Lithium nickel potassium oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames:

Under the experimental conditions chosen here, it is concluded that KDLNO is not a mutagenic
test substance in the bacterial reverse mutation test in the absence and the presence of
metabolic activation.

 

Mouse Lymphoma Assay:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

 

Micronucleus:

The test item, Lithium nickel potassium oxide (KDLNO) was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. 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. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

The details of the donors used are:
Preliminary Toxicity Test: female, aged 35 years
Main Experiment: female, aged 35 years
Cell Culture
Cells (whole blood cultures) were grown in 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), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Cytokinesis block (if used):

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{(CBPIT – 1) / (CBPIC – 1)}
Where: CBPI = (Number of monucleate cells + (2*Number of Binucleate cells) + (3*Number of multinucleate cells) / (Total Number of cells)

Key:
T = test chemical treatment culture
C = vehicle control culture

Scoring of Micronuclei
The micronucleus frequency in 1000 binucleated cells was analyzed per cell culture, i.e. 2000 binucleate cells for the test item dose levels and the positive control and 4000 binucleate cells for the vehicle in each exposure group. Cells with 1, 2 or more micronuclei were recorded and included in the total. In the 4-hour exposure group in the presence of S9 positive control ‘A’ culture did not demonstrate a satisfactory response after the first 1000 cells were scored so to add weight to the data and confirm the response, a further 1000 cells were scored from each of the positive control replicates in this exposure group.

Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.

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. Both nuclei could be attached by a fine nucleoplasmic bridge approximately no greater than one quarter of the nuclear diameter.

Metabolic activation:

with and without

Metabolic activation system:

Culture conditions
Duplicate lymphocyte cultures (A and B) for the test item dose levels and quadruplicate cultures for the vehicle control were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.06 mL MEM,
10% (FBS) 0.1 mL Li-heparin;
0.1 mL phytohaemagglutinin;
0.74-0.75 mL heparinized whole blood.

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.

After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.

After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.

The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The Preliminary Toxicity Test was performed using the exposure conditions as described for the Main Experiment but using single cultures for the test item dose levels and duplicate cultures for the vehicle controls, whereas the Main Experiment used duplicate cultures for the test item and quadruplicate cultures for the vehicle controls.

Test concentrations with justification for top dose:

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 2.5, 5, 10, 20, 40, 80, 160, 320 and 640 μg/mL.

Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the exposure groups of the main experiment.

Vehicle / solvent:

Controls
Vehicle and positive controls were used in parallel with the test item.
The vehicle control used was as follows:
Identity:
Minimal Essential Medium (MEM)
Supplier:
Gibco
Preliminary Toxicity Test
Batch number:
2041445
Expiry Date:
30 November 2019
Main Experiment
Batch Number:
2049804
Expiry Date:
31 December 2019

The positive control items were as follows:

Absence of S9-mix:

Identity:
Mitomycin C (MMC)
CAS No.:
50-07-7
Supplier:
Sigma Aldrich
Batch Number:
SLBN5647V
Purity:
100%
Expiry Date:
01 October 2019
Solvent:
Minimal Essential Medium
Concentration:
0.2 μg/mL for 4-hour exposure

Identity:
Demecolcine (DC)
CAS No.:
477-30-5
Supplier:
Sigma-Aldrich
Batch Number:
BCBV3422
Purity:
99%
Expiry Date:
02 July 2023
Solvent:
Sterile distilled water
Concentration:
0.075 μg/mL for 24-hour continuous exposure

Presence of S9-mix:
Identity:
Cyclophosphamide (CP)
CAS No.:
6055-19-2
Supplier:
Acros Organics
Batch Number:
A0389646
Purity:
99.9%
Expiry Date:
01 October 2022
Solvent:
DMSO
Concentration:
5 μg/mL for 4-hour exposure

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium (MEM)

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Demecolcine (DC)

Details on test system and experimental conditions:

Test Item Preparation
The test item is a UVCB, therefore the maximum proposed dose level in the solubility test was initially set at 5000 μg/mL, the maximum recommended dose level, and no correction for the purity of the test item was applied. The test item formed a suspension in culture medium at 50 mg/mL, which was considered acceptable for dosing in the solubility checks.
The solubility of the test item was investigated in the Mouse Lymphoma Assay, Study Number MS22GN.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm .

The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Due to test item precipitate being observed in the solubility test at and above 312.5 μg/mL, the maximum dose level selected for the Preliminary Toxicity Test was limited to 640 μg/mL.

Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Batch No PB/βNF S9 28/10/18 was used in this study. A copy of the S9 Certificate of Efficacy is presented in Appendix 2.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.

