Reaction mass of 2-[[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]methyl]oxirane and 1,3-bis[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]propan-2-ol and 1-[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]-3-[4-[[4-[3-[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]-2-hydroxy-propoxy]-3,5-dimethyl-phenyl]methyl]-2,6-dimethyl-phenoxy]propan-2-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Initially, three in vitro genotoxicity assays were conducted including an AMES test (OECD 471), a chromosome aberration test (OECD 473) and a mouse lymphoma assay (OECD 476).

All three in vitro tests showed a positive response (Salmonella strains TA100/TA1535 with activation in OECD 471, structural and numerical aberrations with and without activation in OECD 473, and mutagenicity without activation for the OECD 476 test).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Properly maintained
- Periodically checked for Mycoplasma contamination
- Periodically checked for karyotype stabilit
- Periodically "cleansed" against high spontaneous background

The clones used for this assay were stored at -60 to -80°C before use. When required these were defrosted overnight in Oxoid nutrient broth. Optical density readings were performed on all strains used to check the level of growth.

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Pre-incubation with S9 mix

Test concentrations with justification for top dose:

Initial and confirmatory assay: 0 (vehicle), 25, 50, 100, 250, 500, 1000, 2500 and 5000 ug/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

ICR-191 acridine, 2-nitrofluorene, sodium azide, 4-nitroquinoline-N-oxide and 2-aminoanthracene, for various strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: Pre-incubation

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 plates per dose (triplicate)

Confirmatory analysis was conducted on the stock test material solutions and this confirmed acceptable test material concentrations.

Evaluation criteria:

The test substance was judged to have mutagenicity (positive) when the test substance induced a dose-dependant increase in the number of the
revertant colonies (mean) to a level equal to or greater than 2-fold of the negative (solvent) control value (mean value) in the tester strains with high spontaneous levels (e.g. TA100) and three times for those with low spontaneous levels (e.g. TA1537, TA98, TA1535, and WP2 urvA). Other results were judged to be negative.

Statistics:

For each concentration level and for each condition, the mean revertant count and standard deviation was determined.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitates were observed at >/= 250 ug/plate without metabolic activation.

Fold increases indicative of a positive response were observed at ≥250 μg/plate in TA1535 with metabolic activation (≥4.8-fold) and at ≥1000 μg/plate in TA100 with metabolic activation (≥2.4-fold).

Conclusions:

Mean increases in the number of revertant colonies indicative of a positive response were observed with the test substance in the Salmonella strains TA100 and TA1535 with metabolic activation, under the conditions of this assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's complete medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (free of contamination)
- Periodically checked for karyotype stability: yes (karyotype shown to have a modal chromosome number of 21 and 12 o 14 hour cycling time).

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254- induced rat liver S9 fraction

Test concentrations with justification for top dose:

Range-finding assay: 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 ug/mL.
Aberration assay (concentrations evaluated for chromosomal aberrations):
3h without metabolic activation: 100, 250 and 350 ug/mL.
20h without metabolic activation: 30, 40, 55, 65, and 75 ug/mL.
3h with metabolic activation: 140, 150 and 160 ug/mL.

Test concentrations were analysed and confirmed as near nominal by a validated analytical method.

Vehicle / solvent:

Negative control and test substance vehicle: Dimethyl Sulfoxide (DMSO) was used.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Cyclophosphamide and mitocycin C were chosen as metabolism dependant and direct acting positive controls, respectively.

Details on test system and experimental conditions:

Cells were cultured in McCoy's complete media ((McCoy’s medium supplemented with a final concentration of 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 100 units/mL penicillin and 100 μg/mL streptomycin). All cultures were incubated at 37 ± 1 °C in vented flasks in a humidified atmosphere of 5 ± 1% CO2 for approximately 24 hours prior to the initiation of treatment.

