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in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference Type:
Modified In Vivo Comet Assay Detects the Genotoxic Potential of 14-Hydroxycodeinone, an α,β-Unsaturated Ketone in Oxycodone
Pant K, Roden N, Zhang C, Bruce S, Wood C, and Pendino K
Bibliographic source:
Environmental and Molecular Mutagenesis 56: 777 - 787

Materials and methods

Test guidelineopen allclose all
equivalent or similar to guideline
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Modification of the standard method to identify cross-linking.
equivalent or similar to guideline
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Test animals

Details on test animals or test system and environmental conditions:
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: 27.8 - 34.2 g
- Diet: Certified laboratory rodent chow ad libitum
- Water: Tap water ad libitum
- Acclimation period: 5 - 6 days

- Temperature (°C): 22.2 ± 0.7 °C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle.

Administration / exposure

Route of administration:
oral: unspecified
- Vehicle/solvent used: Arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose volume was 10 mL/kg in arachis oil.
Duration of treatment / exposure:
Once daily for 3 consecutive days.
Frequency of treatment:
Once daily for 3 consecutive days.
Post exposure period:
Animals were euthanized following three days of dosing.
Doses / concentrationsopen allclose all
Dose / conc.:
80 mg/kg bw/day
Dose / conc.:
160 mg/kg bw/day
Dose / conc.:
320 mg/kg bw/day
320 mg/kg is the maximum tolerated dose.
Control animals:
yes, concurrent vehicle
Positive control(s):
Methyl methanesulfonate (MMS, the positive control for the standard in vivo comet assay)
Hexamethyl phosphoramide (HMPA, the positive control for the modified comet assay)

- Route of administration
MMS: P.o.
HMPA: P.o.
- Doses / concentrations
MMS: 40 mg/kg/day in 0.9 % saline as a single 10 mL/kg dose 15 minutes prior to tissue collection.
HMPA: 500, 1 000 and 2 000 mg/kg/day in 0.9 % saline, 20 mL/kg dose administered once daily for 3 consecutive days, with samples taken 3 - 4 hours after dosing.


Tissues and cell types examined:
Liver, stomach and duodenum were removed and collected for cell suspension preparation. These three organs were selected as they are the main site of contact and / or metabolism following oral administration (the clinical route of administration).
Small sections of the organs were retained for possible histopathology analysis, however since there were no gross lesions present upon visual inspection, histopathological examination was not performed.
Details of tissue and slide preparation:
A dose range finding study was conducted for each test chemical according to the OECD test guidelines. Doses for the main assays were selected based on the results of the dose range finding assays such that they included the Maximum Tolerated Dose and at least two additional lower and appropriately spaced dose levels for each sampling time. The two additional doses were evaluated at one-half and one-fourth of the top dose.

The animals were dosed once daily for 3 consecutive days. Samples were taken 3 - 4 h after dosing.

Sections of liver were placed in 3 mL cold mincing buffer (20 mM EDTA and 10 % DMSO) and minced with scissors to release the cells. Stomach and duodenum samples were placed in 1 mL cold mincing buffer, and then scraped with a plastic spatula to release the cells. Each cell suspension was strained through a 40 mm cell sieve into a conical tube and then used in preparation of comet slides. Slides were labelled with codes prior to preparation and only the study number and slide code was written on the slides for blind scoring and to remove any scorer’s bias.
At least four slides per animal were prepared per organ/tissue (2 for scoring and 2 as back-ups). From each liver, stomach and duodenum suspension, an aliquot of 5 mL (liver) or 10 – 15 mL (stomach and duodenum) were mixed with 75 mL low melting agarose (0.5 %). The cell/agarose suspension was applied to glass microscope slides previously coated with 1 % normal melting agarose. The slides were kept at 2–8 °C for at least 15 min to allow the gel to solidify.