The dose range of test item used for all three exposure groups was 5, 10, 20, 40, 80, 160 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Preparation of Microscope Slides
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.

Data Evaluation
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.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions (i.e. S9 concentration or S9 origin)) could be useful.

Test items that induce micronuclei in the MNvit test may do so because they induce chromosome breakage, chromosome loss, or a combination of the two. Further analysis using anti-kinetechore antibodies, centromere specific in situ probes, or other methods can be used to determine whether the mechanism of micronucleus induction is due to clastogenic and/or aneugenic activity.

Rationale for test conditions:

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:

Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.

Coding
The slides were coded before analysis using a computerized random number generator.

Scoring of Micronuclei
The micronucleus frequency in 1000 binucleated cells was analyzed per cell culture, i.e. 2000 binucleate cells for the test item dose levels and the positive control and 4000 binucleate cells for the vehicle in each exposure group. Cells with 1, 2 or more micronuclei were recorded and included in the total. In the 4-hour exposure group in the presence of S9 positive control ‘A’ culture did not demonstrate a satisfactory response after the first 1000 cells were scored so to add weight to the data and confirm the response, a further 1000 cells were scored from each of the positive control replicates in this exposure group.

Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.

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.

Statistics:

Statistical Analysis
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.

Major Computerized Systems
The following computerized system was used in this study:
• An in-house validated statistical package (UK0279)

Key result

Species / strain:

lymphocytes: This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Conclusion

The test item, Lithium nickel potassium oxide (KDLNO) was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

SUMMARY

Introduction

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

Methods

Duplicate cultures of human lymphocytes, treated with the test item, and quadruplicate cultures for the vehicle were evaluated for micronuclei in binucleate cells at 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 precipitate.

 

Results

All vehicle (Minimal Essential Medium) 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 did not demonstrate any marked toxicity and did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the lowest precipitating dose level.

 

Conclusion

The test item, Lithium nickel potassium oxide (KDLNO) was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Metabolic activation:

with and without

Metabolic activation system:

Microsomal Enzyme Fraction

Lot No. PB/βNF S9 28/10/18 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test.
S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.
20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test.

Test concentrations with justification for top dose:

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. Due to the precipitate observed in the solubility check, the dose range used in the preliminary toxicity test was 2.44 to 625 μg/mL for all three of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL.
The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent solvent control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA .

Vehicle / solvent:

Solvent and positive controls were used in parallel with the test item. R0 medium exposure groups were used as the solvent controls. Ethylmethanesulphonate (EMS) (Sigma batch BCBW8635, purity treated as 100%, expiry 10/01/24) at 400 μg/mL and 150 μg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation. Cyclophosphamide (Acros Organics batch A0389646, purity 99.9%, Expiry 01/10/22) at 1.5 μg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Cleansing:
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Test Item Preparation:
The test item is a UVCB, therefore the maximum proposed dose level in the solubility test was initially set at 5000 μg/mL, the maximum recommended dose level, and no correction for the purity of the test item was applied. The test item formed a suspension in R0 medium at 50 mg/mL, which was considered acceptable for dosing in the solubility checks. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Control Preparation
Solvent and positive controls were used in parallel with the test item. R0 medium exposure groups were used as the solvent controls. Ethylmethanesulphonate (EMS) (Sigma batch BCBW8635, purity treated as 100%, expiry 10/01/24) at 400 μg/mL and 150 μg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation. Cyclophosphamide (Acros Organics batch A0389646, purity 99.9%, Expiry 01/10/22) at 1.5 μg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO.

Mutagenicity Test
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic 30 mL universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (2.5 to 160 μg/mL for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 1.25 to 80 μg/mL for the 24-hour exposure group in the absence of metabolic activation), solvent and positive controls. To each plastic 30 mL universal was added 2 mL of S9 mix if required, 2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).
The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Evaluation criteria:

Measurement of Survival, Viability and Mutant Frequency
At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured every 24 hours by counting and dilution to 2 x 105 cells/mL for the expression period of two days. On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the solvent control, and when combined with the Viability (%V) data, a Relative Total Growth (RTG) value.

Statistics:

Data Evaluation
The Historical Solvent and Positive Control data is generated by the Mutant 240C program on a rolling system of the last twenty sets of archived data. The program combines the 4-hour and 24-hour data in the absence of metabolic activation as the acceptability criteria is the same for all three of the exposure groups.
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the solvent control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test item is then considered able to induce mutation in this test system.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The dose range of the test item used in the preliminary toxicity test was 2.44 to 625 μg/mL.
There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all three of the exposure groups, when compared to the concurrent solvent control groups. Precipitate of the test item was observed at and above 156.25 μg/mL in the 4-hour exposure groups in both the absence and presence of metabolic activation, and at and above 78.13 μg/mL in the 24-hour exposure group in the absence of metabolic activation, at the end of the exposure period. It should be noted that the most marked reductions in %RSG values were observed at dose levels at or beyond the onset of test item precipitate. Therefore, following the recommendations of the OECD 490 guideline, the maximum dose level in the Mutagenicity Test was limited by a combination of test item-induced toxicity and precipitate in the 4-hour and 24-hour exposure groups in the absence of metabolic activation, and precipitate in the 4-hour exposure group in the presence of metabolic activation.