Cultures were treated with the test article or positive control or vehicle in the presence and absence of metabolic activation for short incubations (3 hours) and in the absence of activation for the long incubation (20 hours). Positive control concentrations were mitomycin C at 0.050, 0.10, 0.50, and 0.75 μg/mL in cultures without activation and cyclophosphamide at 5.0 and 7.5 μg/mL in cultures with activation. After 3 hours, the short treatments were washed and returned to the incubator until harvest at 20 hours. Approximately 2 hours prior to harvest, Colcemid® (0.1 μg/mL, final concentration in culture) was added to each culture to arrest cells in metaphase.

At 20 hours, the culture medium was removed and the cells were washed with phosphate buffered saline. The exposed monolayer of cells was trypsinized and an aliquot was removed for assessment of cell viability via Coulter counter. Cells were then concentrated by centrifugation and resuspended in hypotonic solution (0.075 M KCl). They were fixed in two changes of fixative (using an approximately 3:1 methanol:glacial acetic acid mix) before being stored in the refrigerator. These fixed cells were washed twice more with fresh fixative prior to slide preparation. The final concentrated cell suspension was dropped on clean, cold wet glass slides, dried prior to staining with DifQuik solution at room temperature, and coverslipped. At least two slides were prepared for each culture.

Cytotoxicity, as assessed using cell count reduction from Coulter counts was measured in all cultures. Cells treated with test article or positive controls were compared with vehicle control cultures (Galloway et al., 1994). Cytotoxicity was also assessed by measurement of the reduction in mitotic index compared to the vehicle control for each aberration assay. The number of mitotic cells in 1000 cells from each culture was counted and the mitotic index was calculated. In addition, all cultures were examined visually for signs of cytotoxicity (i.e., visible cell abnormalities), changes in cell morphology, pH change and precipitates at the time of dosing, at the end of the 3-hour treatment (prior to wash), and prior to harvest.

Evaluation criteria:

Criteria for a positive response: As a guideline, the test article would be considered positive for inducing chromosomal aberrations if a significant increase (p ≤ 0.01) in the number of cells with chromosomal aberrations is observed at one or more dose levels, or if a significant increase (p ≤ 0.05) in the number of cells with chromosomal aberrations is observed at two or more dose levels. If a significant increase is seen at one or more dose levels, a dose-response should be observed.

Criteria for a negative response: The test article would be considered negative for inducing chromosomal aberrations if no statistically significant increase (p ≤ 0.05) is observed in the number of cells with chromosomal aberrations at any of the test concentrations.

Criteria for an equivocal response: Cases which do not clearly fit the positive or negative criteria may be judged equivocal. As a general rule the biological relevance of any result would be considered first.

Statistics:

After completion of microscopic analyses, data were decoded and a Fisher’s Exact test, was performed on the mean percentage of cells with structural
aberrations and the mean percentage of cells with more than one chromosome aberration comparing the treated cells to the results obtained from the relevant control group. The same statistical analysis was performed for cells exhibiting polyploidy and/or endoreduplication.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

3h treatment without metabolic activation:
A statisitically significant increase in the percent of cells with aberrations was observed at 350 μg/mL. No statistically significant test article-related increase in the percent of cells with > 1 aberration) was noted. In addition, no statistically significant increase in the percent cells with numerical aberrations (polyploidy and/or endoreduplication) was observed in any test article treated group.

20h treatment without metabolic activation:
A statistically significant increase in the percent of cells with aberrations was observed at ≥ 30 μg/mL. A statistically significant increase in the percent of cells with >1 aberration was observed at ≥ 55 μg/mL. A statistically significant increase in the percent of polyploid cells was observed at ≥ 40 μg/mL. No statistically significant increases were observed in the percent of endoreduplicated cells.

