei are round or oval in shape.
3. Micronuclei are nonrefractile and can therefore be readily distinguished from artefacts such as staining particles.
4. Micronuclei are not linked or connected to the main nuclei.
5. Micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary.
6. Micronuclei usually have the same staining intensity as the main nuclei but occasionally staining is more intense.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation Index
- Any supplementary information relevant to cytotoxicity: Quantitative evaluation of cytotoxicity was performed using the CBPI. The CBPI indicates the average number of cell cycles per cell during the period of exposure to cytochalasin B. The CBPI was determined from at least 500 cells per slide and was used to calculate cell proliferation and to estimate the percentage of cytotoxicity by comparing values in the treated and negative (solvent) control cultures.
The CBPI was calculated for treated (selected doses) and control cultures as a measure of cell cycle delay. If observed, the concurrent measures of cytotoxicity (cell density on the slides, signs of apoptosis or necrosis) were recorded for all treated and negative (solvent) control cultures. Based on the evaluation of cytotoxicity, analysis of micronucleus formation was carried out on four analyzable concentrations of the test substance, together with the solvent and the positive control cultures. Where cytotoxicity occurred, the concentrations selected aimed to cover a range producing 55 ± 5% cytotoxicity, moderate and little or no cytotoxicity. At least two thousand binucleated cells per concentration (500 cells per slide, two slides per culture, two cultures per concentration) were examined for the presence of micronuclei.


Evaluation criteria:
The study was considered valid if
- the positive controls demonstrated a statistically significantly increase in the number of binucleated cells containing micronuclei
- the response of the positive controls was compatible with historical positive control data
- the negative (solvent) controls were within the historical range and /or comparable to the data presented in the literature
- proliferation criteria in the negative (solvent) controls were fulfilled
- adequate numbers of cells and concentrations were analysable

A response was considered positive if all of the following criteria were met:
- at least one of the test concentrations exhibited a statistically significant increase compared to the concurrent negative control
- the increase was concentration-related in at least in one experimental condition when evaluated with an appropriate trend test
- any of the results were outside the distribution of the historical solvent control data

A response was considered negative if all of the following criteria were met:
- none of the test concentrations exhibited a statistically significant increase compared to the concurrent negative control
- there was no concentration-related increase when evaluated with an appropriate trend test
- all results were inside the distribution of the historical negative control data

In case the response was neither clearly negative or clearly positive as described above and/or in order to assist in establishing the biological relevance of a result, justification should be provided or the results should be further evaluated (e.g. scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing).

A test substance was considered equivocal if the response was neither positive nor negative, even after further investigation.

Biological relevance was taken into account for interpretation of the results.
Statistics:
The frequencies of micronuclei were compared with those of the concurrent solvent controls using the Chi-Square test (one sided). The results were considered statistically significant if the P-value was less than 0.05.
Key result
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects observed up to 500 μg/mL
- Effects of osmolality: no effects observed up to 500 μg/mL
- Precipitation: Yes, see “any other information on results inc. tables”. The lowest concentration analyzed for micronuclei did not show any precipitation in the culture medium in both experiments.
- Other confounding effects: In the first experiment, as a result of the test substance precipitation, a green cell pellet was observed during the fixation of the cells at final concentrations ranging from 500 to 62.5 μg/mL in the presence of S9 mix and at 500 μg/ml in the absence of S9-mix. In addition, in the pulse treatment group in the presence of S9-mix, the highest concentration (500 μg/mL) appeared to be slightly haemolytic. The second experiment, i.e. continuous treatment group in the absence of S9-mix, precipitation of the test substance and a green cell pellet was observed during the fixation of the cells at final concentrations ranging from 62.5 to 500 μg/mL

VALIDATION OF THE TEST
- In both experiments, the solvent controls (1% DMSO in the first experiment and 2% DMSO in the second experiment) within the range of historical data of the test facility. In the second experiment, the CBPI index and micronuclei frequency were of the solvent control 2% DMSO was comparable to 1% DMSO. Therefore, it was justified to use 2% DMSO as solvent control. - Treatment with the positive controls Cyclophosphamide and Vinblastine sulphate resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the concurrent solvent control cultures. Therefore, the test was considered valid.

HISTORICAL CONTROL DATA (ranges, means and standard deviation, number of tests)
- Positive historical control data: Cyclophosphamide (1.80 – 6.30, 3.52 ± 1.02, 38); Vinblastine sulphate (2.20 – 6.05, 4.49 ± 1.44, 6)
- Negative (solvent) historical control data: 4 hr, with S9 (0.40 – 1.20, 0.70 ± 0.17, 38); 4 hr, without S9 (0.20 – 1.15, 0.69 ± 0.20, 38); 24 hr, without S9 (0.65 – 1.15, 0.88 ± 0.15, 11); 24 hr, without S9, 2% DMSO (0.90 – 1.65, 1.11 ± 0.27, 7)

Table: Changes with respect to the test substance in culture medium without serum (prior to conduct of the study and after 100-fold dilution in culture medium).

Stock concentrations in DMSO (mg/mL)

Final concentrations in culture medium (μg/mL)

Appearance of the stock concentrations *

Appearance of the final concentrations #

200

Not tested

Viscous suspension, not feasible for use

Not tested

100

Not tested

Dark green suspension with small clumps, not feasible for use

Not tested

50

500

Dark green suspension with small test substance particles, feasible for use

Culture medium turbid, test substance precipitation

25

250

Dark green suspension with small test substance particles

Culture medium turbid, slight test substance precipitation

12.5

125

Dark green solution with few test substance particles

Culture medium turbid

6.25

62.5

Dark green solution

Culture medium turbid

3.13

31.3

Green to light green solution.

