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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: JaCVAM protocol version 14.2

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]propan-2-ol; 1-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-3-{4-[(4-{3-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-2-hydroxypropoxy}-3,5-dimethylphenyl)methyl]-2,6-dimethylphenoxy}propan-2-ol; 2-{[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]methyl}oxirane

EC Number:

941-357-0

Molecular formula:

Not applicable for multi-constituent substances

IUPAC Name:

1,3-bis[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]propan-2-ol; 1-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-3-{4-[(4-{3-[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]-2-hydroxypropoxy}-3,5-dimethylphenyl)methyl]-2,6-dimethylphenoxy}propan-2-ol; 2-{[4-({3,5-dimethyl-4-[(oxiran-2-yl)methoxy]phenyl}methyl)-2,6-dimethylphenoxy]methyl}oxirane

Test material form:

other: Liquid

Details on test material:

Batch number: AL745-24
Storage: at room temperature protected from light

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.