There was evidence of marked dose related toxicity following exposure to the test item in the 4-hour and 24-hour exposure groups in the absence of metabolic activation, and modest toxicity in the 4-hour exposure group in the presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any significant reductions in viability (%V) in any of the three exposure groups, indicating that residual toxicity had not occurred. At the end of the exposure periods, the onset of test item precipitate was observed at 120 μg/mL in the 4-hour exposure groups in both the absence and presence of metabolic activation, and at 60 μg/mL in the 24-hour exposure group in the absence of metabolic activation. Therefore, following the recommendations of the OECD 490 guideline, the lowest precipitating dose level was plated for viability and 5-TFT resistance and the subsequent dose levels were discarded as they were surplus to requirements. Acceptable levels of toxicity were seen with the positive control substances.

The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, using a dose range that included the lowest precipitating dose level in all three of the exposure groups, as recommended by the OECD 490 guideline.

CONCLUSION
The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Conclusions:

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

SUMMARY

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with solvent (R0 medium), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

 

Results

The maximum dose level in the Mutagenicity Test was limited by a combination of test item-induced toxicity and precipitate in the 4-hour and 24-hour exposure groups in the absence of metabolic activation, and precipitate in the 4-hour exposure group in the presence of metabolic activation, as recommended by the OECD 490 guideline. The solvent control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, using a dose range that included the lowest precipitating dose level in all three of the exposure groups.

 

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

STUDY COMPLETED
22 Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy
(his-) to histidine prototrophy (his+) is determined. The tester strains TA 1535, TA 1537,
TA 98 and TA 100 selected by Ames and coworkers are derivatives of Salmonella typhimurium
LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision
repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and
this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all
strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an
increase in permeability to lipophilic substances.
The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by
the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and
TA 98 are strains for the detection of frameshift mutagens. These strains carry different
frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type
his D 3052 in the case of TA 98.
The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 and, in addition to having
genes resistant to antibiotics, they have a modified postreplication DNA repair system, which
increases the mutation rate by inducing a defective repair in the DNA; this again leads to a
considerable increase in sensitivity.

Escherichia coli
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a
derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which
induce base pair substitutions. The rate of induced back mutations from tryptophan
auxotrophy (trp-) to tryptophan independence (trp+) is determined.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: Defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and results in greatly enhanced sensitivity of some mutagens.

Remarks:

Strains also have a reduced hydrophilic polysaccharide layer (rfa), resulting in increase permeability to lipophilic substances.

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with

Metabolic activation system:

S9 fraction
The S9 fraction was prepared at BASF SE in an AAALAC-approved
laboratory in accordance with the German Animal Welfare Act and the effective
European Council Directive.
At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany
GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally (both supplied
by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days.
During this time, the animals were housed in polycarbonate cages: central air conditioning with
a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 30 - 70%. The
day/night rhythm was 12 hours: light from 6 am to 6 pm and darkness from 6 pm to 6 am.
Standardized pelleted feed and drinking water from bottles were available ad libitum.
24 hours after the last administration, the rats were sacrificed, and the livers were prepared
using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and
washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three
volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at
+4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

S9 mix
The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient
amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with
9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on
ice until used. The concentrations of the cofactors in the S9 mix were:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM
The phosphate buffer is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution
in a ratio of about 4:1.
To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with
benzo(a)pyrene.

Test concentrations with justification for top dose:

DOSES
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were
generally selected as maximum test dose at least in the 1st Experiment. However, this
maximum dose was tested even in the case of relatively insoluble test compounds to detect
possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be
tested in repeat experiments for further clarification/substantiation.
In this study, due to the purity of the test substance 5.2 mg/plate was used as top dose in all
experiments.

Vehicle / solvent:

Choice of the vehicle
Due to the insolubility of the test substance in water, ethanol was used as vehicle, which had
been demonstrated to be suitable in bacterial reverse mutation tests and for which historical
control data are available.

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle control The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.

True negative controls:

yes

Remarks:

Each experiment included negative controls in order to check for possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control).

