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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity In-Vitro: OECD 471 Bacterial reverse mutation assay, the test substance considered to be non-mutagenic

Genetic Toxicity In-Vitro: OECD 473 Chromosome abberation test, the test substance considered to be clastogenic.

Genetic Toxicity In-Vitro: OECD 476 Mouse lymphona assay, the test substance considered to be mutagenic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 June 2018 - 19 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test substance: 6-methyl-3,4-dihydro-2H-1,4-benzoxazine
CAS Number: 71472-57-6
Physical state/Appearance: Amber coloured viscous liquid
Batch Number: 107562
Purity: 98.8%
Expiry Date: 01 January 2019
Retest Date: 07 March 2023
Storage Conditions: Approximately 4 °C in the dark
Formulated concentrations were adjusted to allow for the stated water/impurity content (1.2%) of the test item.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

S9: 5.0 mL
1.65 M KCl/0.4 M MgCl2 : 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP:2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
Test concentrations with justification for top dose:
During experiment 1 the test item was tested using the following method. The maximum concentration was 5000 g/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 g/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 g/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 toxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
Test for Mutagenicity: Experiment 1 – Plate Incorporation Method:

Dose selection
The test item was tested using the following method. The maximum concentration was 5000 g/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 g/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 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 between 48 and 72 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).


Test for Mutagenicity: Experiment 2 – Pre-Incubation Method:

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 g/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 toxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of 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 between 48 and 72 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).
Rationale for test conditions:
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

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

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical 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.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative 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:
not specified
Vehicle controls validity:
valid
Untreated negative 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:
not specified
Vehicle controls validity:
valid
Untreated negative 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:
not specified
Vehicle controls validity:
valid
Untreated negative 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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Experiment 1 (plate incorporation):
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate. 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).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Experiment 2 (pre-incubation):
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate).
The test item induced a toxic response employing the pre-incubation modification with weakened bacterial background lawns noted to all of the tester strains in both the absence and presence of metabolic activation at 5000 µg/plate.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).
Conclusions:
In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test substance did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test, the substance was considered to be non mutagenic.
Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1)guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

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 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 mg/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 toxicity of the test item following the change in test methodology.

Results

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

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence ofmetabolic activation(S9-mix), in the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (pre-incubation method). Results from the second mutation test showed that the test item induced a toxic response employing the pre-incubation modification with weakened bacterial background lawns noted to all of the tester strains in both the absence and presence ofmetabolic activation at 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). 

Conclusion

The test substance was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 June 2018 - 17 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 473 "In Vitro Mammalian Chromosome Aberration Test" adopted 29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Test substance: 6-methyl-3,4-dihydro-2H-1,4-benzoxazine
CAS Number: 71472-57-6
Physical state/Appearance: Amber coloured viscous liquid
Batch: 107562
Purity: 98.8%
Expiry Date: 01 January 2019
Storage Conditions: Approximately 4°C the dark
Intended use/Application: Research and development
Formulated concentrations were adjusted to allow for the stated water/impurity content (1.2%) of the test item.
Species / strain / cell type:
other: Human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/NF S9 29 March 2018 was used in this study.

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%.
Test concentrations with justification for top dose:
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 47.5, 95, 190, 380, 760 and 1140 µg/mL.

ii)4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 180, 360, 720, 810, 1080, 1260 and 1492 µg/mL.

iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 1.48, 2.97, 5.94, 11.88, 23.75, 47.5 and 95 µg/mL.

The molecular weight of the test item was given as 149.19, therefore, the maximum dose level was 1492 µg/mL, which was the maximum recommended dose level.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test item data sheet indicated that the test item was immiscible in water/aqueous vehicles but was miscible in DMSO at 149.2 mg/mL in solubility checks performed in house. Prior to each experiment, the test item was accurately weighed, formulated in DMSO and appropriate serial dilutions prepared.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
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 0.1 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. 1mL 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 Main Experiment.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, 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 original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.

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 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged and 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. The cells were then returned to the incubator for a further 20 hours.