3h treatment with metabolic activation:
A statistically significant increase in the percent of cells with aberrations and in the percent of endoreduplicated cells was observed at ≥ 140 μg/mL. A statistically significant increase in the percent of polyploid cells was observed at 160 μg/mL. No statistically significant test article-related increase in the percent of cells with > 1 aberration) was noted.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The test substance was considered to have the potential to induce chromosomal aberrations in CHO-WBL cells (both with and without metabolic activation) under the conditions employed in the study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Basic medium - RPMI 1640 hepes buffered medium (Dutch modification).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 Homogenate prepared from male sprague dawley rats dose with phenobarbitol and B-napthoflavone

Test concentrations with justification for top dose:

Dose range finding phase: 3 to 333 μg/ml (3 hours, with and without metabolic activation)
Mutation experiment: 1, 3, 10, 33, 66, 100, 150, 333 μg/ml (without activation), and 1, 3, 10, 33, 66, 100, 150, 200, 250 and 300 μg/ml (with activation)

Vehicle / solvent:

The test substance was dissolved in Dimethyl sulfoxide (DMSO).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

MMS used as a direct acting mutagen, and CP used as an indirect acting mutagen (requiring metabolic activation)

Details on test system and experimental conditions:

Exposure medium consisted of Basic medium (RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin) supplemented with with 5% (v/v) heat-inactivated horse serum (R-5 medium; inactivated at 56°C for at least 30 minutes).

Dose range finder:
Cell cultures were exposed to the test material in exposure medium for 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix. After exposure, the cells were separated from treatment solutions by 2 centrifugation steps each followed by removal of the supernatant. The cells in the final suspension were counted with the coulter particle counter. The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to
determine an appropriate dose range for the mutagenicity tests.

Test and control materials:
The test substance was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves until the test substance was completely dissolved. Amber-coloured glassware or tubes wrapped in aluminium-foil were used when preparing the test solutions. Test substance concentrations were used within 1 hour after preparation. The final concentration of the solvent in the exposure medium was 0.8% (v/v).

The positive controls were prepared as follows:
In the absence of S9: Methyl methanesulfonate (MMS). MMS was used as a direct acting mutagen at a concentration of 15 μg/ml. MMS was dissolved in dimethyl sulfoxide. The stock solutions of MMS were prepared immediately before use.
In the presence of S9: Cyclophosphamide (CP). CP was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 7.5 μg/ml with 4% (v/v)) S9 fraction. CP was dissolved in Hanks’ balanced salt solution (HBSS) without calcium and magnesium. The stock solutions of CP were stored in aliquots at ≤-15°C in the dark and one sample was thawed immediately before use.

Mutagenicity test:
Eight doses of the test substance were tested in the mutation assay, both in the absence and presence of S9-mix. The highest doses that were tested gave a cell survival of approximately 10-20%. The test material was tested below and beyond the limit of the solubility to obtain adequate mutagenicit data. The survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.

Treatment of cells:
Per culture 8 x 10^6 cells (10^6 cells/ml) were used. The cell cultures were placed in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 spm for 3 hours. Solvent and positive controls were included and the solvent control was tested in duplicate. After exposure, the cells were separated from treatment solutions by 2 centrifugation steps (216 g, 8 min) each followed by removal of the supernatant. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the final centrifugation step the cells were resuspended in R10 medium. The cells in the final suspension were counted with the coulter particle counter.

Expression period:
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test
substance the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

Determination of mutant frequency:
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. 1 cell was added per well (2 x 96-well microtiter plates/concentration) in non selective medium. For determination of the MF a total number of 9.6 x 10^5 cells/concentration were plated in five
96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection) (total number of 471 to 480 wells, with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

Determination of mutant colonies: The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

Evaluation criteria:

A test substance is considered positive on test (potentially mutagenic), if it induces a mutation frequency of more than the mutation frequency for thecontrols (+ 126) in a dose dependant manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered negative (not mutagenic) if in the mutation assay if:
1) None of the tested concentrations reaches a mutation frequency of the mutation frequency of the controls (+ 126).
2) The results are confirmed in an independantl y repeated test.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Dose levels >250 ug/mL (not used for mutation frequency measurements)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Solubility: The test material precipitated in the exposure medium at concentrations of 100 μg/ml and above. The test substance was tested beyond the limit fo solubility to obtain adequate cytotoxicity data.