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: With S9 mix • 2-aminoanthracene (2-AA) (Sigma-Aldrich; 96%) Without S9 mix • N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Fluka; 97%) • 4-nitro-o-phenylenediamine (NOPD) (Sigma-Aldrich; 98%)

Details on test system and experimental conditions:

TEST SYSTEM
For testing, deep-frozen (-70°C to -80°C) bacterial cultures (Salmonella typhimurium TA 1535,
TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) were thawed at room temperature, and 0.1 mL
of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth
+ 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The
optical density of the fresh bacteria cultures was determined. Fresh cultures of bacteria were
grown up to late exponential or early stationary phase of growth (approximately 109 cells per
mL). These cultures grown overnight were kept in iced water from the beginning of the
experiment until the end in order to prevent further growth.
The use of the strains mentioned was in accordance with the current scientific
recommendations for the conduct of this assay.
The Salmonella strains TA 1535, TA 100, TA 1537 and the Escherichia coli strain were
obtained from Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA on 02 Dec 2014. The
Salmonella strain TA 98 was obtained from Moltox Molecular Toxicology on 07 Jan 2015.

Controls
Negative controls / Vehicle controls
Each experiment included negative controls in order to check for possible contaminants
(sterility control) and to determine the spontaneous mutation rate (vehicle control).
• Sterility control
Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance
but without the addition of tester strains (see Appendix 3).
• Vehicle control
The vehicle control with and without S9 mix only contains the vehicle used for the test
substance at the same concentration and volume for all tester strains.

Positive controls
The following positive controls were used to check the mutability of the bacteria and the activity
of the S9 mix:
With S9 mix
• 2-aminoanthracene (2-AA) (Sigma-Aldrich; 96%)
- 2.5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Fluka; 97%)
- 5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD) (Sigma-Aldrich; 98%)
- 10 μg/plate, dissolved in DMSO
- strain: TA 98
• 9-aminoacridine (AAC) (Sigma-Aldrich; 98%)
- 100 μg/plate, dissolved in DMSO
- strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO) (Sigma-Aldrich; 98%)
- 5 μg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA
The stability of the selected positive controls was well-defined under the selected culture
conditions, since they were well-established reference mutagens.

Mutagenicity tests
Standard plate test
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL
agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino
acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept
in a water bath at about 42 - 45°C, and the remaining components were added in the
following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular
Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants)
were counted. The colonies were counted using the Sorcerer Image Analysis System with
the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK).
Colonies were counted manually, if precipitation of the test substance hinders the counting
using the Image Analysis System.

Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL
agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino
acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath
at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular
Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants)
were counted. The colonies were counted using the Sorcerer Image Analysis System with
the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK).
Colonies were counted manually, if precipitation of the test substance hinders the counting
using the Image Analysis System.

Preincubation Test

0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with
metabolic activation) or phosphate buffer (without metabolic activation) were incubated at
37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was
added and, after mixing, the samples were poured onto the agar plates within approx.
30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The
colonies were counted using the Sorcerer Image Analysis System with the software program
Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted
manually, if precipitation of the test substance hindered the counting using the Image Analysis
System.

Rationale for test conditions:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the
historical negative control data for each tester strain (see Appendix 5).
• The sterility controls revealed no indication of bacterial contamination (see Appendix 3).
• The positive control substances both with and without S9 mix induced a distinct increase in
the number of revertant colonies within the range of the historical positive control data or
above (see Appendix 5).
• Fresh bacterial culture containing approximately 109 cells per mL were used.

Solubility
If precipitation of the test material was observed, it would be recorded and indicated in the
tables. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was
generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at
least in the 1st Experiment even in the case of relatively insoluble test compounds to detect
possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat
experiments for further clarification/substantiation.

Evaluation criteria:

Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard
deviations were given for all dose groups as well as for the positive and negative (vehicle)
controls in all experiments. In general, six doses of the test substance were tested with a
maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the
1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat
studies or further experiments were based on the findings of the 1st Experiment.

Toxicity
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated
in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose
groups.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least
doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli
WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and
TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix
or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative
control data under all experimental conditions in at least two experiments carried out
independently of each other.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True 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:

no cytotoxicity

Vehicle controls validity:

valid

True 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:

no cytotoxicity

Vehicle controls validity:

valid

True 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:

no cytotoxicity

Vehicle controls validity:

valid

True 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:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the experimental conditions chosen here, it is concluded that KDLNO is not a mutagenic
test substance in the bacterial reverse mutation test in the absence and the presence of
metabolic activation.

Executive summary:

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard plate test and preincubation assay).
Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria.
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.

Under the experimental conditions chosen here, it is concluded that KDLNO is not amutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The in-vitro studies all gave negative results.