24-Hour Exposure Without Metabolic Activation (S9)
As the exposure was continuous the cultures were established, at a nominal volume of 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.

The preliminary toxicity test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
The dose range of test item used was 0, 5.83, 11.66, 23.31, 46.63, 93.25, 186.5, 373, 746 and 1492 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 mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the Main Experiment.

Main Experiment
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 47.5, 95, 190, 380, 760 and 1140 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 180, 360, 720, 810, 1080, 1260 and 1492 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 1.48, 2.97, 5.94, 11.88, 23.75, 47.5 and 95 µg/mL.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) between two and 2.5 hours (depending on experiment number) 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.

Preparation of Metaphase Spreads
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.

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.
Rationale for test conditions:
Test conditions are based on the preliminary toxicity test.
Evaluation criteria:
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:
other: Human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
The test item was considered to be clastogenic to human lymphocytes in vitro.
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
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 the maximum dose level of test item in the 4(20)-hour exposure group in the presence of S9 (1492 µg/mL) and the 24-hour exposure group (95 µg/mL). In the 4(20)-hour exposure in the absence of S9, the maximum dose level of the test item with metaphases suitable for scoring was 760 µg/mL.

No precipitate was observed at the end of exposure in the blood cultures at any dose level tested.

The mitotic index data for the Main Experiment confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed in the 4(20)-hour exposure groups and a plateau of toxicity was observed in the 24 hour exposure group.

In the 4(20)-hour exposure in the absence of S9, 23%, 35% and 52% mitotic inhibition was achieved at 190, 380 and 760 µg/mL, respectively. Above this dose level, there were no metaphases available for analysis. Therefore, the maximum dose level selected for metaphase analysis was 760 µg/mL because this dose level achieved optimum toxicity as determined by the OECD 473 Test Guideline (55±5%).

In the presence of S9, 28% and 87% mitotic inhibition was achieved at 1260 and 1492 µg/mL, respectively. However, the lower concentrations of the test item analyzed returned elevated Mitotic Index values. Consequently, the maximum dose level selected for metaphase analysis was 1492 µg/mL because optimum toxicity was not achieved with the lower dose levels, and although this dose level exceeded the OECD 473 Test Guideline optimum toxicity, it was the maximum recommended dose level.

In the 24-hour continuous exposure group, a plateau of toxicity was observed where an inhibition of mitotic index of 24%, 39%, 31% and 68% was noted at 11.88, 23.75, 47.5 and 95 µg/mL, respectively. The maximum dose level selected for metaphase analysis was , therefore, 95 µg/mL because this dose level only slightly exceeded the OECD 473 Test Guideline for optimum toxicity which was not achieved with the other doses.
Remarks on result:
other:

Chromosome Aberration Test – Main Experiment

The dose levels of the controls and the test item are given in the table below:

Group

Final concentration of 6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine (µg/mL)

4(20)-hour without S9

0*, 47.5, 95, 190*, 380*, 760*, 1140, MMC0.4*

4(20)-hour with S9 (2%)

0*, 180, 360, 720, 810*, 1080*, 1260*, 1492*, CP2*

24-hour without S9

0*, 1.48, 2.97, 5.94, 11.88*, 23.75*, 47.5*, 95*, MMC0.1*


*            = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP           = Cyclophosphamide

Discussion

In the Preliminary Toxicity Test, microscopic assessment of the slides suggested that the toxicity curve in the absence of S9-mix was shallow and drawn out over several dose levels. However, in the presence of S9, toxicity seemed to be abrupt where elevated Mitotic Index values were observed prior to complete cell loss of the human lymphocytes, indicating that possible cell cycle delay had occurred resulting in the synchronizing of many more metaphases after exposure.

In the main test, the 4(20)-hour exposure in the absence of S9, achieved optimum toxicity (52%) as determined by the OECD 473 Test Guideline (55±5%). No statistically significant increases in the frequency of cells with chromosome aberrations were observed.