Culture medium very slightly turbid

1.56

15.6

Green to light green solution.

No abnormalities

0.78

7.8

Green to light green solution.

 

 * Results shortly after preparation at ambient temperature.

# Results shortly after preparation at ambient temperature and after three hours incubation at ca. 37 °C

and ca. 5% CO2.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Reverse bacterial mutation assay (Ames test)

The test substance was examined for possible mutagenic activity in the bacterial reverse mutation test using the histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiring Escherichia coli strain WP2uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix) (Triskelion B.V., 2017). This experiment was performed according to OECD TG 471 and GLP. A single test was performed. All strains, both in the absence and presence of S9-mix, were treated with five concentrations of the test substance. Stock solutions of the test substance of 50 mg/mL and 5.56 mg/mL in dimethyl sulfoxide (DMSO) were prepared, resulting in a green, turbid suspension and a green, clear solution with a few lumps, respectively. Negative controls (solvent) and positive controls were run simultaneously with the test substance. The mean numbers of his+and trp+revertant colonies of the negative controls used were within the acceptable range in all strains and the positive controls gave the expected increase in the mean numbers of revertant colonies. Therefore, the test was considered valid. No toxicity was observed in any strain, which was evidenced by absence of a clearing of the background lawn of bacterial growth compared to the negative controls, absence of a decrease in the mean number of revertants was observed and absence of pinpoint colonies. The test substance did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control with strains TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA, in both the absence and presence of S9-mix. It is concluded that the results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2uvrA, in both the absence and presence of the S9-mix, indicate that the substance isnot mutagenicunder the conditions used in this study.

In a supporting study the mutagenic activity of the substance was evaluated similar to OECD TG 471 and according to GLP principles (HRC, 1985). The test was performed in the absence and presence of S9-mix. The dose levels (50 -5000 µg/plate) were selected based on the dose range finding experiment (5-5000 µg/plate). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA 1538, TA98, TA100) in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic with and without metabolic activation.

In vitro mammalian cell gene mutation test (MLA)

The test substance was examined for its potential to induce gene mutations at the Tk-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of an exogenous metabolic activation system (S9-mix), in a study according to OECD TG 490 and GLP (Triskelion B.V., 2018a). Two independent experiments were performed. In the first experiment, the treatment period was 4 hours in the presence and absence of S9-mix. In the second experiment, the treatment period was 24 hours in the absence of S9-mix. Dimethyl sulfoxide (DMSO) was used as a solvent for the test substance. The maximum concentration of the test substance was limited by precipitation of the test substance. In both experiments the final concentrations tested ranged from 500 to 3.9 μg/mL. Single cultures were used. Negative (solvent, duplicate cultures) and positive (single cultures) controls were run simultaneously with the test substance. 3-Methylcholanthrene (MCA) and Methyl methanesulphonate (MMS) were used as positive control substances for the 4 hours treatment in the presence of S9-mix and the 24 hours treatment in the absence of S9-mix, respectively, and DMSO served as the solvent control. All acceptance criteria for the positive and negative control substances were met. Therefore, the study was considered valid. In both the first and second experiment, the test substance was slightly cytotoxic to the cells resulting in a reduction of the relative total growth (RTG). At the highest concentration tested (500 μg/mL) the RTG was 70% and 76% after 4 hours treatment in the absence and presence of S9-mix, respectively, and 43% after 24 hours treatment in the absence of S9-mix. In both the absence and presence of S9-mix no increase in mutant frequency (MF) by more than 126 mutants per 1,000,000 clonable cells, i.e. no positive response, compared to the negative control was observed at any of the concentrations tested. It is concluded that under the conditions used in this study, the test substance isnot mutagenicat the TK-locus of mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix).

In vitro micronucleus test (MN)

The test substance was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix) in a study according to OECD TG 487 and GLP (Triskelion B.V., 2018b). Two independent experiments were performed. In the first experiment the treatment/recovery time was 4/20 hours (pulse treatment), both in the presence and absence of S9-mix. In the second experiment, the treatment/recovery time was 24/0 hours in the absence of S9-mix (continuous treatment). Dimethyl sulfoxide (DMSO) was used as a solvent for the test substance. The maximum concentration of the test substance was limited by precipitation of the test substance. In both experiments the final concentrations tested ranged from 500 to 1 μg/mL. Duplicate cultures were used. Cytotoxicity was determined from the Cytokinesis-Block Proliferation Index (CBPI). Negative controls (solvent, at the same concentration as used for the test substance) and positive controls were run simultaneously with the test substance. In both experiments, the solvent control was within the range of historical data of the test facility. Treatment with the positive controls Cyclophosphamide and Vinblastine sulphate resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the concurrent solvent control cultures. Therefore, the test was considered valid. No cytotoxicity was observed at any concentration analysed when compared to the concurrent solvent control cultures in both experiments. For each treatment group, four concentrations (i.e. 500, 125, 31.3 and 7.8 μg/mL) were selected for micronuclei analysis. In both experiments, the test substance did not show a statistically significant increase in the number of binucleated cells containing micronuclei at any of the concentrations analyzed when compared to the concurrent solvent control cultures. From the results obtained in this in vitro micronucleus test, it is concluded that the test substance wasnot clastogenic and/or aneugenicto cultured human lymphocytes, under the conditions used in this study.

Justification for classification or non-classification

Based on the results of the available studies, classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.