Preliminary (dose range finding phase): In the absence of S9 mix, the relative suspension of growth was 41% at the test substance concentration of 333 μg/ml compared to the solvent control. In the presence of S9, no toxicity in the relative suspension of growth was observed up to test substance concentrations of 100 μg/ml compared to the solvent control. Little cell survival was observed at 333 μg/ml.

Main phase (mutation experiment):
Toxicity: In the absence of S9 mix, no severe toxicity was observed and all dose levesl evaluated. In the presence of S9 mix, the dose levels of 250 to 300 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
Mutagenicity: In the absence of S9 mix, the test substance induced an up to 6.4 fold increase in the mutation frequency at the TK-locus. The test material showed up to 7.0 and 4.1 fold increases in the mutation frequency of the small and large colonies respectively, compared with the solvent controls. In the presence of S9 mix, the test material did not induce an increase in the mutation frequency at the TK locus.

Formulation analysis:
No test substance was detected in the solvent controls and test concentrations analysed with in agreement with nominal concentrations.

The spontaneous mutation frequencies in the solvent control treated cultures were between the minimum and maximum value of the HCD. Mutation frequencies in cultures treated with positive control chemicals were increased by 7.7-fold for MMS in the absence of S9-mix, and by 32-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

It is concluded that the test material is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report. The mutagenicity was confined only to incubations without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The in vitro tests were followed by two in vivo tests (micronucleus and comet assay (OECD 474/489 respectively)) to clarify the results obtained in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: JaCVAM protocol version 14.2

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 489

Deviations:

no

Principles of method if other than guideline:

Study conducted to JaCVAM protocol. The JaCVAM formally validated the method in conjunction with ECVAM, ICCVAM and NICEATM. This study predates the recently issued OECD 489 guideline, but is considered to use equivalent methodology.

GLP compliance:

yes

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (Frederick, MD)
- Age at study initiation: 6 or 7 weeks for the initial and repeat definitive phases respectively.
- Weight at study initiation: 185-208.7 (initial definitive), and 174.1 - 197.4 (repeat definitive)
- Assigned to test groups randomly: yes (weight variation of animals did not exceed ± 20% of the mean weight).
- Housing: Up to 5 rats in micro-barrier cages equipped with micro-vent full ventilation in a HEPA filtered system.
- Diet (e.g. ad libitum): Certified rodent laboratory chow ad libitum.
- Water (e.g. ad libitum): Ad libitum and meeting USEPA drinking water standards.
- Acclimation period: 6 or 12 days for the initial and repeat definitive phases respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20% relative humidity
- Air changes (per hr): Uninterrupted positive air flow
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Route of administration:

oral: gavage

Vehicle:

Corn oil (stored at 2-8°C before use)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

An appropriate amount of test article was added to a container. An appropriate amount of vehicle was then added and the formulation was mixed via amagnetic stirrer until homogeneous in appearance. All formulations were stored at room temperature until use and used within one day of preparation.

Duration of treatment / exposure:

Animals were dosed once on each of three consecutive days. The second dose occured approximately 24 hours after the first dose, with the third dose occuring 21 hours after the second. All animals were euthanized between 3-4 hours after the third dose.

Frequency of treatment:

Daily for 3 days.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per group

Control animals:

yes, concurrent vehicle

Positive control(s):

- Ethyl methanesulfonate dosed on Study Days 2 and 3 only
- Route of administration: Oral gavage
- Doses / concentrations: 200 mg/kg/day

Tissues and cell types examined:

Sections of liver and duodenum.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The MTD/MFD provided.

TISSUE DETAILS:
The liver which is the most active organ in metabolism of substances, and also a frequently a target for carcinogenicity and the duodenum as an early site of first contact (and more relevant to humans than the rodent glandular stomach), were selected as tissues in which to conduct the comet assay in this test. Sections of the liver and duodenum were placed in chilled mincing solution (Hanks Balanced salt solution with EDTA and DMSO). Liver was minced with fine scissors to release the cells. Duodenum was scraped with a plastic spatula to release the cells. The cell suspension was strained into a pre-labeled conical polypropylene tube through a Cell Strainer.

