p-tert-butylphenyl 1-(2,3-epoxy)propyl ether

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: gene mutation

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:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

p-tert-butylphenyl 1-(2,3-epoxy)propyl ether

EC Number:

221-453-2

EC Name:

p-tert-butylphenyl 1-(2,3-epoxy)propyl ether

Cas Number:

3101-60-8

Molecular formula:

C13H18O2

IUPAC Name:

2-[(4-tert-butylphenoxy)methyl]oxirane

Constituent 2

Reference substance name:

p-tert-butylphenyl-1-(2,3)-epoxypropyl ether

IUPAC Name:

p-tert-butylphenyl-1-(2,3)-epoxypropyl ether

Constituent 3

Reference substance name:

Oxirane, 2-((4-(1,1-dimethylethyl)phenoxy)methyl)-

IUPAC Name:

Oxirane, 2-((4-(1,1-dimethylethyl)phenoxy)methyl)-

Test material form:

other: Liquid at room temperature.

Details on test material:

As per IUCLID5 Sections 1.1. 1.2. and 4.1.

Specific details on test material used for the study:

Identification: p-tert-butylphenyl 1-(2,3-epoxy)propyl ether
Batch No.: DG4K20088 (Sample 0001)
CAS No.: 3101-60-8
Purity: 100% (Per Protocol)
Molecular Weight: 206.2808 g/mol
Description: Clear yellow liquid (Sample 0001)
Clear whitish yellow liquid (Sample 0002)
Storage Conditions: Room temperature, protected from light
Receipt Date: 19 April 2016 (Sample 0001)
05 July 2016 (Sample 0002)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

This species has been routinely used as an animal model of choice for the mammalian bone marrow erythrocyte Micronucleus and Comet assays. This strain is an outbred strain that maximizes genetic heterogeneity, and therefore, tends to eliminate strain-specific response to the test substance.
The Micronucleus assay and the Comet assay were combined in a single study using one set of animals. This combined three daily dose study design allows the assessment of the test substance using two genotoxicity end points and markedly reduces the number of animals, the time for generation of the relevant data, as well as the amount of the test substance required.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Welfare Provisions
This study is not duplicative or unnecessary. The number of animals, procedures, and design used for this study, has been reviewed and were approved by the BioReliance Institutional Animal Care and Use Committee. Procedures involving animals performed at BioReliance follow the specifications recommended in the most current version of The Guide for the Care and Use of Laboratory Animals adopted by BioReliance (National Academy Press, Washington, D.C., 2011).

Animal Receipt and Acclimation
Virus antibody-free (VAF) animals were acclimated as noted above and were judged to be healthy prior to utilization in the study.

Housing
Animals were housed in a controlled environment at 72 ± 3F and 50 ± 20% relative humidity with a 12-hour light/dark cycle. The light cycle was not interrupted for study related activities. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air per hour. Animals of the same sex were housed up to five per Micro-Barrier cage. Cages were placed on racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system.

Bedding, Food and Water
Heat treated hardwood chips (P.J. Murphy Forest Products) were used for bedding to absorb liquids. A certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients. Animals had free access to tap water, which met U.S. EPA drinking water standards [Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens. The results of bedding, food and water analyses are on file at BioReliance. There were no contaminants in the bedding, feed and water that were expected to interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn Oil

Details on exposure:

Animals initially were treated (on Day 1 at t = 0) with the test or control substances and euthanized at the appropriate time as described below. The following procedures were used for the Micronucleus - Comet assays as appropriate.

Group Treatment Dose Level Dose VolumeA Dose RouteB Male Rats Euthanasia Time
(mg/kg/day) (mL/kg/day) (Hrs after Treatment)
1 Corn oil 0 10 Oral Gavage 6 3-4
2 Heloxy Modifier AQ 125 10 Oral Gavage 6 3-4
3 Heloxy Modifier AQ 250 10 Oral Gavage 6 3-4
4 Heloxy Modifier AQ 500 10 Oral Gavage 6 3-4
5 EMS 200 10 Oral Gavage 3 3-4
ABased upon individual body weight
BUsing appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The route has been routinely used and is widely-accepted for use in the mammalian toxicology testing.

