Registration Dossier

Administrative data

in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31st January 2019 - 4th April 2019
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Information as requested in ECHA Decision TPE-D-2114360326-52-01/F.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
other: OECD 489 In vivo mammalian alkaline comet assay
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Specific details on test material used for the study:
Identification: 2,2-dimethylpropane-1,3-diyl cyclohex-4-ene-1,2-dicarboxylate
Batch/Lot No.: Colkb03
CAS No.: 41026-17-9
Purity: 92% (per substance identification report)
Molecular Weight: 408.44 g/mol
Description: Clear pale whitish yellow solid

Test animals

Details on species / strain selection:
Sprague-Dawley (Hsd:SD) rats were received from Envigo RMS, Inc., Frederick, MD on 08 February.

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.
Details on test animals and environmental conditions:
- Source: Envigo RMS Inc Frederick MD
- Age at study initiation: 6 weeks
- Weight at study initiation: 178.4 – 191.9 g
- Assigned to test groups randomly: yes. . A randomization function within Provantis™ (Instem) was used to achieve random placement of animals throughout all groups. Following randomization, animals were identified by sequentially numbered ear tags.

- Housing and Environmental Conditions: 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 temporarily 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. Animals were provided with environmental enrichment (such as Nylabones®)

- Diet (e.g. ad libitum) and Water (e.g. ad libitum): 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. Based on historical test results, there are no contaminants in the bedding, feed and water that are expected to interfere with the study.

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

8th February to 4th April 2019

Administration / exposure

Route of administration:
oral: gavage
The vehicle used to deliver the test substance to the test system was propylene glycol CAS #57-55-6. This was supplied by Sigma-Aldrich, lot # MKCC5403 which has a retest/expiry date of 31st.December 2020
Details on exposure:
Preparation of Control Article
The neat EMS was prepared in 0.9% saline. The dosing formulation was prepared at a concentration of 20.0 mg/mL, just prior to use.
Preparation of Test Substance Dose Formulations
The test substance dose formulations were prepared at least once on each day of use. A correction factor of 1.087 was used for dose formulations. Each concentration was prepared by calibrating a suitable size amber vial to appropriate volume with a PTFE stir bar to the target batch size that holds the pre-weighted amount of test substance. The appropriate test substance vial with the pre-weighed amount was selected and approximately (~) 70% of the total volume of the vehicle was added into the vial then stirred magnetically. The formulation was stirred magnetically for an appropriate amount of time. The formulation needed additional manipulations to achieve a soluble formation. The formulation was sonicated, homogenized and stirred for an appropriate amount of time until homogenous in appearance. The final dose formulation was stored at room temperature. The formulations were stirred prior to and during dosing.
All dose formulations were administered at a volume of 10 mL/kg/dose by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). This route of administration has been routinely used and is widely accepted for use in the mammalian alkaline Comet assay.
Duration of treatment / exposure:
Comet Assay
Animals initially were treated with the test substance or control articles and euthanized at the appropriate time as described below.
Group Treatment Dose Level (mg/kg/dose) Dose VolumeA (mL/kg/dose) Male Animals Euthanasia Time (Hrs) after Final Treatment
1 Vehicle 0 10 6 3-4
2 TS 500 10 6 3-4
3 TS 1000 10 6 3-4
4 TS 2000 10 6 3-4
5 EMS 200 10 3 3-4
ABased upon individual body weight
The outline of the study was as follows for the Comet assay:
• All animals in Groups 1 through 4 and 6 through 9 were dosed once per day on two consecutive days (Days 1 and 2) with the vehicle and with test substance. The second dose occurred approximately 21 hours after the first dose.
• Animals in Group 5 were dosed with the positive control (EMS) on day 1, and again on day 2 approximately 3 to 4 hours prior to euthanasia.
Frequency of treatment:
Daily for two days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
other: EMS 3 males treated at 200 mg/kg/d


Tissues and cell types examined:
All animals were euthanized 3 to 4 hours after the last dose (Day 2) by CO2 inhalation, and then, the following was performed:
• Animals were dissected and the liver, stomach and duodenum were extracted (removed) and collected.
• A section of the liver, stomach and duodenum were cut and placed in formalin for possible histopathology analysis.
• Another section of the liver, stomach and duodenum were placed in chilled mincing solution (Hanks’ balanced salt solution with EDTA and DMSO) and was used in preparation of cell suspensions and Comet multi-well slides.
Details of tissue and slide preparation:
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, stomach and duodenum cell suspensions were placed on wet-ice. An aliquot of the suspensions were used to prepare the comet multi-well slides.