In the 24-hour continuous exposure group, the plateau of toxicity observed resulted in selecting dose levels that were either side of the OECD 473 Test Guideline for optimum toxicity. Consequently, 95 µg/mL (68% mitotic inhibition) was considered acceptable for selection as the maximum dose level for metaphase analysis because it only slightly exceeded optimum toxicity and the lower dose levels approached optimum toxicity. The test item induced statistically significant increases in the frequency of cells with aberrations at 47.5 µg/mL and 95 µg/mL.

In the presence of S9, a previous experiment had been attempted where the dose range was the same as theshort exposure in the absence of S9. However, this experiment mirrored the Preliminary Toxicity Test by confirming the test item had a steep toxicity curve, confounded by elevated Mitotic Index values and then excessive toxicity. Therefore, the exposure group was repeated with an extended but narrowed dose range in an effort to achieve optimum toxicity (where the maximum dose level was the maximum recommended dose level). Again, elevated Mitotic Index values were observed prior to the onset of test item toxicity but optimum toxicity was still not achieved. Consequently, the toxicity observed resulted in selecting dose levels that were either side of the OECD 473 Test Guideline for optimum toxicity. The test item induced a statistically significant increase in the frequency of cells with aberrations in the presence of metabolic activation at 1260 µg/mL but with only 28% mitotic inhibition. The increase in aberrations at 1492 µg/mL (87% mitotic inhibition) are considered to be an artefact of the excessive toxicity observed at this dose level and, therefore, the statistical significance at this dose level will not be reported.

Therefore, the test item was considered to have been adequately tested although two of the three exposure groups did not quite meet the optimum toxicity range as defined byby the OECD 473 test guideline (55±5%) because

  • In the case of the 24-hour continuous exposure group, 68% mitotic inhibition was considered to be acceptable as this only slightly exceeded optimum toxicity and the dose before (which approached optimum toxicity) induced a statistically significant increase in the frequency of cells with aberrations.
  • For the exposure group in the presence of S9, the test item dose range was quite narrow but the elevation in mitotic index meant that the test item toxicity was synchronising the cells to enter metaphase at the same time. This was the main contributing factor of the failure to achieve optimum toxicity in this exposure group.
Conclusions:
The test substance did not induced a statistically significant increase in the frequency of cells with chromosome aberrations in the 4(20)-hour exposure group in the absence of a liver enzyme metabolizing system.
However, the substance did induce statistically significant increases in the frequency of cells with chromosome aberrations in both the presence of a liver enzyme metabolizing system and the 24-hour continuous exposure group. The test item was therefore considered to be clastogenic to human lymphocytes in vitro.
Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems in so far as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1991). 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) 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 where the results indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Experiment were as follows:

Group

Final concentration of test item 6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine (µg/mL)

4(20)-hour without S9

0, 47.5, 95, 190, 380, 760, 1140

4(20)-hour with S9 (2%)

0, 180, 360, 720, 810, 1080, 1260, 1492

24-hour without S9

0, 1.48, 2.97, 5.94, 11.88, 23.75, 47.5, 95

Results

All vehicle (dimethyl sulphoxide (DMSO)) 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 and did induce statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that either approached or achieved 55±5% mitotic inhibition.

Conclusion

The test substance was considered to be clastogenic to human lymphocytes in vitro under the conditions of this study.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 November 2018 - 04 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Test substance: 6-methyl-3,4-dihydro-2H-1,4-benzoxazine
Batch Number: 107562
CAS Number: 71472-57-6
Purity: 98.8%
Physical State / Appearance: Amber coloured viscous liquid
Expiry Date: 01 January 2019
Storage Conditions: Approximately 4°C, in the dark
Target gene:
Thymidine Kinase Gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
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:
Following solubility checks performed in-house for the Micronucleus Test in Human Lymphocytes in vitro performed on the same test item (Envigo Study No. PX68RL), the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared. The molecular weight of the test item was 149.19, therefore, the maximum recommended dose was set at 1492 µg/mL, the 10 mM limit dose, and a correction for the purity of 98.8% was applied to the formulations.

The dose range used in the preliminary toxicity test was 5.83 to 1492 µg/mL for all three of the exposure groups.