DETAILS OF SLIDE PREPARATION:
From each single cell suspension, an aliquot was mixed with 75 μL of low melting agarose (0.5%; maintained in a dry bath set at 37 to 42°C). The cell/agarose suspension was applied to microscope slides, previously coated with normal melting agarose (1%). The slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify. Four slides were prepared per organ/tissue for each animal. Two slides were used in scoring and the remaining two slides were designated as backups. Following solidification of agarose, the slides were placed in jars containing lysis solution.

Lysis:
Following solidification of agarose, the slides were submerged in a cold lysis solution composed of 100 mM EDTA (disodium), 2.5 M sodium chloride, 10 mM tris hydroxymethyl aminomethane in purified water; pH 10; 1% Triton X-100 and 10% DMSO. The slides were kept in this solution at least overnight at 2-8°C.

Unwinding:
After cell lysis, slides were washed with neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, pH ~7.5) and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with alkaline buffer composed of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water. The pH was > 13. All slides remained in the buffer for 20 minutes at 2-1°C and protected from light, allowing DNA to unwind.

Electrophoresis:
Using the same buffer, electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-10°C and protected from light. The electrophoresis time was constant for all slides.

Neutralization:
After completion of electrophoresis, the slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides (gels) were then dehydrated with 100% ethanol for at least 5 minutes, air dried and stored at room temperature with desiccant.

Staining:
Slides were stained with a DNA stain (i.e., Sybr-gold) prior to scoring. The stain solution was prepared by diluting 1 ∝L of Sybr-gold stain in 15 mL of 1xTBE (tris-boric acid EDTA buffer solution).

METHOD OF ANALYSIS:
Two slides/animal/tissue were used. Fifty randomly selected, non-overlapping cells per slide were scored resulting in a total of 100 cells evaluated for DNA damage.

DNA damage was assessed by the software system by measuring:
- Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail.
- % Tail DNA; (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
- Tail Moment (also known as Olive Tail moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/ 100.

Each slide was also examined for indications of cytotoxicity, i.e. presence of “clouds”. The “clouds”, also known as “hedgehogs”, are a morphological indication of highly damaged cells often associated with severe genotoxicity, necrosis or apoptosis. A “cloud” is produced when almost the entire cell DNA is in the tail of the comet and the head is reduced in size, almost nonexistent. The rough estimate of the percentage of “clouds” was determined by scanning 200 cells per animal (percentage of “clouds” was calculated by adding the total number of clouds for all slides scored, dividing by the total number of cells scored and multiplying by 100). “Clouds” with visible gaps between the nuclei and the comet tail were excluded from comet image analysis.

Evaluation criteria:

The test article was considered to have induced a positive response if it induced a statistically significant and dose-dependent increase in % tail DNA in a particular tissue in any dose group. If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.

Statistics:

- Means of 100 counts of % tail DNA, Tail moment and Tail migration were presented for each animal and each organ. The mean and standard deviation of the mean values for % tail DNA were presented for each treatment group.
- Statistical analyses were performed only for % tail DNA. The group variances for % tail DNA generated for the vehicle and test article groups were compared using Levene’s test (significant level of p δ 0.05). The differences and variations between groups were found not to be significant, therefore, a parametric one-way ANOVA followed by a Dunnett post-hoc test was performed (significant level of p < 0.05).
- Linear regression analysis was used to determine a dose response relationship (p < 0.01).
- Pair-wise comparison (Student’s t-test, p δ 0.05) was used to compare the data from the positive control group against the vehicle control group.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Duodenum – Dose dependent decrease in % tail DNA of the test article dose animals was detected. Because the protocol was not optimized to predict the decrease due to any DNA- DNA or DNA-protein crosslinking activity of the test article, the decrease could not be categorized. However, the test article did not show signs of % tail DNA increase and thus was evaluated as non-DNA damaging in duodena cells of test article treated animals. Positive and negative control mean values were within the expected ranges.