Preparation of Multi-well slides
From each liver suspension an aliquot of 2.5 µL and for each stomach and duodenum suspension an aliquot of 7.5 µL was mixed with 75 µL (0.5%) of low melting agarose. The cell/agrose suspension was applied to microscope multi-well slides commercially available pre-treated multi-well slides. Commercially purchased Trevigen® multi-well slides were used and these slides have 20 individual circular areas, referred to as wells in the text below. The multi-well slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify. Multi-well slides were identified with a random code that reflects the study number, group, animal number, and organ/tissue. Three wells were used in scoring and the other wells were designated as a backup. Following solidification of agarose, the multi-well slides were placed in jars containing lysis solution.
Following solidification of agarose, the multi-well slides were submerged in a commercially available lysis solution supplemented with 10% DMSO on the day of use. The multi-well slides were kept in this solution at least overnight at 2-8oC.
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 multi-well slides remained in the buffer for approximately 20 minutes at 2-10C and protected from light, allowing DNA to unwind.
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 multi-well slides.
After completion of electrophoresis, the multi-well slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The multi-well 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.
Multi-well 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).
Evaluation criteria:
Three wells per organ/animal were used. Fifty randomly selected, non-overlapping cells per slide/well were scored resulting in a total of 150 cells, when possible, evaluated per animal for DNA damage using the fully validated automated scoring system Comet Assay IV from Perceptive Instruments Ltd. (UK). If 150 cells are not available, then the calculations were performed using the number of scorable cells.

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 multi-well slides scored, dividing by the total number of cells scored and multiplying by 100). Every effort was made to score at least 150 cells; otherwise, the total number of scorable cells was used for calculations. 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 median value of at least 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).
• 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.

Results and discussion

Test results
Vehicle controls validity:
Positive controls validity:

Applicant's summary and conclusion

Under the conditions of this study, the administration of EC 255-180-5 at doses up to and including a dose of 2000 mg/kg/dose did not cause a biologically significant increase in DNA damage in liver, stomach and duodenum relative to the concurrent vehicle control. Therefore, the substance was concluded to be negative (non-DNA damaging) in the in vivo Comet Assay.
Executive summary:

The test substance, 2,2-dimethylpropane-1,3-diyl cyclohex-4-ene-1,2-dicarboxylate also known as NPG-THPA Hemiester (TK 12094), was evaluated for its genotoxic potential in the Comet assay to induce DNA damage in liver, stomach, and duodenum cells of male rats. Propylene glycol was selected as the vehicle. Test and/or control article formulations were administered at a dose volume of 10 mL/kg/dose by oral gavage.

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

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

None of the test substance treated animal multi-well slides had significant increases in the % Tail DNA compared to the respective vehicle controls for duodenum or liver. There was a statistically significant increased observed in the high dose for stomach. A dose response was also observed. However, all individual animal % Tail DNA values and the group mean % Tail DNA value are within the historical control range, thus the increase was considered to be not biologically relevant. The vehicle controls % Tail DNA was within the Testing Facility’s historical range, and the positive controls had statistically significant increases in % Tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver, stomach and duodenum.

Under the conditions of this study, the administration of NPG-THPA Hemiester (TK 12094) at doses up to and including a dose of 2000 mg/kg/dose did not cause a biologically significant increase in DNA damage in liver, stomach and duodenum relative to the concurrent vehicle control. Therefore, NPG-THPA Hemiester (TK 12094) was concluded to be negative (non-DNA damaging) in the in vivo Comet Assay.