Reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis ((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-hydroxymethyl butane

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

In vivo Mammalian Alkaline Comet Assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 13 July 2016 and Experiemental Completion date: 06 February 2017.

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

Updated guidance adopted 26 September 2014.

Deviations:

yes

Remarks:

please see Principles of method if other than guideline below.

Principles of method if other than guideline:

The following deviations from the protocol occurred during the conduct of this study:
Event No. 251943: In the Dose Range Finding Assay, the time elapsed between first dosing of animals until the second dosing of animals was between 24 and 25 hours for all test article concentrations. Protocol requires the second dose be approximately 21 hours following the first dose. Group 1 was dosed from 1245 to 1247 on study day 1 and from 1314 to 1317 on study day 2. Group 2 was dosed from 1249 to1251 on study day 1 and from 1319 to 1322 on study day 2. Group 3 was dosed from 1253 to 1255 on study day 1 and from 1324 to 1327 on study day 2. This is a deviation from the protocol. The Study Director has determined that there was no impact on the assay outcome or validity. Because the range finding portion of the assay assesses mortality and clinical signs for the selection of dose levels in the definitive assay. Both parameters were able to be assessed in the selection of dose levels for the definitive assay.

Event No. 252964: During electrophoresis, the buffer temperature at the end of electrophoresis was outside of the protocol range of 2 to 10 ºC. Electrophoresis Run #2 temperature was 11.1ºC. This is a deviation from the protocol. The Study Director has determined that the results for the negative and positive controls were within the expected range and all the criteria for a valid assay were met. Thus having slightly out of range buffer temperature had no effect on the study outcome.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vivo Mammalian Alkaline Comet Assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis ((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-hydroxymethyl butane

EC Number:

701-135-4

Cas Number:

30499-70-8

Molecular formula:

C15H26O6;C15H22O5

IUPAC Name:

Reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis ((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-hydroxymethyl butane

Test material form:

other: Liquid

Details on test material:

Intended use/Application: resin for coating systems

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Justification for the Test System
This species has been routinely used as an animal model of choice for the mammalian alkaline Comet assay. This strain was an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to the test substance.
Sprague-Dawley (Hsd:SD) rats were used.

Sex:

male

Details on test animals or test system and environmental conditions:

Sprague-Dawley (Hsd:SD) rats were received from Envigo RMS, Inc., Frederick, MD on 13 July 2016 (DRF) and 27 July 2016 (definitive assay).
The age at time of initiation, as well as the body weights and days of acclimation of the rats assigned to the study groups at randomization are indicated below:

Dose Range Finding: Male (between 167.2-181.4); Age at initiation:6 weeks; days of acclimation: 6.
Dose Range Finding: Female (between 133.7-151.7); Age at initiation:6 weeks; days of acclimation: 6
Definitive Test: Male (between 169.6-187.5); Age at initiation:6 weeks; days of acclimation: 7

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 three 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.

Randomization and Animal Identification
Animals were assigned to groups using a randomization procedure. At the time of randomization, the weight variation per sex of all animals assigned to the study did not exceed ±20% of the mean weight. A randomization function within Excel™ was used to achieve random placement of animals throughout all groups. Following randomization, animals were identified by sequentially numbered ear tags. The cage card contained, at least, the animal number(s), sex, study number, treatment group number, dose level, test substance ID and route of administration. Cage cards were color coded by treatment group. Raw data records and specimens were also identified by the unique animal number.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

The vehicle used to deliver the test item to the test system was Polyethyene glycol 400 (PEG 400). The following reagents were used in preparation of the vehicle:
Component: PEG 400
CAS N°: 25322-68-3
Supplier/Source: Sigma Aldrich
Lot/Batch N°: BCBF6930V
Expiration/Retest Date: 28 Feb 2018.

Details on exposure:

Dose Administration
All dose formulations were administered at a volume of 10 mL/kg/day by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The route has been routinely used and is widely-accepted for use in the comet assay.

Preparation of Test Substance Dose Formulations
The test substance dose formulations were prepared fresh on each day of use. Each concentration was prepared by calibrating a suitable size amber vial with a PTFE stir bar to the target batch size. An appropriate amount of the test substance was transferred into the calibrated container and then the approximately 70% of the vehicle was added and stirred with the PTFE stir bar. The formulation was QS to the final volume and mixed (stirred) magnetically until homogenous in appearance. The final dose formulation was stored at room temperature.
Residual dose formulations were discarded after use.