Liver – According to the mean values for each group, there was no increase in the DNA damage (% tail DNA) and positive and negative control mean values were within the expected ranges in the initial experiment. However, when the individual animal data were reviewed, it became evident that one animal in the highest dose group 2000 mg/kg/day) exhibited a significant increase in % tail DNA and one animal in the positive control group had no increase. As a result of this discrepancy, the phase was repeated for liver cells in which the test article gave a negative response and was evaluated as negative (non-DNA damaging) in the liver comet assay.

Dose formulations were analysed on study using a validated method.

Concentration analysis from the first formulation analysis indicated that the actual mean concentrations of the analyzed dose levels were between 96.7 % and 101.8% of their respective targets with ≤ 0.96 % relative standard deviation (RSD) and from the second formulation analysis, actual mean concentrations of the analyzed dose levels were between 70.4% and 106.6% of their respective targets with ≤ 2.22% relative standard deviation (RSD). All test article formulations met the protocol specified acceptance criteria for concentration and homogeneity except for the 10 mg/mL formulation from the second formulation analysis which was found to be below the acceptable range (70.4% of target concentration) but was within ≤ 5.00% RSD. However, the 10 mg/mL formulation from the first formulation analysis met the protocol specified criteria.

No test article was detected in the vehicle control samples.

Stability analysis indicated that the test article in Corn Oil at concentrations of 10.1 and 195 mg/mL was stable for 3.5 hours at room temperature.

Conclusions:

Under the conditions of this study, the test substance at dose levels up to and including 2000 mg/kg/day for three consecutive days administered once daily did not cause a significant increase in DNA damage in liver or duodena cells in male Hsd:SD rats. Therefore, the test substance was concluded to be negative in the in vivo Comet assay.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (Frederick, MD)
- Age at study initiation: 6 or 7 weeks for the initial and repeat definitive phases respectively.
- Weight at study initiation: 185-208.7 (initial definitive), and 174.1 - 197.4 (repeat definitive)
- Assigned to test groups randomly: yes (weight variation of animals did not exceed ± 20% of the mean weight).
- Housing: Up to 5 rats in micro-barrier cages equipped with micro-vent full ventilation in a HEPA filtered system.
- Diet (e.g. ad libitum): Certified rodent laboratory chow ad libitum.
- Water (e.g. ad libitum): Ad libitum and meeting USEPA drinking water standards.
- Acclimation period: 6 or 12 days for the initial and repeat definitive phases respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20% relative humidity
- Air changes (per hr): Uninteruppted positive air flow
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Route of administration:

oral: gavage

Vehicle:

Corn oil (stored at 2-8°C before use)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

An appropriate amount of test article was added to a container. An appropriate amount of vehicle was then added and the formulation was mixed via amagnetic stirrer until homogeneous in appearance. All formulations were stored at room temperature until use and used within one day of preparation.

Duration of treatment / exposure:

Animals were dosed once on each of three consecutive days. The second dose occured approximately 24 hours after the first dose, with the third dose occuring 21 hours after the second. All animals were euthanized between 3-4 hours after the third dose.

Frequency of treatment:

Daily for 3 days.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per group.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Ethyl methanesulfonate dosed on Study Days 2 and 3 only
- Route of administration: Oral gavage
- Doses / concentrations: 200 mg/kg/day

Tissues and cell types examined:

Femoral bone marrow from all animals was collected for the micronucelus assay.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The MTD/MFD provided.

DETAILS OF SLIDE PREPARATION: The bone marrow cells were transferred to a labeled centrifuge tube containing approximately 2 mL of fetal bovine serum. The tubes were centrifuged at about 100x g for about 5 minutes. The supernatant was drawn off leaving a small amount of fetal bovine serum with the remaining cell pellet. The cells were resuspended by manually flicking the tube and a small drop of bone marrow suspension was spread onto a clean glass slide.

