[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): not mutagenic up to limit or cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 & E.coli WP2uvrA-.

- Micronucleus test in vitro (OECD 487, GLP, K, Rel.1): not clastogenic and not aneugenic up to cytotoxic concentrations in human peripheral blood lymphocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 23 to October 10, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 471 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 21 August 2018 / signed on 19 November 2018)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Storage condition of test material: Approximately 4 °C in the dark under nitrogen
- Formulated concentrations were adjusted to allow for the stated water/impurity content (4%) of the test item.

Target gene:

Histidine and tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

Experiment 1 (Pre-incubation method):
TA1535 [absence of S9-mix] & all strains [with S9-mix] : 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
TA 100, TA98, TA1537 & E.coli Wp2uvrA [absence S-9 mix] : 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate.

Experiment 2 (Pre-Incubation Method):
All tester strains (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
TA100 and TA1535 (presence of S9 mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
WP2uvrA (presence of S9 mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
TA98 and TA1537 (presence of S9 mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration in solubility checks performed in-house. DMSO was therefore selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; (preincubation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 37 ± 3 °C for 20 minutes (with shaking)
- Exposure duration/duration of treatment: 37 ± 3 °C for between 48 and 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: background growth inhibition

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Possibility of evaporation from medium: The test item was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls. For volatile substances, the pre-incubation method may increase exposure of the bacteria to the test item in comparison to the standard plate incorporation method.
- Water solubility: Test item was immiscible in sterile distilled water at 50 mg/mL
- Precipitation and time of the determination: No precipitate of the test item was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- Signs of toxicity
Experiment 1: The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. However, the test item induced excessive toxicity to tester strains TA100, TA98 and TA1537 and E.coli strain WP2uvrA dosed in the absence of S9-mix and, therefore, part of the experiment was repeated using an amended dose range of 0.05 to 150 μg/plate.
The test item induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 50 μg/plate (TA100, TA98, TA1537and WP2uvrA) and 150 μg/plate (TA1535). In the presence of S9-mix, weakened bacterial background lawns were noted from 150 μg/plate (TA100 and TA1535), 500 μg/plate (TA98 and TA1537) and 1500 μg/plate (WP2uvrA).
Experiment 2: The maximum dose level of the test item in the second experiment was 5000 μg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.
The test item induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 15 μg/plate (TA100) and 50 μg/plate (TA1535, TA98, TA1537and WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted from 150 μg/plate (TA100, TA1535 and TA1537), 500 μg/plate (TA98) and at 5000 μg/plate (WP2uvrA).
- Individual plate counts / Mean number of revertant colonies per plate and standard deviation (cf. attached document):
Experiment 1: There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).
Experiment 2: No biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). A statistically significant value was noted (WP2uvrA at 15 μg/plate in the absence of S9-mix; not identified as statistically significant on Table 4, as per the requirements of the Study Plan), however this response was not dose-related, not reproducible, and was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Cf. attached document.

Conclusions:

Under the test condition, the test item was not mutagenic in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA- at any of the dose levels used either with or without metabolic activation.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames pre-incubation method at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The original dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. However, the test item induced excessive toxicity to tester strains TA100, TA98 and TA1537 and E.coli strain WP2uvrA dosed in the absence of S9-mix and, therefore, part of the experiment was repeated using an amended dose range of 0.05 to 150 μg/plate. Experiment 2 was performed on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and ranged between 0.05 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve four non-toxic dose levels and to achieve the toxic limit of the test item. Both experiments were performed using the pre-incubation methodology because the volatility of the test item was considered to be incompatible with the plate incorporation method.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Depending on bacterial strain type and presence or absence of S9-mix, the maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate or the toxic limit. In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawn of all of the tester strains, initially from 50 and 150 μg/plate in the absence and presence of metabolic activation (S9-mix), respectively.

Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) or the toxic limit was employed in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. The test item once again induced a toxic response with a visible reduction in the growth of the bacterial background lawns noted to all of the tester strains, initially from 15 and 150 μg/plate in the absence and presence of metabolic activation (S9-mix), respectively.

No precipitate of the test item was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1.

Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. One statistically significant value was noted (WP2uvrA at 15 μg/plate in the absence of S9-mix; not identified as statistically significant on Table 4, as per the requirements of the Study Plan), however this response was not dose-related, not reproducible, and was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.