Duration of treatment / exposure:

Two dose administrations (Days 1 and 2) Second dose ~ 21 hours after the second dose..

Frequency of treatment:

one dose per day during 2 days and the second dose occured approximately 21 hours after the first dose.,

Post exposure period:

not applicable

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Dose for the Dose Range Finder (DRF) and dose also used in the Comet Assay. The Dose volume in mL/kg/day: 10

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Dose for the Dose Range Finder (DRF) and dose also used in the Comet Assay. The Dose volume in mL/kg/day: 10

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Dose for the Dose Range Finder (DRF) and dose also used in the Comet Assay. The Dose volume in mL/kg/day: 10

No. of animals per sex per dose:

- In the Dose Rnage Finding: 3 animals/sex were exposed to 2000/1000 and 500 mg/kg/day.
- In the Comet Assay: 6 animals (Male) were exposed to 500 /1000 and 2000 mg/kg/day.

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control used was ethyl methanesulfonate (EMS).
The neat EMS was prepared in 0.9% saline. The dosing concentration of 20 mg/mL, just prior to use.
EMS at dose level of 200 mg/kg/day and at dose volume 10 mL/kg/day by Oral gavage was used. 3 animals were used.
The positive control (EMS) animals were dosed once approximately 3 to 4 hours prior to euthanasia on day 2.

Examinations

Tissues and cell types examined:

All animals were euthanized 3 to 4 hours after the last dose (day 2) by CO2 asphyxiation, and thenthe following was performed:
- Animal were dissected and the liver, stomach and duodenum were extracted (removed) and collected.
- A section of the liver, stomach and duodenum was cut and placed in formalin for possible histopathology analysis.
- Another section of the liver, glandular stomach and duodenum was placed in chilled mincing solution (Hank's 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 Cell Suspensions and Comet Slides
A portion of each dissected liver was placed in 3 mL of 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 glandular stomach and duodenum were placed in 1 mL of cold mincing buffer, then the glandular stomach and duodenum were 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 and the resulting liver, glandular stomach and duodenum cell suspensions were placed on wet-ice. An aliquot of the suspensions were used to prepare the comet slides.

Preparation of Slides
From each liver, glandular stomach, and duodenum suspension, an aliquot of 2.5 μL, 7.5 μL and 7.5 μL, respectively, was mixed with 75 μL (0.5%) of low melting agarose. The cell/agrose suspension was applied to microscope slides commercially available pre-treated multi-well slides. Commercially purchased multi-well slides were used and these slides have 3 individual circular areas, referred to as wells in the text below. The slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify. At least four Trevigen, Inc. 3-well slides were prepared per animal per tissue. Three slides/wells were used in scoring and the other wells were designated as a backup. Following solidification of 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 2-8°C.

Unwinding
After cell lysis, slides/wells were washed with neutralization buffer (0.4M 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 (see deviations) and protected from light. The electrophoresis time was constant for all slides.

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 μL of Sybr-gold stain in 15 mL of 1xTBE (tris-boric acid EDTA buffer solution).

Evaluation criteria:

Evaluation of DNA Damage
Three slides/wells per organ/animal were used. Fifty randomly selected, non-overlapping cells per slide/well were scored resulting in a total of 150 cells evaluated per animal for DNA damage using the fully validated automated scoring system Comet Assay IV from Perceptive Instruments Ltd. (UK).

The following endpoints of DNA damage were assessed and measured:
• 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; Olive et al. 1990)].

Each slide was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was determined by scanning 150 cells per animal, when possible (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). 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 (Collins et. al., 2004). “Clouds” with visible gaps between the nuclei and the comet tail were excluded from comet image analysis.

The comet slides, which are not permanent (the slides can be affected/damaged by environmental storage conditions), were discarded prior to report finalization.

Statistics:

The median value of 150 counts of % Tail DNA, Tail moment and Tail migration were determined and presented for each animal in each treatment group for each organ. The mean and standard deviation of the median values only for % Tail DNA were presented for each treatment group. Statistical analysis was performed only for % Tail DNA.
In order to quantify the test substance effects on DNA damage, the following statistical analysis was performed:

• The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for % tail DNA generated for the vehicle and test substance groups were compared using Levene’s test (significant level of p ≤ 0.05). If the differences and variations between groups were found not to be significant, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p < 0.05). If Levene’s test indicated heterogeneous group variances (p ≤ 0.05), the suitability of a transformation of the original data was evaluated (e.g. using logarithm transformed values of the original data) in an attempt to meet the normality criteria. Afterwards, statistical analysis was performed using the parametric tests described above. If parametric tests were not acceptable, non-parametric statistical methods, (Kruskal Wallis and/or Mann Whitney test) may have been used in evaluation of data.

