Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was found to be mutagenic in a bacterial mutageneicty study only in Escherichia coli strain WP2uvrA, when utilizing the pre-incubation method.

In an in vitro test for gene mutation, the test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

When tested in an in vitro chromosome aberration study using human lymphocytes, there was no statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system . The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19/09/2017 - 05/02/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Chemring/ batch 0727/16
- Expiration date of the lot/batch: 24 April 2019
- Purity test date: 99%
- Physical state/Appearance: Off white powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable.
- Solubility and stability of the test substance in the solvent/vehicle: The test item was soluble in MEM at 8.5 mg/mL and in dimethyl sulphoxide (DMSO) at 85 mg/mL in solubility checks performed in-house. MEM was chosen as the solvent for the study.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prior to each experiment, the test item was accurately weighed, formulated in MEM and appropriate serial dilutions prepared. 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 (Scott et al., 1991).
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
Target gene:
Chromosome aberrations
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 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: male, aged 34 years
Main Experiment: female, aged 23 years
Main Experiment (repeat) female, aged 28 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 % foetal 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).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Microsomal Enzyne Fraction and S9-Mix
Test concentrations with justification for top dose:
Group Final concentration of Triazolone (µg/mL)
4(20)-hour without S9 0, 13.25, 26.56, 53.13, 106.25, 212.5, 425, 850, MMC 0.4
4(20)-hour wit S9 (2%) 0, 26.56, 53.13, 106.25, 212.5, 425, 850, CP 2
24-hour without S9 0, 13.25, 26.56, 53.13, 106.25, 212.5, 425, 850, MMC 0.2

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 3.32 to 850 μg/mL. The maximum dose was the maximum recommended dose level, 10 mM concentration.No precipitate of the test item was observed in any of the parallel blood-free cultures at the end of the exposure.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 850 μg/mL in all three exposure groups. The mitotic index data are presented in Table 1 of the study report. The test item induced evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on the maximum recommended dose level 10 mM concentration for all three exposure groups
Vehicle / solvent:
Identity: Eagle's minimal essential medium with HEPES buffer (MEM)
Supplier: Sigma
Batch number (purity):
-Preliminary Toxicity Test: RNBF9655 (Not applicable) 09/2018
-Main test: 1892415 (Not applicable) 30/11/2017
-Main test repeat (4-hour+S9): 1928996 (Not applicable) 31/10/2018
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48hours
- Exposure duration:
4-hour exposure with Metabolic Activation (S9)
4-hour exposure without Metabolic Activation (S9)
24-Hour exposure without Metabolic Activation (S9)
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent): 20-hour culture in treatment-free media prioor to cell harvest for the 2 4-hour exposure.
- Fixation time (start of exposure up to fixation or harvest of cells): 2.5 hours before the required harvest time.

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):
demecolcine
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) 2.5 hours before the required harvest time.
After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
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. 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 EVALUATED:
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated.
If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985) (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported. Endoreduplicated cells were recorded separately and are included in the polyploid cell total number. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range is shown in Appendix 1.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: Yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No

- OTHER:
Rationale for test conditions:
According to guideline
Evaluation criteria:
The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures 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.
• 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 were analyzed.

Criteria for determining the Study Conclusion

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The assay was considered valid as it met all of the following criteria:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.
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 were analyzed.
Remarks on result:
other: NA

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 3.32 to 850 μg/mL. The maximum dose was the maximum recommended dose level, 10 mM concentration.

No precipitate of the test item was observed in any of the parallel blood-free cultures at the end of the exposure.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 850 μg/mL in all three exposure groups. The mitotic index data are presented in Table 1 of the study report. The test item induced evidence of toxicity in all of the exposure groups.

The selection of the maximum dose level for the Main Experiment was based on the maximum recommended dose level 10 mM concentration for all three exposure groups.

Chromosome Aberration Test – Main Experiment

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 850 μg/mL in all three exposure groups.

No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in any of the exposure groups tested.