Each slide was identified by the harvest date, study and animal number. Two slides were prepared for each animal. The slides were allowed to air dry and then fixed by dipping in methanol and allowed to air dry prior to staining.
One set of the slides was stained with acridine orange, a nucleic specific stain, and used for microscopic evaluation. The second set of slides (not stained) was kept as a backup.

METHOD OF ANALYSIS: Two thousand Polychromatic erythrocytes (PCEs)/animal were scored for the presence of micronuclei (MnPCEs). In addition, 1000 total erythrocytes (PCEs + Normochromatic erythrocytes (NCEs)) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity. PCE proportions <20% was considered excessively cytotoxic and were excluded fromevaluation.

Evaluation criteria:

A test article was considered to have induced a positive response if it induced a significant increase in MnPCE frequency (p ≤ 0.05) at any dose level or sampling time. The test article was considered to be negative if no significant increase in MnPCE frequency was observed (p> 0.05). If criteria for either a positive or negative clastogenic response were not met, the results were judged as equivocal.

Statistics:

The frequency of MnPCEs and the proportion of PCEs to total erythrocytes were determined for each animal and treatment group. Statistical significance (p ≤ 0.05) was determined using the binomial distribution (Kastenbaum-Bowman tables).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

There was no significant increase in the micronucleus formation in test article samples when compared to the vehicle control. The test substance gave a negative result (no significant increase in micronuclei induction compared to the vehicle control) response in this assay. Thus, the test article has been evaluated as non-clastogenic.

The positive control EMS induced a statistically significant increase in the incidence of MnPCEs relative to the concurrent vehicle control article (p ≤ 0.05, binomial distribution, Kastenbaum-Bowman Tables).

The results indicated that all criteria for a valid Micronucleus assay were met.

Dose formulations were analysed on study using a validated method.

Concentration analysis from the first formulation analysis indicated that the actual mean concentrations of the analyzed dose levels were between 96.7 % and 101.8% of their respective targets with ≤ 0.96 % relative standard deviation (RSD) and from the second formulation analysis, actual mean concentrations of the analyzed dose levels were between 70.4% and 106.6% of their respective targets with ≤ 2.22% relative standard deviation (RSD). All test article formulations met the protocol specified acceptance criteria for concentration and homogeneity except for the 10 mg/mL formulation from the second formulation analysis which was found to be below the acceptable range (70.4% of target concentration) but was within ≤ 5.00% RSD. However, the 10 mg/mL formulation from the first formulation analysis met the protocol specified criteria.

No test article was detected in the vehicle control samples.

Stability analysis indicated that the test article in Corn Oil at concentrations of 10.1 and 195 mg/mL was stable for 3.5 hours at room temperature.

Conclusions:

Under the conditions of the study, the test material at dose levels of up to 2000 mg/kg/day for three consecutive days, administered once daily, did not cause a significant increase in the micronucleus formation in treated animal samples, compared with controls. As such the test material gave a negative response in this assay, and evaluated as non-clastogenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

All three in vitro tests showed a positive response (Salmonella strains TA100/TA1535 with activation in OECD 471, structural and numerical aberrations with and without activation in OECD 473, and mutagenicity without activation for the OECD 476 test).

The follow-up in vivo tests which tested for both the test substance's ability to induce DNA damage in liver and duodena cells, and also clastogenic activity and/or disruption of the mitotic apparatus (by detecting micronuclei in polychromatic erythrocytes cells) in rat bone marrow, both showed a clear negative response for genotoxic effects.

Justification for selection of genetic toxicity endpoint
Key In Vivo genotoxicity study alongside an in vivo rat micronucleus test.

Justification for classification or non-classification

Two in vivo tests conducted to clarify the positive results for genotoxicity found in vitro, showed that the in vitro results were not reproducible in vivo under assays investigating both the test substances ability to induce DNA damage and also clastogenic activity.

As such the test substance is considered not to cause mutagenicity under the conditions described, and is not classified under the CLP regulation (EC No. 1272/2008, as amended).