• A linear regression analysis was conducted to assess dose responsiveness in the test substance treated groups (p ≤ 0.01).

• A pair-wise comparison (Student’s T-test, p ≤ 0.05) was used to compare the positive control group to the concurrent vehicle control group. If needed, non-parametric statistical methods (Kruskal Wallis and/or Mann Whitney test) may be used in evaluation of data.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

valid

Remarks:

Polyethylene glycol 400 (PEG 400)

Positive controls validity:

valid

Remarks:

Ethyl methane sulfonate (EMS)

Remarks on result:

other: Please see Remarks field.

Remarks:

A dose responsive, statistically significant increase in % tail DNA was observed in the 1000 and 2000 mg/kg/day dose of the liver samples over the vehicle control. The 2000 mg/kg/day increase was outside our current historical control and 95% confidence range. A dose responsive, statistically significant increase in % tail DNA was observed in the 2000 mg/kg/day dose of the duodenal samples over the vehicle control. No increases in % tail DNA were observed in the stomach samples. The vehicle and positive control % tail DNA values were within expected ranges for all three organs. All valid assay criteria were met.

Additional information on results:

Dose Range Finder (DRF):
No significant bodyweight loss was observed. Piloerection was observed in male and female animals at all dose levels. A crusty nose was observed in a male animal at the 1000 mg/kg/day dose level only. Following the last observation, animals were euthanized by exposure to CO2 and discarded without further examination. No mortality or significant differences in clinical observations were seen between the sexes, therefore only males were used in the definitive assay.

The following clinical signs were observed:
- At 500 mg/kg/day; Piloerection for Males and females
- At 1000 mg/kg/day: Piloerection, rusty nose in males and piloerection in females.
- At 2000 mg/kg /day: Piloerection in males and piloerection in females.

The high dose for the Comet assay was the limit dose of 2000 mg/kg/day as determined in the DRF assay. Two additional doses were evaluated, generally at one-half and one-fourth of the top dose.
No mortality or significant clinical observations were seen during the dose range finding assay, the maximum tolerated dose for the definitive comet assay was set at 2000 mg/kg.

Definitive Assay:
- Mortality was observed at the following concentration in the definitive assay:
Vehicle:PEG 400 1/6 male died

No mortality occurred at any test article dose levels or in the positive control group during the course of the definitive assay. Appreciable reductions in mean group body weights were seen in the high test substance treated group during the course of the study.
The following clinical signs were observed in males rats (only male used in the definitive assay):
- PEG 400 (vehicle): Normal.
- At 500 mg/kg/day: Normal
- At 1000 mg/kg/day: Piloerection, Diarrhea
- At 2000 mg/kg/day: Lethargy, Piloerection, Crrusty nose, Diarrhea
- Positive Control: EMS (200 mg/kg): Normal.

Any other information on results incl. tables

Comet Assay

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

• The presence of ‘clouds’ in the test substance groups was ≤ 2.8%, which was lower than the % of clouds in the vehicle control group (4.0%).

• 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 significant difference in the group variance (p < 0.05); therefore, the suitability of a transformation of the original data was evaluated (e.g. using logarithm transformed values of the original data) in an attempt to meet the normality criteria. Afterwards, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

• Statistically significant responses in the % Tail DNA (DNA damage) was observed in the 1000 and 2000 mg/kg/day test substance groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p < 0.05).

• A dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p < 0.01).

• 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.

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

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

• The presence of ‘clouds’ in the low and mid test substance groups was ≤ 10.5%, which was higher than the vehicle control group (9.4%) and the high test substance group was 7.3% which was lower than the % of clouds in the vehicle control group (9.4%).

• 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.

• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in glandular stomach cells as compared to the vehicle control groups (Mann-Whitney test, p ≤ 0.05).

• In the vehicle control group, % Tail DNA was within the historical vehicle control range for the glandular stomach.

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

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

• The presence of ‘clouds’ in the test substance groups was ≤ 28.2%, which was lower than the % of clouds in the vehicle control group (48.0%).