The mitotic index data for the Main Experiment are given in Table 2 and Table 3. They confirm the qualitative observations in that no marked dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, 23% mitotic inhibition was achieved at 850 μg/mL. In the presence of S9, a inhibition of mitotic index was observed where 32% and 14% inhibition was observed at 425 and 850 μg/mL, respectively. An inhibition of mitotic index of 19% was noted at 850 μg/mL in the 24-hour continuous exposure group.

The maximum dose level selected for metaphase analysis of all three exposure groups was the maximum recommended dose level the 10 mM concentration dose level (850 μg/mL).

The chromosome aberration data are given in Table 4, Table 5 and Table 6 of the study report.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation

Conclusions:
Triazolone did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

A Chromosome aberration test in Human Lymphocytes in vitro, Lacey F.E., 2018, has been performed according to the 473 OECD guideline and in GLP compliance.

Duplicate culture of human lymphocytes, treated with the test item were evaluated for chormosome aberrations at 3 dose levels, together with vehicle and positive controls, at 3 exposure conditions : 4 hours exposure in the presence of an induce rat liver homogenate metabolizing system (S9), 4 hours expposure in the absence of metabolic activation (S9) both with a 20-hour expression period and a 24 -hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity test, depending on the exposure group 0, 13,28, 26,56, 53,13, 106,25, 212,5, 425 and 850 µg/mL : concentrations tested for the 4 -hour exposure without S9, 0, 26,56, 53,13, 106,25, 212,5, 425 and 850 µg/mL : concentrations tested for the 4 -hour exposure with S9 and 0, 26,56, 53,13, 106,25, 212,5, 425 and 850 µg/mL: concentrations used for the 24 -hour exposure without S9.

All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

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

The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberration either in the presence or absence of metabolic activation at any dose level in any of the exposure groups.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in all of the exposure groups.

The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20/07/2017-05/10/2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Chemring /atch 0727/16
- Expiration date of the lot/batch: 28 April 2019
- Purity test date: 99 %
- Description/Appearance: Off white powder
- Molecular Weight: 162,2 g/mol

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark
- Stability under test conditions: No analysis was conducted 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.
- Solubility and stability of the test substance in the solvent/vehicle: Following solubility checks performed in-house for the Micronucleus Test in Human Lymphocytes in vitro performed on the same test item (Envigo Study No. FH77HP), the test item was accurately weighed and formulated in MEM medium prior to serial dilutions being prepared.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Following solubility checks performed in-house for the Micronucleus Test in Human Lymphocytes in vitro performed on the same test item (Envigo Study No. FH77HP), the test item was accurately weighed and formulated in MEM medium prior to serial dilutions being prepared.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
Target gene:
The hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).

The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:

Group Final concentration of Triazolone (µg/mL)
4-hour without S9 0, 50,63, 101,25, 202,5, 405, 810, 1620, EMS 500, 750
4-hour with S9 (2%) 0, 50,63, 101,25, 202,5, 405, 810, 1620, DMBA 1,0 and 2,0

A concentration range of 6.33 to 1620 μg/mL was used in the preliminary cytotoxicity test. The maximum concentration tested was the 10 mM limit dose level.
At the end of the exposure period, precipitate of the test item was not observed at any of the dose levels in either the absence or presence of metabolic activation.
The results of the individual flask counts and their analysis are presented in Table 1. There was no evidence of marked or concentration related reductions in cloning efficiency in either the absence or presence of metabolic activation.
The maximum concentration selected for the main mutagenicity experiment was therefore the 10 mM limit dose level of 1620 μg/mL as recommended by the OECD 476 guidelines.
Vehicle / solvent:
Identity: MEM
Supplier: Sigma Aldrich
Batch numbers: 1838993 and 1928996
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
MEM
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 1 x 107 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items. This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with eight test item concentrations, and vehicle and positive controls. Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.

The concentration range of test item was 50.63 to 1620 μg/mL in both the absence and presence of metabolic activation.

At the end of the treatment period the flasks were washed twice with PBS, detached from the flasks with trypsin and the cells suspended in MEM with 10% FBS. A sample of each concentration group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 106 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask to obtain the cloning efficiency, for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10% FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.

Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 and 5 to maintain logarithmic growth. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:

i) In triplicate at 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 105 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 μg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.