During the main experiment 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 (11.63 to 372 µg/mL for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.75 to 30 µg/mL in the 24-hour exposure group in the absence of metabolic activation), vehicle and positive controls.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO


Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
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. The dose range used in the preliminary toxicity test was 5.83 to 1492 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, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.
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, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained. 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 vehicle 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 experiment. 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 (Moore et al., 2002).

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 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 (11.63 to 372 µg/mL for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.75 to 30 µg/mL in the 24-hour exposure group in the absence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.2 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.

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 for the expression period of two days, by counting and dilution to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.
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 vehicle control, and when combined with the Viability (%V) data, a Relative Total Growth (RTG) value.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after eleven days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
Evaluation criteria:
Please see evaluation criteria under materials and methods section.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary Cytotoxicity Test

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, with the greatest reductions observed in the 24-hour exposure group in the absence of metabolic activation. Precipitate of the test item was observed at 1492 µg/mL in all three of the exposure groups at the end of the exposure periods. Therefore, following the recommendations of the OECD 490 guideline, the maximum dose levels in the Mutagenicity Test were limited by test item induced toxicity.

Mutagenicity Test

Please see Table 1 in the results section for a summary of results.

As was seen previously, there was evidence of marked dose-related toxicity in cells treated with the test item in all three of the exposure groups, as indicated by the RTG and / or %RSG values (Tables 3, 6, and 9). Based on the RTG and / or %RSG values observed, optimum levels of toxicity were considered to have been achieved in all three of the exposure groups (Tables 3, 6, and 9). There was also evidence of reductions in viability (%V) in all three of the exposure groups, therefore indicating that residual toxicity had occurred (Tables 3, 6, and 9). The excessive toxicity observed at 372 µg/mL in the 4-hour exposure groups in both the absence and presence of metabolic activation, and at 30 µg/mL in the 24-hour exposure group in the absence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 248 and 310 µg/mL in the 4-hour exposure group in the absence of metabolic activation, and at 310 µg/mL in the 4-hour exposure group in the presence of metabolic activation, exceeded the upper acceptable limit of 90%, therefore, these doses were excluded from the statistical analysis. Acceptable levels of toxicity were seen with the positive control substances. No precipitate of the test item was observed in any of the three exposure groups at the end of the exposure periods.
The vehicle 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 induced toxicologically significant and dose related increases in the mutant frequency x 10-6 per viable cell in all three of the exposure groups, with the most marked increases observed in the 4-hour exposure group in the presence of metabolic activation, and the 24-hour exposure group in the absence of metabolic activation. The GEF was exceeded in all three of the exposure groups, there was evidence of marked increases in the absolute numbers of mutant colonies, and there was also a shift towards small colony formation indicating clastogenic activity

Table1            Summary of Results

 

Main Experiment

Concentration

(µg/mL)

4-Hours-S9

Concentration

(µg/mL)

4-Hours+S9

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

0

 

100

1.00

192.02

 

0

 

100

1.00

138.76

 

11.63

Ø

85

 

 

 

11.63

Ø

92

 

 

 

23.25

 

78

0.95

173.93

 

23.25

 

78

0.85

156.52

 

46.5

 

75

0.76

200.04

 

46.5

 

60

0.61

215.14

*

93

 

49

0.47

324.84

*

93

 

39

0.39

409.65

*

186

 

20

0.13

236.92

 

186

 

22

0.22

449.58

*

248

X

13

0.08

257.51

 

248

 

17

0.10

252.65

*

310

X

11

0.05

386.50

 

310

X

10

0.07

260.55

 

372

Ø

7

 

 

 

372

Ø

4

 

 

 

MF threshold for a positive response = 318.02

MF threshold for a positive response = 264.76

Linear trend **

Linear trend ***

Positive control

 

 

Positive control

 

 

EMS

 

 

 

 

 

CP

 

 

 

 

 

400

 

75

0.56

1335.56

 

1.5

 

75

0.60

904.90

 

Concentration

(µg/mL)

24-Hours-S9

 

%RSG

RTG

MF§

0

 

100

1.00

183.04

 

0.75

Ø

83

 

 

 

1.5

 