• 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.

• Statistically significant response in the % Tail DNA (DNA damage) was observed in the 2000 mg/kg/day test substance groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p < 0.05).

• A dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p < 0.01).

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in duodenal 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 duodenum (Appendix I).

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

Dosing Formulation Analysis

Dosing formulations were analyzed by the analytical laboratory at BioReliance using the validated method AD90MA.GTCHEM.BTL. A copy of the analytical report is included in Appendix IV. The results of the analysis indicate that the actual mean concentrations of the analyzed formulation samples were between 98.2 and 134.0% of their respective target concentrations with S/L ratios of > 0.925. The 200 mg/mL formulation was found to be above the acceptable range for concentration (85.0 to 115.0% of target) but met the S/L ratio of > 0.925. This indicates that the formulations were accurately prepared, except as indicated above.

No test substance was detected in the vehicle control sample. Additionally, TK30174 in PEG 400, at concentration of 49.1 mg/mL, was stable at room temperature for at least 5.17 hours. Also, TK30174 in PEG 400, at concentration of 199 mg/mL, was stable at room temperature for at least 4.10 hours. TK30174 in PEG 400, at a concentration of 196 mg/mL, was stable at room temperature for at least 5.04 hours.

Histopathology Analysis

Based on the histopathological evaluation of liver and duodenum in this study, there was no evidence of test article induced toxicity.

Male Rat Historical Control Data

2012 to 2015

Electrophoresis performed refrigerated (2 to 10ºC), protected from light

Organs harvested at ~3 hour post last dose

VEHICLE (NEGATIVE) CONTROL1

		Tail moment	Tail migration (microm)	% Tail DNA
Organ	Parameter	Males	Males	Males
Liver	Mean 3	0.073	11.42	0.34
Liver	Standard Deviation	0.17	10.18	0.74
Liver	Range 4	0.00012	0.00	0.00075
Liver	Range 4	1.29	53.04	5.84
Liver	95% Confidence 5	0.00	0.00	0.00
Liver	95% Confidence 5	0.41	31.77	1.83

POSITIVE CONTROL2

		Tail moment	Tail migration (microm)	% Tail DNA
Organ	Parameter	Males	Males	Males
Liver	Mean 3	4.25	42.00	21.45
Liver	Standard Deviation	2.26	10.53	9.17
Liver	Range 4	0.4991	17.00	2.430
Liver	Range 4	15.56	76.48	56.62
Liver	95% Confidence 5	0.00	20.94	3.10
Liver	95% Confidence 5	8.77	63.07	39.80

1. Negative control articles: all vehicles used; Route of administration: oral gavage (PO), intraperitoneal (IP), subcutaneous (SC), or intravenous (IV)

2. 2Positive control article: Ethyl methanesulfonate (200 mg/kg)

3. Average (mean) of the median Comet Assay parameters measured per aimal for the total number of animals used in studies during 2012 to 2015.

4. Minimum and Maximum range of median Comet Assay measurments.

5. 95% Confidence is calculated by the mean of the median Comet parameter +/- 2 standard deviations.

Male Rat Historical Control Data

2011 to 2015

Electrophoresis performed refrigerated (2 to 10ºC), protected from light

Organs harvested at ~3 hour post last dose

VEHICLE (NEGATIVE) CONTROL1

		Tail moment	Tail migration (microm)	% Tail DNA
Organ	Parameter	Males	Males	Males
Stomach	Mean 3	1.40	36.09	6.98
Stomach	Standard Deviation	0.90	10.78	3.62
Stomach	Range 4	0.23	12.40	0.94
Stomach	Range 4	4.31	62.60	16.88
Stomach	95% Confidence 5	0.00	14.53	0.00
Stomach	95% Confidence 5	3.20	57.66	14.23

POSITIVE CONTROL2

		Tail moment	Tail migration (microm)	% Tail DNA
Organ	Parameter	Males	Males	Males
Liver	Mean 3	7.04	51.23	31.79
Liver	Standard Deviation	2.79	9.88	9.47
Liver	Range 4	1.13	28.79	5.37
Liver	Range 4	15.42	76.97	56.12
Liver	95% Confidence 5	1.47	31.47	12.84
Liver	95% Confidence 5	12.62	71.00	50.74

1. Negative control articles: all vehicles used; Route of administration: oral gavage (PO), intraperitoneal (IP), subcutaneous (SC), or intravenous (IV)

2. 2Positive control article: Ethyl methanesulfonate (200 mg/kg)

3. Average (mean) of the median Comet Assay parameters measured per aimal for the total number of animals used in studies during 2012 to 2015.