The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

Calculations
The cloning efficiency (CE), % control, mutant plate counts, mutant frequency/106 (MF10-6) and mutant frequency/106 survival rate (MFSV) were calculated using the following formulae:
CE% = (xCE counts/200)x100 % Control = (CE% of Dose IDx/CE% of Dose ID0)x100 MF 10-6 for each dose = Total mutant plate counts/2 MFS 10-6 for each dose = (MF 10-6/Day 7 CE%)x100
Where:
Concentration ID0 = Vehicle control values Concentration IDx = Concentration values
Small errors may occur when calculating mean cell concentrations and volumes for diluting; and in the calculation of means for cloning efficiency and mutant frequency; if these errors are ≤5% they are regarded to be within reasonable experimental error and considered not to affect the integrity of the study.
Rationale for test conditions:
Acceptable
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly 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 judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful.
Statistics:
When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Test Item Preparation
Following solubility checks performed in-house for the Micronucleus Test in Human Lymphocytes in vitro performed on the same test item (Envigo Study No. FH77HP), the test item was accurately weighed and formulated in MEM medium prior to serial dilutions being prepared. The molecular weight of the test item was 85, therefore, the maximum proposed concentration level in the solubility test was set at 850 μg/mL, the 10 mM limit dose level, and no correction for the purity of the test item was applied.
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 at the concentration levels investigated (Scott et al., 1991).
Remarks on result:
other: NA

Preliminary Cytotoxicity Test

A concentration range of 6.33 to 1620 μg/mL was used in the preliminary cytotoxicity test. The maximum concentration tested was the 10 mM limit dose level.

At the end of the exposure period, precipitate of the test item was not observed at any of the dose levels in either the absence or presence of metabolic activation.

The results of the individual flask counts and their analysis are presented in Table 1. There was no evidence of marked or concentration related reductions in cloning efficiency in either the absence or presence of metabolic activation.

The maximum concentration selected for the main mutagenicity experiment was therefore the 10 mM limit dose level of 1620 μg/mL as recommended by the OECD 476 guidelines.

Mutagenicity Test – Main Experiment

At the end of the exposure period, precipitate of the test item was not observed at any of the dose levels in either the absence or presence of metabolic activation.

The Day 0 and Day 7 cloning efficiencies for the exposure groups in the absence and presence of metabolic activation are presented in Tables 2 and 3. There were no marked concentration-related reductions in the Day 0 cloning efficiency values in either the absence or presence of metabolic activation. There was also no evidence of any reductions in the Day 7 cloning efficiencies in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred.

The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 2 and Table 3. The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test (including the 10 mM limit dose level as recommended by the OECD 476 guidelines), in either the absence or presence of metabolic activation

The vehicle control values were all considered to be within an acceptable range, and the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

Conclusions:
The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.
Executive summary:

In a V79 HPRT Gene Mutation Assay (Flanders L., 2018), performed according to the OECD guideline 476 and in compliance with GLP requirements, Chinese hamster (V79) cells were treated with the test item at six concentrations, in duplicate, together with vehicle (MEM medium) and positive controls in both the absence and presence of metabolic activation.

The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be the 10 mM limit dose level of 850 μg/mL, as recommended by the OECD 476 guidelines. The concentrations of test item plated for cloning efficiency and expression of mutant colonies were 53.13, 106.25, 212.5, 425, 637.5 and 850 either with and without S9 mix.

The vehicle (MEM medium) controls gave mutant frequencies within the range expected of V79 cells at the HPRT 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 or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.

The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30/08/2017-24/10/2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Chemring /Batch 0727/16
- Expiration date of the lot/batch: 28 April 2019
- Purity test date: 99%
- Physical state/Appearance: Off-white powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
- Solubility and stability of the test substance in the solvent/vehicle: The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in-house.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in-house.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel: The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer and sonication for 5 minutes at 40 °C.
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
Target gene:
The histidine or tryptophan locus in the genome of bacteria
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: trp-; uvrA-
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: Genotype : his C 3076; rfa-; uvrB-
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: his D 3052; rfa-; uvrB-; R-factor
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: his G 46; rfa-; uvrB-
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: his G 46; rfa-; uvrB-; R-factor
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fractions
Test concentrations with justification for top dose:
Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500, 5000 μg/plate.
Six test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Vehicle / solvent:
- Vehicule : Sterile distilled water
- Justification for choice of solvent/vehicle: The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene : Positive control in the absence of S9-mix
Details on test system and experimental conditions:
Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously (see 3.3.2.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A number of manual counts were performed due to colonies spreading, thus distorting the actual plate count.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
With Metabolic Activation
The procedure was the same as described previously (see 3.3.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate.
The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.