 

Under the test condition, the test item was not mutagenic in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA- at any of the dose levels used either with or without metabolic activation.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 6 October 26, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD TG 487 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected from 18 to 20 july 2017 / signed on November 28, 2017)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- Storage conditions: Approximately 4 ºC in the dark
Formulated concentrations were adjusted to allow for the stated water/impurity content (4 %) of the test item.

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human peripheral blood lymphocyte
- Suitability of cells: The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Normal cell cycle time (negative control): approximately 16 hours

For lymphocytes:
- Sex, age and number of blood donors: sex not reported, one donor, 18-35 years old
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: not applicable
- Mitogen used for lymphocytes: phytohemagglutinin

MEDIA USED
- Type and composition of media: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS).
- CO2 concentration: 5%
- Humidity level: humidified air
- Temperature: approximately 37 ºC

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

cytochalasin B (4.5 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

Preliminary Toxicity Test: , 7.51, 15.02, 30.05, 60.09, 120.19, 240.38, 480.75, 961.5 and 1923 μg/mL.
4 h exposure without S9: 0, 10, 20, 40, 60, 70, 80, 90, 100 and 120 μg/mL.
4 h exposure with S9 (2%): 0, 10, 20, 40, 60, 80, 90, 100, 110, and 120 μg/mL.
24 h continuous exposure without S9,: 0, 10, 20, 40, 60, 70, 80, 90, 100 and 120 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxyde (DMSO)
- Justification for choice of solvent/vehicle: The test item was insoluble in Minimal Essential Medium at 19.2 mg/mL but was soluble in Dimethyl sulphoxide (DMSO) at 192.3 mg/mL in solubility checks performed in-house

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: demecolcine. 0.075 μg/mL in sterile distilled water for 24-hour continuous exposure

Remarks:

Absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h (± S9) and 24 h continuous exposure (-S9) in preliminary toxicity test; 4 h (± S9) and 24 h continuous exposure (-S9) in main experiment
- Harvest time after the end of treatment (sampling/recovery times): At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B before harvest.

FOR MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: Cytochalasin B, 4.5 μg/mL, 24 hour
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry with gentle warming. Each slide was permanently labeled with the appropriate identification data. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
The micronucleus frequency in at least 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). In circumstances where the response was not as expected, as in a poor positive control response a further 1000 binucleated cells were selected for scoring.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not necessary and not performed in cases where no significant increase in micronucleus frequency is observed.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis block proliferation index (CBPI).
A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis by the following formula:
% Cytostasis = 100 – 100 [CBPI(T)-1] / [CBPI(C)-1]
Where Cytostasis = [No. mononucleated cells + 2 x No. binucleated cells + 3 x No. multinucleated] cells / Total number of cells
T = test chemical treatment culture
C = vehicle control culture

ACCEPTABILITY CRITERIA:
The following criteria were used to determine a valid assay:
• The concurrent negative control was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• Cell proliferation criteria in the solvent control were considered to be acceptable.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations was analyzed.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There was no significant change in pH when the test item was dosed into media
- Data on osmolality: The osmolality did not increase by more than 50 mOsm
- Precipitation and time of the determination: Precipitate observations were recorded at the beginning and end of the exposure periods.
Preliminary toxicity test: A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, at and above 120.19 μg/mL in the absence of S9 and at and above 240.38 μg/mL in the presence of S9.
Main test: No precipitate of test item was observed at the end of the exposure period in any of the exposure groups