The outline of the study was as follows:
• All animals in Groups 1 through 4 were dosed on three consecutive days (Study Days 1, 2, and 3) with the vehicle and with test substance. The second dose occurred approximately 24 hours after the first dose, the third approximately 21 hours after the second and approximately 3 – 4 hours before euthanasia.
• Animals in Group 5 were dosed with the positive control (EMS) once on Study Day 3; approximately 3 to 4 hours prior to euthanasia.
• Six animals in groups 2-4 were bled prior to euthanasia (See Deviations). Animals in groups 1 and 5 and all other animals in groups 2-4 were euthanized 3-4 hours after the last dose by CO2 inhalation without collection of blood samples.

Duration of treatment / exposure:

3 days

Frequency of treatment:

once per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate (EMS)
The neat EMS was prepared in 0.9% sodium chloride for injection (saline). The dosing formulation was prepared at a concentration of 20 mg/mL, just prior to use.

Examinations

Tissues and cell types examined:

Micronucleus Assay Bone Marrow Collection and Processing
Femoral bone marrow was collected, at approximately 3-4 hours after the final dose, as indicated. Animals were euthanized by carbon dioxide inhalation. Immediately following euthanasia, the femurs from Groups 1 to 4 were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum.

Comet Assay Tissue Collection and Processing
All animals were euthanized 3 to 4 hours after the last dose (Study Day 3) by CO2 asphyxiation, and then, the following was performed:
• Animals were dissected and the liver and stomach were extracted (removed) and collected.
• A section of the liver and stomach was cut and placed in formalin for possible histopathology analysis.
• Another section of the liver and stomach was placed in chilled mincing solution (Hanks’ balanced salt solution with EDTA and DMSO) and was used in preparation of cell suspensions and Comet slides.

Details of tissue and slide preparation:

Preparation of Micronucleus Slides
The bone marrow was transferred to a centrifuge tube containing 2 mL fetal bovine serum, the cells were pelleted by centrifugation, and the supernatant was drawn off leaving a small amount of fetal bovine serum with the pellet. Cells were re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. At least four slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of two slides (including at least 5 positive control slides) was stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup and will be archived at report finalization. Each slide was identified by the harvest date, study number, and animal number. Slides were coded using a random number table by an individual not involved with the scoring process.
Preparation of Cell Suspensions and Comet Slides
A portion of each dissected liver was placed in cold mincing buffer, then the liver was finely cut (minced) with a pair of fine scissors to release the cells. A portion of each dissected stomach was placed in cold mincing buffer; then, the stomach was scraped using a plastic spatula to release the cells. Each cell suspension was strained through a Cell Strainer into a pre-labeled 50 mL polypropylene conical tube. An aliquot of the suspension was used to prepare the Comet slides.
Preparation of Slides
From each liver and stomach suspension, an aliquot of 2.5 µL (liver) or 7.5 µL (stomach) was mixed with 75 µL (0.5%) of low melting agarose (0.5%). The cell/agarose suspension was applied to microscope slides commercially available pre-treated. The slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify. At least two Trevigen, Inc 3-well slides were prepared per animal per tissue. Two wells were used in scoring and the other wells were designated as a backup. Following solidification of the agarose, the slides were placed in jars containing lysis solution.
Lysis
Following solidification of agarose, the slides were submerged in a commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution at least overnight at 2-8oC.
Unwinding
After cell lysis, slides/wells 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 2-10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 200-proof ethanol for at least 5 minutes, then air dried for at least 4 hours 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).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Initial Dose Range Finding Assay

Clinical signs are presented inTable 1. Mean group body weight data are found inTable 2.

Mortality was observed at the 1000 and 2000 mg/kg/day dose levels.