Evaluation criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983), Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
Combined historical negative and solvent control ranges for 2015 and 2016 are presented in Appendix 1.
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive control reference items for 2015 and 2016 are presented in Appendix 1.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
NA
Remarks on result:
other: NA

RESULTS on CYTOTOXICITY for dose selection

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level was used as the maximum dose in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method).

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

MAIN TEST : Experiment 1 (Plate Incorporation)

In the first mutation test (plate incorporation method) there were no 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).

MAIN TEST : Experiment 2 (Pre-Incubation)

However, after employing the pre-incubation modification in Experiment 2, large, dose-related and statistically significant increases in WP2uvrA revertant colony frequency were noted from 1500 μg/plate in the absence of S9-mix and 500 μg/plate in the presence of S9-mix. The increases at 5000 μg/plate noted in the absence of S9-mix and at 500, 1500 and 5000 μg/plate in the presence of S9-mix were in excess of the in-house untreated/vehicle

control maxima for the tester strain and showed clear evidence of a dose-related response. Furthermore, the fold increase for the 5000 μg/plate exposure group was 4.3 and 5.9 times the concurrent vehicle controls which are above the two-fold criteria for judging a positive response.

No significant increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).

Conclusions:
Triazolone was considered to be mutagenic under the conditions of this test only in Escherichia coli strain WP2uvrA when utilizing the pre-incubation method.
Executive summary:

In a Reverse Mutation Assay 'Ames test', Wisher M., 2018, performed according to the OECD Guideline 471, in compliance with GLP requirements, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to 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 dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

The vehicle (sterile distilled water) 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 or without metabolic activation.

Results for the negative controls(spontaneous mutation rates) were considered to be acceptable.

Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level was used as the maximum dose in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method).

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

In the first mutation test (plate incorporation method) there were no 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).

However, after employing the pre-incubation modification in Experiment 2, large, dose-related and statistically significant increases in WP2uvrA revertant colony frequency were noted from 1500 μg/plate in the absence of S9-mix and 500 μg/plate in the presence of S9 - mix. The increases at 5000 μg/plate noted in the absence of S9-mix and at 500, 1500 and 5000 μg/plate in the presence of S9-mix were in excess of the in-house untreated/vehicle control maxima for the tester strain and showed clear evidence of a dose-related response. Furthermore, the fold increase for the 5000 μg/plate exposure group was 4.3 and 5.9 times the concurrent vehicle controls which are above the two-fold criteria for judging a positive response. No significant increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).

Triazolone was considered to be mutagenic under the conditions of this test only in Escherichia coli strain WP2uvrA when utilizing the pre-incubation method.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In a Comet assay test performed according to the OECD 489 Guideline, GLP complaince, with male animals at the Maximum Recommended Dose (MRD) 7 rats of the 2000 mg/kg dose level, 5 rats of the 1000 mg/kg dose level and 5 rats of the 500 mg/kg were dosed by oral gavage at time 0 and 24 hours after the initial dosing and were killed 4 hours after the second dose administration, at a sampling time of 28 hours. The glandular stomach and liver tissues were sampled and processed, the slides were then prepared prior to scoring for the presence of Comets. Further groups of rats were given a double oral dose of arachis oil (5 rats) or methyl nitrosourea (3 rats), to serve as vehicle and positive controls respectively.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05/12/2017 -20/02/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
The number of animals used for the positive control group was reduced to 3 rather than 5 as stated in the study plan. This was to reduce the number of animals used in the study and it complies with the OECD 489 guideline which states 3 animals are sufficient for the positive control group.
Deviations:
yes
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / Batch :0727/16
- Expiration date of the lot/batch: 28 April 2019
- Purity test date: 99%
- Physical state/Appearance: Off white powder