73

1.00

166.97

 

3

 

73

0.86

195.25

 

6

 

65

0.90

178.95

 

12

 

41

0.59

347.67

*

18

 

22

0.31

457.31

*

24

 

17

0.24

730.13

*

30

Ø

9

 

 

 

MF threshold for a positive response = 309.04

Linear trend ***

Positive control

 

 

EMS

 

 

 

 

 

150

 

44

0.47

1968.69

 

Conclusions:
The test item induced increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be mutagenic in this assay.
Executive 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 vehicle (dimethyl sulfoxide (DMSO)), 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:

Mutagenicity Test

Group

Concentration of 6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine (µg/mL) plated for viability and mutant frequency

4-hour without S9

23.25, 46.5, 93, 186, 248, 310

4-hour with S9 (2%)

23.25, 46.5, 93, 186, 248, 310

24-hour without S9

1.5, 3, 6, 12, 18, 24

1.3                  Results……..

The maximum dose levels in the Mutagenicity Test were limited by test item‑induced toxicity in all three of the exposure groups, asrecommended by the OECD 490 guideline. The vehicle 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 induced toxicologically significant and dose related increases in the mutant frequency x 10-6per viable cell in all three of the exposure groups, with the most marked increases observedin the 4-hour exposure group in the presence of metabolic activation, and the 24-hour exposure group in the absence of metabolic activation. The Global Evaluation Factor (GEF) was exceeded in all three of the exposure groups, there was evidence of marked increases in the absolute numbers of mutant colonies, and there was also a shift towards small colony formation indicating clastogenic activity.

Conclusion

The test item induced increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be mutagenic in this assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Genetic Toxicity In-Vivo: OECD 474 Micronucleous assay in bone marrow cells of mice. The test substance considered to be non-mutagenic.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 April 2019 - 21 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
9th Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 474, adopted July 29, 2016, „Mammalian Erythrocyte Micronucleus Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Part B: Methods for the determination of toxicity and other health effects: Mutagenicity – In vivo Mammalian Erythrocyte Micronucleus Test, No B.12; No L 142.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA 712-C-98-226, August 1998.
Version / remarks:
Environmental Protection Agency, Health Effects Test Guidelines OPPTS 870.5395 “Mammalian Erythrocyte Micronucleus Test“, EPA 712-C-98-226, August 1998.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
Test system: Mice, NMRI
Rationale: Recognised as the recommended test system.
Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Number of animals for the pre-test: 2 males and 2 females for each pre-test
Number of animals for the main study: 36 males
Age (beginning of treatment): 6 – 8 weeks
Body weight: 31.3(g) - 37.2(g)
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type II / III, with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 + 2°C
relative humidity approx. 45-65%
artificial light 6.00 a.m. - 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: On the day of the experiment, the test item was formulated in corn oil. The vehicle was chosen due to its relative non-toxicity for the animals and ability to form a suitable dosing formulation.
- Amount of vehicle (if gavage or dermal): All animals received a single volume of 10 mL/kg b.w. once orally by gastric gavage.
Details on exposure:
All animals received a single volume of 10 mL/kg b.w. once orally by gastric gavage.
The preparations were made freshly before the dosing occasion.
Duration of treatment / exposure:
24 - 48 hours
Frequency of treatment:
The animals received the test item, the vehicle or the positive control item once orally.
Post exposure period:
The animals of all dose groups, except the positive control group, were examined for acute toxic symptoms at time intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and/or 48 h after administration of the test item.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Medium dose
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
High dose
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Positive control
No. of animals per sex per dose:
Six males were treated per dose group and sampling time.
Control animals:
yes
Positive control(s):
Cyclophosphamide
Purity: 97% dissolved in sterile water.
Route and frequency of administration: orally once by gastric gavage
Concentration: 40 mg/kg b.w.
Volume administered: 10 mL/kg b.w.
Tissues and cell types examined:
micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
Details of tissue and slide preparation:
Main Study Dose Selection
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly, or 2000 mg/kg b.w. as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes visually noticeable toxic reactions but not death or evidence of pain, suffering or distress necessitating humane euthanasia.
The administered volume was 10 mL /kg b.w. each treatment.
Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken 48 h after treatment.