The following clinical signs were observed:

Dose Level (mg/kg)	Males	Females
500	Piloerection	Piloerection
1000	Piloerection, Lethargy, Prostration	Piloerection, Lethargy
2000	None	None

Due to mortality occurring in the middle and high dose groups, a repeat DRF was conducted.

Repeat Dose Range Finding Assay

Clinical signs are presented inTable 3. Mean group body weight data are found inTable 4.

No mortality occurred at any dose level during the course of the dose range finding assay. No appreciable reductions in mean group body weights were seen in the test substance treated groups during the course of the study.

All rats appeared normal throughout the observation period. Due to the mortality and observations seen during the dose range finding assay, the maximum tolerated dose for the definitive Micronucleus - Comet assay was set at 500 mg/kg/day.

Micronucleus - Comet Assay

Clinical signs are presented inTable 5. Mean group body weight data are found inTable 6.

No mortality occurred at any dose level during the course of the definitive assay. No appreciable reductions in mean group body weights were seen in the test substance treated groups during the course of the study.

All rats appeared normal throughout the observation period.

Micronucleus Assay

The incidence of MnPCEs per 24,000 PCEs scored (4000 PCEs/animal) and the proportion of polychromatic erythrocytes per total erythrocytes are summarized and presented for each treatment group by sacrifice time inTable 7. Individual animal data are presented inTable 8.

The scoring results and a statistical analysis of data indicated the following:

·        No appreciable reductions in the PCEs/EC ratio was observed in the test substance groups compared to the vehicle control group, indicating the test substance did not induce cytotoxicity.

·        Group variances for mean of means of the micronucleus frequency in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

·        No statistically significant increase in the incidence of MnPCEs was observed in the test substance treated groups relative to the vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05). 

·        The positive control, CP, induced a statistically significant increase in the incidence of MnPCEs (20,000 PCEs scored, 4000 PCEs/animal; Student’s t‑test, p ≤ 0.05). 

·        The number of MnPCEs in the vehicle control groups did not exceed the historical control range (Appendix I).

Based upon this, all criteria for a valid test were met as specified in the protocol.

Comet Assay

The % Tail DNAissummarized for each treatment group and presented inTable 9(liver cells) andTable 11(stomach cells). Median valuesfor the % Tail DNA,Tail moment and Tail migration (µm) for were calculated per 150 cells for each animal and are presented inTable 10(liver cells) and inTable 12(stomach cells).

The scoring results and a statistical analysis of data indicated the following:

Liver

·        The presence of ‘clouds’[ML1] in the test substance groups was ≤ 1.8%, which was higher than the % of clouds in the vehicle control group (0.5%).

·        Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

·         

·         

·        The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the vehicle control groups (Student’s t‑test, p ≤ 0.05). 

·        In the vehicle control group, % Tail DNA was within the historical vehicle control range for the liver (Appendix I). 

These results indicate that all criteria for a valid test, as specified in the protocol, were met.

Stomach

·        The presence of‘clouds’[ML2] in the test substance groups was ≤ 45.3%, which was higher than the % of clouds in the vehicle control group (36.5%).

·        Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

·         

·         

·        The positive control, EMS, induced a statistically significant increase in the % Tail DNA in stomach cells as compared to the vehicle control groups (Student’s t‑test, p ≤ 0.05). 

·        In the vehicle control group, % Tail DNA was slightly higher than the 95% confidence interval for the stomach but was within the historical vehicle control range for this tissue (Appendix I).   

These results indicate that all criteria for a valid test, as specified in the protocol, were met.

The Common Technical Document (CTD) Summary Table is included inAppendix V.