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions:
No analysis was carried out to determine the stability of the test item formulation. The test item was formulated within 2 hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.
- Solubility and stability of the test substance in the solvent/vehicle: test item was freshly prepared as required as a suspension at the appropriate concentration in arachis oil.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test item was freshly prepared as required as a suspension at the appropriate concentration in arachis oil :
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
Species:
rat
Strain:
Wistar
Details on species / strain selection:
HsdRccHan™WIST
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (UK)
- Age at study initiation: 8-10 weeks old
- Weight at study initiation: 184.4 to 209.4 g
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding.
- Diet and Water : at libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%relative humidity
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: 200, 100 and 500 mg/mL
- Amount of vehicle (if gavage or dermal): 10mL
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required):
Identification: Arachis Oil
Supplier: W M Hodgson & Co
Label Information: ZZZ08613
Purity: Treated as 100%
Expiry Date: 12 May 2018
Storage Conditions: Room temperature
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:
Duration of treatment / exposure:
24h
Frequency of treatment:
Groups of male rats were dosed twice with a 24 hour interval
Post exposure period:
4 hours following the second administration
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals for the 1000 and 500 mg/kg bw dose level
7 animals for the 2000 mg/kg bw dose level
5 animals for the vehicle control
3 animals for the positive control
Control animals:
yes, concurrent vehicle
Positive control(s):
N-Nitroso-N-methylurea (MNU)
- Justification for choice of positive control(s):
- Route of administration: oral route, gavage. MNU is a positive control item that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test.
- Doses / concentrations: 25 mg/kg bw


Identification: N-Nitroso-N-methylurea (MNU)
Supplier: AstaTech Inc
Supplier’s Lot Number: 4486-027
Physical state/ Appearance: Pale orange solid
Purity: 90%
Expiry: 05 December 2018
Storage Conditions: Approximately -20 ºC
Solvent: Distilled water

For the purpose of this study the positive control item was freshly prepared as required as a solution at a concentration of 2.5 mg/mL in distilled water (Laboratoire Aguettant batch no. 3012436).
Tissues and cell types examined:
Humane euthanasia was performed on the animals at the end of the exposure period using a method that did not affect the integrity of the required tissues (carbon monoxide asphyxiation). Samples of liver and glandular stomach were obtained from each animal for comet processing.
Sub-samples of the liver and glandular stomach were taken from the vehicle control animals and the dose group animals and preserved in 10% buffered formalin for possible histopathology investigations.
Assessment of cytotoxicity by histopathology may be conducted if the results from the Comet assay, or other observations, suggest cytotoxicity may be confounding the interpretation of the Comet assay.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A range-finding test was performed to find suitable dose levels of the test item following a double oral administration at zero and 24 hours. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The tissue samples were processed under subdued lighting and over ice to provide single cell suspensions, providing sufficient cells for scoring for the comet assay as follows:
Liver - A small piece of liver (approximately 1 cm3) was washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered through gauze to provide a single cell suspension.
Glandular Stomach– The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for possible histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension was obtained by scraping the underlying glandular stomach tissue and suspending it in stomach buffer. The resulting cell suspension was filtered through gauze prior to use for the comet slides.

DETAILS OF SLIDE PREPARATION:
Approximately 30 μL of the cell suspension was added to 270 μL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension mix was immediately covered with a glass cover-slip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once the LMP agarose had set, the cover-slips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
Following lysis, the slides were removed from the solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis bath, which was filled with chilled electrophoresis buffer (pH>13), until the slide surface was just covered. The slides were then left for 20 minutes to allow the DNA to unwind, after which they were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
At the end of the electrophoresis period, the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer and left for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer was performed twice. The slides were further drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry.
Once dry the slides were stored prior to scoring. Two of the four processed slides were scored and the remaining slides were stored as backup slides.