Treatment
At the beginning of the treatment and at the end of the in-life phase the animals (including the controls) were weighed and the individual dose volume was adjusted to the animal’s body weight. The animals received the test item, the vehicle or the positive control item once orally. Six males were treated per dose group and sampling time.
The animals of all dose groups, except the positive control group, were examined for acute toxic symptoms at time intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and/or 48 h after administration of the test item. Sampling of the bone marrow was done 24 h after treatment for all treatment groups including the positive control, and at 48 h after treatment for the high dose group only.

Preparation of the Animals
The animals were sacrificed using CO2 followed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Analysis of Cells
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 4000 polychromatic erythrocytes (PCE) per animal were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per total erythrocytes. The analysis was performed with coded slides.
All animals per test group were evaluated as described.
Evaluation criteria:
A test substance is classified as positive in the assay if
a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
b) This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
c) Any of these results are outside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits).
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.
A test substance that fails to produce a biologically relevant increase in the number of micronucleated polychromatic erythrocytes, applying above mentioned criteria, is considered negative in this system, given that there is evidence for bone marrow exposure (e.g., the substance can be detected in the blood by bioanalytical methods).
Statistical methods were used as an aid in evaluating the results.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test using the validated statistical program RScript Wilcoxon_2.Rnw. The Holm-Bonferroni Adjustment method was used to correct for the familywise error rate of the multiple comparisons.
A linear regression (least squares, calculated using the validated statistical program RScript LM_v02.Rnw) was performed to assess a possible dose dependent increase of mean micronuclei values. The mean number of micronuclei obtained for the groups treated with the test item was compared to the vehicle control group. A trend is judged as significant whenever the p-value (probability value) is below 0.05. A p-value of 0.4072 was obtained, demonstrating that there was a no dose dependent increase of mean micronuclei values.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
The test substance was dissolved in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w..
At time intervals of 24 h and 48 h after a single oral administration of the test item the bone marrow cells were collected for micronuclei analysis.
Six males per test group were evaluated for the occurrence of micronuclei. Per animal 4000 polychromatic erythrocytes (PCEs) were scored for micronuclei.
To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 500 erythrocytes.
The following dose levels of the test item were investigated:
24 h preparation interval: 50, 100, and 200 mg/kg b.w..
48 h preparation interval: 200 mg/kg b.w..
As estimated by two pre-experiments treatment at 200 mg BN-02 per kg b.w. (the maximum tolerated dose) was suitable as the highest treatment dose.
Clinical symptoms in the main experiment included partially closed eyes, piloerection, and decreased activity for all dose levels. Additionally, some animals of the mid and high dose showed discoloration of the urine (orange) and the symptoms closed eyes and lachrymation were observed in the high dose group only.
The observed systemic toxicity at the tested doses and the discoloration of the urine are indicative of a systemic distribution of the test item. Thus, bioavailability of the test item to the bone marrow under the tested conditions reported is assumed.
The mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control, indicating that the test substance did not have any cytotoxic properties in the bone marrow.
In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation time interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group and well within historical vehicle control values.
A linear regression (least squares, calculated using the validated statistical program RScript LM_v02.Rnw) was performed to assess a possible dose dependent increase of mean micronuclei values. The mean number of micronuclei obtained for the groups treated with the test item was compared to the vehicle control group. A trend is judged as significant whenever the p-value (probability value) is below 0.05. A p-value of 0.4072 was obtained, demonstrating that there was a no dose dependent increase of mean micronuclei values.
Treatment with 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In the main experiment for each test item dose group 6 males received the test item dissolved in corn oil once orally. The volume administered was 10 mL/kg b.w.. The clinical symptoms observed following treatment are shown in the following table for each dose group, which indicates the number of animals with findings.