Dosing Formulation Analysis

Dosing formulations were sent to the analytical chemistry laboratory at BioReliance foranalysis. A copy of the draft analytical report is included inAppendix III. The results of the analysis indicate that the actual mean concentrations of the analyzed formulation samples (l2.5, 25 and 50 mg/mL) were 111.9, 126.3 and 120.3% of target, respectively,withS/L ratios of > 0.925. The 25 and 50 mg/mL formulations were found to be above the acceptable range for concentration (85.0 to 115.0% of target) but metthe S/L ratio of > 0.925. This indicates that the formulations were accurately prepared, except as indicated above. The actual concentrations of the mid and high dose formulations were higher than the protocol-specified acceptance criterion, indicating that the test guideline recommended limit dose was exceeded. The overall results of the assay were concluded to be negative. Therefore, the Study Director has concluded that the difference in nominal concentration at the mid and high doses had no adverse impact on the study results or validity of the study conclusion. No test article was detected in the vehicle control sample. Additionally, Heloxy Modifier AQ in Corn Oil, at concentrations of 14.0 and 60.2 mg/mL, was stable at room temperature for at least 3 hours.

Bioanalysis

Plasma samples were sent to the analytical chemistry laboratory at BioReliance for analysis. A copy of the draft bioanalysis report is included inAppendix IV. All plasma samples were received for analysis of Heloxy Modifier AQ concentration in plasma by LC/MS/MS and were successfully analyzed. All matrix standards and QC samples met acceptance criteria. Measurable concentrations of Heloxy Modifier AQ were detected in the plasma samples ofGroup 1[ML3] from the initial DRF, Groups 4-6 from the repeat DRF and Groups 2-4 from the definitive assay.

 [ML1]Need a definition for this term here and in the tables where it is used.

 [ML2]Need a definition somewhere in the document

 [ML3]Add nominal dose levels for each group

Applicant's summary and conclusion

Conclusions:

Under the conditions of the assay described in this report, Heloxy Modifier AQ was considered to be negative for the induction micronucleated polychromatic erythrocytes in rat bone marrow or of DNA damage in liver and stomach cells.

Executive summary:

The test substance,Heloxy Modifier AQ, was evaluated for its genotoxic potential in bone marrow of male rats and in the Comet assay to induce DNA damage in liver and stomach cells. Corn oil was selected as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg/day by oral gavage once per day for three consecutive days. 

In the initial dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg/day. The dose levels tested were 500, 1000, and 2000 mg/kg/day in 3 animals/sex. Due to mortality occurring in the mid and high dose groups, a repeat DRF assay was conducted.

In the repeat DRF assay, the maximum dose level tested was 500 mg/kg/day. The dose levels tested were 125, 250, and 500 mg/kg/day in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 500 mg/kg/day, which was estimated to be the maximum tolerated dose (MTD). 

The definitive assay dose levels tested were 125, 250, and 500 mg/kg/day. Since nosignificant differences[ML1] in male and female rats were seen in the dose rage finder portion, only male rats were used in the definitive comet/micronucleus assay.

In the Micronucleus assay,no statistically significant increase in the incidence ofMnPCEs[ML2] was observed in the test substance treated groups relative to the vehicle control groups. Thus, the test substance was negative (non-clastogenic). The positive control induced a statistically significant increase in the incidence of MnPCEs. The number of MnPCEs in the vehicle control groups did not exceed the historical control range. 

In the Comet assay, the test substance gave a negative (non-DNA damaging) response in liver and stomach cells for males in % Tail DNA. None of the test substance treated animal slides had significant increases in the % Tail DNA compared to the respective vehicle controls. For the liver, the vehicle control % Tail DNA was within the Testing Facility’s historical range.For the stomach, the vehicle control % Tail DNA was slightly higher than the Testing Facility’s historical range.[ML3] In both the stomach and liver, the positive control had a statistically significant increase in % Tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met.

Under the conditions of this study, the administration ofHeloxy Modifier AQ, at doses up to and including a dose of 500 mg/kg/day,did not induce a significant increase in the incidence of MnPCEs relative to the concurrent vehicle control or induce a significant increase in DNA damage inliver and stomach cells. Therefore,Heloxy Modifier AQwas considered to be negative in thein vivoMicronucleus and Comet assays.

 [ML1]Identify endpoints used for this statement

 [ML2]Define

 [ML3]Change the order to match the order of the statements above – 1) Stomach, 2) Liver