METHOD OF ANALYSIS:
The slides were stained just prior to analysis for comets. To each dry slide, 75 μL of propidium iodide (20 μg/mL) was placed on top of the slide and then overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program, i.e. Comet IV.
Two slide gels for each tissue per animal were scored with a maximum of 100 cells per slide gel giving an accumulative total of 200 cells per tissue per animal. Care was taken to guarantee that a cell was not scored twice. The slide score data were processed using the Excel macro program provided in Comet IV. Comparisons between the vehicle control group response and that of the test item dose groups were made. The primary end-points are percentage tail DNA (%Tail intensity) and median percentage tail intensity.
Each slide was assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity. Hedgehogs are cells that exhibit a microscopic image consisting of a small or non-existent head, and large diffuse tails and are considered to be heavily damaged cells, although the etiology of the hedgehogs is uncertain.

OTHER:
Evaluation criteria:
The following criteria will be used to determine a valid assay:
• The concurrent negative control is comparable with the laboratory historical negative control range.
• The positive controls induce responses that are comparable with those in the laboratory historical positive control range.
• Adequate numbers of cells and doses have been analyzed.
• The highest dose level selected meets the requirements of the guideline and the study plan.
Statistics:
A comparison was made between the vehicle control groups and the positive control groups. The individual slide score data for the percentage tail intensity and median percentage tail intensity was compared using a Students t-test with a √1+x transformation. Comparisons between the vehicle control groups and the test item dose groups were made when there was an increase over the vehicle control value.

Statistical analysis was performed on the 1000 mg/kg and the 2000 mg/kg dose groups of the liver compared to the vehicle control group where increases in mean percentage tail intensity and median % tail intensity were observed.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not valid
Additional information on results:
RESULT OF THE RANGE-FINDING TOXICITY TEST /
In animals dosed with test item (2000 mg/kg bw) there were no premature deaths and no clinical signs observed.
Bone marrow slides were prepared for quantitative assessment from the range-finding experiments. The slides were scored per 1000 cells.
The quantitative assessment revealed that moderate bone marrow toxicity was observed at 2000 mg/kg in one of the animals. Although not conclusive since the other animals in the range finding experiments did not demonstrate any marked toxicity it was considered to give an indication of systematic absorption of the test item in the absence of clinical signs.

Based on the above data the maximum recommended dose (MRD) of the test item, 2000 mg/kg, was selected for use in the main test, with 1000 and 500 mg/kg as the lower dose levels. There was no noticeable difference in clinical signs between the male and female animals and therefore only male animals were used for the main test.

RESULTS OF THE BONE MARROW QUANTITATIVE ASSESSMENT :

The quantitative assessment revealed that moderate bone marrow toxicity was observed at 2000 mg/kg in one of the animals. Although not conclusive since the other animals in the range finding experiments did not demonstrate any marked toxicity it was considered to give an indication of systematic absorption of the test item in the absence of clinical signs.

RESULTS OF DEFINITIVE STUDY : COMET ASSAY

Mortality Data and Clinical Observations :
There were no premature deaths seen in any of the test item dose groups and no clinical signs were observed.

The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control range.
The positive control item (MNU) produced a marked increase in the percentage tail intensity and median percentage tail intensity in the liver and glandular stomach, comparable with the laboratory historical control range for these tissues.
The test method itself was therefore operating as expected and was considered to be valid under the conditions of the test.

There were no marked increases in percentage tail intensity or median percentage tail intensity for any of the test item dose levels in the glandular stomach which exceeded the current laboratory historical control range for a vehicle, confirming the test item did not induce DNA damage in the glandular stomach.

The liver did demonstrate a small but statistically significant response at the maximum dose level (2000 mg/kg). The response does appear to be dose related but the values for all three exposure groups are well within the historical laboratory control range for a vehicle. The values for the lowest dose group (500 mg/kg) are less than that for the concurrent vehicle control. In the 2000 mg/kg dose group, animal 14 had higher mean percentage tail intensities than the rest of the group and this skewed the data for this group resulting in the higher response. The ‘B’ replicate slide for animal 12 also had a much higher percentage tail intensity than its partner and was not consistent with the rest of the slides in the dose group. The higher standard deviations for these slides indicates a greater spread in the response which can be attributed to a few cells with higher percentage tail intensities rather than a consistent increase in all cells scored. The response seen in the liver at 2000 mg/kg is therefore considered to be of no toxicological significance. It does not meet the requirements of the study plan for a positive in that the results are not substantially outside the laboratory historical vehicle control range. The test item was therefore considered to be unable to induce DNA strand breakage in the liver under the conditions of the test.