4. Minimum and Maximum range of median Comet Assay measurments.

5. 95% Confidence is calculated by the mean of the median Comet parameter +/- 2 standard deviations.

Male Rat Historical Control Data

2008 to 2015

Electrophoresis performed refrigerated (2 to 10ºC), protected from light

Organs harvested at ~3 hour post last dose

VEHICLE (NEGATIVE) CONTROL1

		Tail moment	Tail migration (microm)	% Tail DNA
Organ	Parameter	Males	Males	Males
Duodenum	Mean 3	0.99	37.71	4.74
Duodenum	Standard Deviation	1.06	14.66	4.34
Duodenum	Range 4	0.018	6.43	0.11
Duodenum	Range 4	5.03	69.37	18.40
Duodenum	95% Confidence 5	0.00	8.40	0.00
Duodenum	95% Confidence 5	3.11	67.02	13.43

POSITIVE CONTROL2

		Tail moment	Tail migration (microm)	% Tail DNA
Organ	Parameter	Males	Males	Males
Duodenum	Mean 3	5.62	55.37	26.11
Duodenum	Standard Deviation	2.23	11.88	7.85
Duodenum	Range 4	1.29	28.48	8.80
Duodenum	Range 4	14.25	79.75	51.33
Duodenum	95% Confidence 5	1.15	31.62	10.42
Duodenum	95% Confidence 5	10.08	79.12	41.80

1. Negative control articles: all vehicles used; Route of administration: oral gavage (PO), intraperitoneal (IP), subcutaneous (SC), or intravenous (IV)

2. 2Positive control article: Ethyl methanesulfonate (200 mg/kg)

3. Average (mean) of the median Comet Assay parameters measured per aimal for the total number of animals used in studies during 2012 to 2015.

4. Minimum and Maximum range of median Comet Assay measurments.

5. 95% Confidence is calculated by the mean of the median Comet parameter +/- 2 standard deviations.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the assay described in this report, TK30174 was concluded to be positive for the induction of DNA damage in liver at doses ≥1000 mg/kg/day and duodenal cells at a dose of 2000 mg/kg/day. TK30174 was concluded to be negative for the induction of DNA damage in stomach cells at doses ≥2000 mg/kg/day .

Executive summary:

The test substance, TK30174, was evaluated for its genotoxic potential in the Comet assay to induce DNA damage in liver, stomach, and duodenum cells of male rats. Polyethylene glycol 400 (PEG 400) was selected as the vehicle. Test and/or control article formulations were administered at a dose volume of 10 mL/kg/day by oral gavage.

In the dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg/day. The other dose levels tested were 500 and 1000 mg/kg/day in 3 of animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/day, which is the highest guideline recommended dose for this assay, which was estimated to be the maximum tolerated dose (MTD).

The definitive assay dose levels tested were 500, 1000, and 2000 mg/kg/day.

150 liver, duodenal and stomach cells/animal were analysed.

A dose responsive, statistically significant increase in % tail DNA was observed in the 1000 and 2000 mg/kg/day dose of the liver samples over the vehicle control. The 2000 mg/kg/day increase was outside our current historical control and 95% confidence range. A dose responsive, statistically significant increase in % tail DNA was observed in the 2000 mg/kg/day dose of the duodenal samples over the vehicle control. No increases in % tail DNA were observed in the stomach samples. The vehicle and positive control % tail DNA values were within expected ranges for all three organs. All valid assay criteria were met.

Under the conditions of this study, the administration of TK30174 was concluded to be positive for the induction of DNA damage in liver at doses ≥1000 mg/kg/day and duodenal cells at a dose of 2000 mg/kg/day. TK30174 was concluded to be negative for the induction of DNA damage in stomach cells at doses ≥2000 mg/kg/day.

TK30174 at doses ≥1000 mg/kg/day did cause a significant increase in DNA damage in liver and duodenal cells relative to the concurrent vehicle control. Therefore, TK30174 was concluded to be positive in the in vivo Comet Assay.

	Dose Level Administred
	500 mg/kg/day	1000 mg/kg/day	2000 mg/kg/day
Liver		+	+
Stomach
Duodenum			+

+ = statistically significant effect observed in % tail DNA