PRELIMINARY TOXICITY TEST
Hemolysis was observed following exposure to the test item at and above 15.02 μg/mL and 30.05 μg/mL in the 4-hour exposure groups in the absence and presence of S9, respectively and at and above 60.09 μg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
A reduced cell pellet was also observed at the end of the exposure period at and above 60.09 μg/mL in the 4-hour exposure in the absence of S9, at and above 120.19 μg/mL in the 4-hour exposure in the presence of S9 and at and above 240.38 μg/mL in the 24-hour exposure. This reduction was considered to be indicative of overall toxicity to the cells, which resulted in reduced cell numbers being present on the slides.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present up to 60.09 μg/mL in all three exposure groups. The CBPI data for the 4-hour exposures in the absence and presence of S9 and the 24-hour exposure are presented in Table 2, Table 3, Table 4, respectively. The test item demonstrated a steep toxicity curve between 60.09 μg/mL and 120.19 μg/mL.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 120μg/mL for all three exposure groups.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The vehicle control cultures had frequencies of cells with micronuclei within the expected range.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- Results from cytotoxicity measurements:
Hemolysis was observed at the end of exposure at and above 40 μg/mL in the 4-hour exposure groups and at and above 60 μg/mL in the 24-hour exposure groups. A reduced cell pellet was also noted at the end of the exposure period at and above 90 μg/mL in the 4-hour exposure group in the absence of S9.
The CBPI data for the 4-hour exposure groups in the absence and presence of S9 and for the 24-hour exposure group are given in Table 5, Table 6 and Table 7, respectively. They confirm the qualitative observations in that a dose-related inhibition of CBPI was observed in all three exposure groups. In the 4-hour exposure group in the absence of S9, 20%, 36% and 70% cytostasis was demonstrated at 60 μg/mL, 70μg/mL and 80μg/mL, respectively. The 4-hour exposure in the presence of S9 demonstrated 35%, 59% and 79% cytostasis at 90 μg/mL, 100 μg/mL and 110 μg/mL, respectively. The 24-hour exposure group demonstrated 28%, 62% and 80% cytostasis at 70 μg/mL, 80 μg/mL and 90 μg/mL, respectively.
The maximum dose level selected for analysis of binucleate cells was 100 μg/mL for the 4-hour exposure in the presence of S9 and 80 μg/mL for the 24-hour exposure group. These dose levels achieved near optimum toxicity with approximately 55±5% cytostasis. The 4-hour exposure group in the absence of S9 achieved slightly less than optimum toxicity at 70μg/mL with 36% cytostasis. This was the maximum dose level selected for scoring for micronuclei in the binucleate cells as the marginally higher next dose level showed 70% cytostasis, which was considered too toxic for scoring. However, since the 24-hour exposure group in the absence of S9 achieved the required level of toxicity, the test item was considered to be adequately tested in the absence of S9
The ‘A’ culture of the cyclophosphamide positive control in the presence of S9 did not demonstrate a satisfactory positive response after the first 1000 binucleate calls were scored for micronuclei and so an additional 1000 cells were scored from the partner slide. The additional scoring was considered to add power to the data and an adequate positive response was demonstrated as a result.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
See attachment

Conclusions:

Under the test conditions, test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Experiment were as follows:

4 h exposure without S9: 0, 10, 20, 40, 60, 70, 80, 90, 100 and 120 μg/mL.

4 h exposure with S9 (2%): 0, 10, 20, 40, 60, 80, 90, 100, 110, and 120 μg/mL.

24 h continuous exposure without S9,: 0, 10, 20, 40, 60, 70, 80, 90, 100 and 120 μg/mL.

 

All vehicle (Dimethyl sulphoxide) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was toxic but did not induce any statistically significant increases in the frequency of cells with micronuclei. The 4-hour exposure in the presence of S9 and the 24-hour exposure group included a dose level that induced an approximate 55±5% cytostasis. The 4-hour exposure group in the absence of S9 achieved slightly less than optimum toxicity with 36% cytostasis at the maximum dose level scored for micronuclei but the dose level with a marginally higher concentration was considered too toxic for scoring with 70% cytostasis.

Under the test conditions, test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

This study is considered as acceptable and satisfies the requirement for in vitro micronucleus endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1  (Envigo, 2019)	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 1538, TA 98, TA 100 TA 102	-S9 +S9	Up to limit or cytotoxic concentration	-S9 : weak mutagenic +S9 : weak mutagenic
2 (Envigo, 2019)	MNT vitro (OECD 487) K, rel.1	Chromosomal aberration	Human lymphocytes	-S9 +S9	Up to cytotoxic concentration	-S9 and +S9 Not clastogenic -S9 and +S9 Not aneugenic

Gene mutation Assay (Test n° 1):

- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

 

Chromosomal aberration (Test n°2)

The clastogenic potential of the substance was determined using an in vitro micronucleus test in human lymphocytes (OECD 487), which measures the potential of a substance to increase the incidence of micronuclei in cultured human lymphocytes. The test item was cytotoxic to human lymphocytes but it did not induce any statistically significant increases in the frequency of cells with micronuclei, in any of the exposure groups, using a dose range that included a dose level that induced a sufficient reduction in the cytokinesis block proliferation index (CBPI). Both positive and negative controls validated the sensitivity of the assay. Therefore, the substance is considered as non-clastogenic and non-aneugenic under the conditions used in this assay.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification for human health according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification:

Based on the available data, no self-classification is proposed regarding germ cell mutagenicity according to the CLP and to the GHS.