There was no marked increase in hedgehog frequency for any of the test item dose levels in either of the tissues investigated.

Mortality data for the Range-Finding Toxicity Study :

Dose Level (mg/kg) Sex Number of Animals Treated Route Deaths on Day
0 1
2000 male 1 oral 0 0
2000 female 1 oral 0 0
2000 male 1 oral 0 0
2000 female 1 oral 0 0

Scored bone marrow slides from the range-finding experiments :

Animal code Sex Dose level (mg/kg) Number of Polychromaatic erythrocytes (PCE) Number of normochromatic erythrocytes (NCE) PCE/NCE (ratio)
1-0 male 2000 313 687 0,46
2-0 female 2000 514 486 1,06
3-0 male 2000 486 514 0,95
3-1 female 2000 542 458 1,18
Conclusions:
The test item did not induce any toxicologically significant increases in the percentage tail intensity or median percentage tail intensity values in the liver or glandular stomach when compared to the concurrent vehicle control group. The test item was considered to be unable to induce DNA strand breakage to the liver and glandular stomach in vivo, under the conditions of the test.
Executive summary:

A range-finding test was performed to find suitable dose levels of the test item and the most appropriate sex.

In a Comet assay test (Morris A., 2018, ENVIGO) performed according to the OECD 489 Guideline, GLP complaince, with male animals at the Maximum Recommended Dose (MRD)

7 rats of the 2000 mg/kg dose level, 5 rats of the 1000 mg/kg dose level and 5 rats of the 500 mg/kg were dosed by oral gavage at time 0 and 24 hours after the initial dosing and were killed 4 hours after the second dose administration, at a sampling time of 28 hours. The glandular stomach and liver tissues were sampled and processed, the slides were then prepared prior to scoring for the presence of Comets.

Further groups of rats were given a double oral dose of arachis oil (5 rats) or methyl nitrosourea (3 rats), to serve as vehicle and positive controls respectively.

The quantitative assessment revealed that moderate bone marrow toxicity was observed at 2000 mg/kg in one of the animals. Although not conclusive since the other animals in the range finding experiments did not demonstrate any marked toxicity it was considered to give an indication of systematic absorption of the test item in the absence of clinical signs.

There was no evidence of an increase in the glandular stomach in the percentage tail intensity or median percentage tail intensity in the test item dose groups when compared to the concurrent vehicle control group.

The liver did demonstrate a small but statistically significant increase in both the percentage tail intensity and the median percentage tail intensity at the maximum dose level (2000 mg/kg). However, the increases were within the current laboratory historical range for a vehicle and much of the increase could be attributed to one animal. As we do not meet the three requirements in the study plan to designate a positive response we consider this to be of no toxicological significance.

The positive control item produced a marked increase in the % tail intensity value in the liver and glandular stomach, indicating that the test method was working as expected. The vehicle control group for the liver and the glandular stomach had % tail intensity values which were consistent with the current laboratory historical range for a vehicle.

The test item did not induce any toxicologically significant increases in the percentage tail intensity or median percentage tail intensity values in the liver or glandular stomach when compared to the concurrent vehicle control group. The test item was considered to be unable to induce DNA strand breakage to the liver and glandular stomach in vivo, under the conditions of the test.

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

Additional information

The test item did not induce any toxicologically significant increases in the percentage tail intensity or median percentage tail intensity values in the liver or glandular stomach when compared to the concurrent vehicle control group. The test item was considered to be unable to induce DNA strand breakage to the liver and glandular stomach in vivo, under the conditions of the test.

Justification for classification or non-classification

The substance is negative in an vivo study (Comet Assay) and therefore is not classified for mutagenicity acording to the classification criteria in EU Regulation

No 1907/2006 (CLP).