 

hours post-treatment (males)

Clinical symptoms

1

2-4

5-6

24

48

High dose: 200 mg/kg b.w. (12 males at 1 to 24 h; 6 males at 48 h)

Partially Closed Eyes

5

1

3

2

0

Closed Eyes

3

2

0

0

0

Piloerection

12

10

6

5

6

Decreased Activity

11

2

0

2

0

Stained Urine (orange)

0

0

7

0

0

Lachrymation

0

0

0

1

0

 

hours post-treatment (males)

Clinical symptoms

1

2-4

5-6

24

48

Medium dose: 100 mg/kg b.w. (6 males at 24 h)

Partially Closed Eyes

2

2

2

0

 

Piloerection

4

4

2

0

 

Decreased Activity

3

1

1

0

 

Stained Urine (orange)

0

0

3

0

 

Low dose: 50 mg/kg b.w.(6 males at 24 h)

Partially Closed Eyes

2

1

0

1

 

Piloerection

4

6

2

1

 

Decreased Activity

3

4

1

1

 

The animals treated with the vehicle control (corn oil) did not express any clinical symptoms.

 

Test
Group

Dose
mg/kg
b.w.

Sampling
time

Mean MN/4000 PCE

SD MN/4000 PCE

Range

Ratio
 PCE /total Ery

% ratio
Vehicle

min

max

Vehicle

0

24

4.8

2.8

2

10

0.632

100.00

Dose 1

50

24

3.0

1.1

2

4

0.637

100.79

Dose 2

100

24

7.3

4.5

3

13

0.629

99.53

Dose 3

200

24

6.5

4.1

1

12

0.626

99.05

Positive

40

24

114.7

60.5

36

216

0.634

100.32

Dose 3

200

48

3.7

2.7

0

7

0.576

91.14

MN = micronuclei

Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, the substance is considered to be non-mutagenic in this in-vivo micronucleus assay.
Executive summary:

This study was peformed to investigate the potential of 6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in corn oil, which was also used as vehicle control. The dose volume administered orally was 10 mL/kg b.w. The administered volume of the positive control was 10 mL/kg.

At time intervals of 24 h and 48 h after a single oral administration of the test item bone marrow cells were collected for micronuclei analysis. Six males per test group were evaluated for the occurrence of micronuclei. Per animal 4000 polychromatic erythrocytes were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes. The following dose levels of the test item were investigated in the main experiment:

24 h preparation interval: 50, 100, and 200 mg/kg b.w.
48 h preparation interval: 200 mg/kg b.w.

The highest dose (200 mg/kg b.w.) was estimated by two pre-experiments to be suitable.

Clinical symptoms in the main experiment included partially closed eyes, piloerection and decreased activity for all dose levels. Additionally, some animals of the mid and high dose showed discoloration of the urine (orange) and the symptoms closed eyes and lachrymation were observed in the high dose group only. The observed systemic toxicity at the tested doses and the discoloration of the urine are indicative of a systemic distribution of the test item. Thus, bioavailability of the test item to the bone marrow under the tested conditions reported is assumed. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test substance did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronulclei at any preparation time interval after administration of the test item and with any dose level used.

A linear regression (least squares, calculated using the validated statistical program RScript LM_v02.Rnw) was performed to assess a possible dose dependent increase of mean micronuclei values. The mean number of micronuclei obtained for the groups treated with the test item was compared to the vehicle control group. A trend is judged as significant whenever the p-value (probability value) is below 0.05. A p-value of 0.4072 was obtained, demonstrating that there was a no dose dependent increase of mean micronuclei values.

Treatment with 40 mg/kg b.w. cyclosphosphamide administered orally was used as positive control which induced a substantial increase in cells with micronuclei.

Conclusion

In conclusion, in can be stated that under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the substance is considered to be non-mutagenic in this in-vivo micronucleus assay.

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

Additional information

In vitro genetic toxicity studies according to OECD guidelines and to GLP quality standards showed evidence of gene mutation and chromosomal aberrations. On the basis of the two positive in vitro results and one negative in-vitro result there was justification for further in vivo genetic toxicology testing. In an in-vivo genetic toxicity study according to OECD guideline and to GLP quality standards showed no evidence of gene mutation.

Justification for classification or non-classification

GLP compliant guideline studies each with a klimisch score of 1