Following solidification of agarose, the slides were placed in jars containing lysis solution. For the Modified Comet Assay, the remaining cell suspensions from vehicle control- and test article-treated mice were placed on ice and X-ray irradiated for 10 min at 105–110 kv (~10 Gy total). After irradiation, a second set of slides was prepared using these cells, which were then processed and scored in the same manner as described below.
Following solidification of agarose, the slides were submerged in a cold lysis solution [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] and kept in this solution at least overnight at 2–8 °C. After cell lysis, slides were washed with neutralization buffer (0.4 M tris-hydroxymethyl aminomethane in purified water, pH ~7.5) and placed in an electrophoresis chamber. The chamber reservoir was slowly filled with alkaline buffer (300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water, pH >13). All slides remained in the buffer for ~ 20 min at 2–10 °C and protected from light, allowing DNA to unwind. Using the same buffer, the slides were subjected to electrophoresis for 30 min at 0.7 V/cm, at 2–10 °C and protected from light. The electrophoresis time was the same for all slides. After electrophoresis, the slides were washed with neutralization buffer for at least 10 min. The slides (gels) were then dehydrated with 200-proof ethanol for at least 5 min, air dried, and stored at room temperature with desiccant.
Slides were stained with the DNA stain, Sybr-gold™, prior to scoring. The stain solution was prepared by diluting 1 µL Sybr-gold stain in 15 mL 13 tris-boric acid EDTA buffer solution. Two slides per animal per tissue were scored. Fifty randomly selected, non-overlapping cells per slide were scored so that a total of 100 cells were evaluated per animal. If one of the two slides did not contain 50 scorable cells, additional cells were scored using the backup slides. DNA damage was assessed by the image analysis software system [The comet Assay IV from Perceptive Instrument Ltd (Bury St Edmunds in Suffolk, UK)] and used to measure:
- 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 compared to the total DNA (head and tail DNA). This can be determined as the fluorescence intensity of the comet tail compared to the total comet intensity (tail and head) [OECD TG 489]. The % DNA in the tail is considered to be the most reliable parameter for quantifying comet assay results;
- 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 3 Tail Length)/100 Each slide was first examined to obtain information regarding the handling and condition of the tissue, according to the percentage of “clouds” in 200 cells per animal. The “clouds,” in which there are visible gaps between the nuclei and the comet tail, are a morphological indication of highly damaged cells often associated with severe genotoxicity, necrosis, or apoptosis.
The handling and condition of the organ tissues were comparable between all the treatment and control groups analysed in these studies, as indicated by the comparable percentage of “clouds” in cells from these groups.
Evaluation criteria:
See below
The mean value of 100 counts of % tail DNA, Olive Tail moment, and Tail migration were determined for each animal. The mean and standard deviation of the mean values for % tail DNA were presented for each treatment group with and without X-ray irradiation. The scoring results for % tail DNA were statistically analysed using the following statistical methods:
- The use of parametric or nonparametric statistical methods in evaluation of data was based on the variation between groups (the data was normally distributed). The group variances were compared using Levene’s test at the significance level of P ≤0.05. Since the differences between group variances were not found to be significant, a parametric one-way ANOVA was performed followed by a Dunnett’s post-hoc test for comparison of the test article groups with the concurrent vehicle controls (ANOVA, significance level of P ≤0.05).
- Linear regression analysis was used to determine the dose response relationship (one sided P<0.01).
- Pair-wise comparison (Student’s t-test, significance level of P ≤0.05) was used to compare the positive control data from the Standard Comet Assay against the vehicle control (without X-ray irradiation given that the positive control for the Standard Comet Assay was not irradiated) to determine and confirm acceptable criteria for a valid test.
All conclusions were based on sound scientific judgment. As a guide to interpretation of the data, the following points were considered (according to the OECD 489 TG [OECD, 2012]).

Results and discussion

Additional information on results:
Vehicle Control Values and the Effect of X-Ray Irradiation
The % tail DNA was lowest in liver cells from vehicle control animals and, as expected, the % tail DNA was highest in stomach cells, since this measurement was obtained from cells scraped from this organ (which is considered to be more damaging to cells than the mincing method used for liver).
The vehicle and positive control values for the stomach and liver were reproducible and within the historical ranges reported for this laboratory. The % tail DNA values in duodenal cells from vehicle control mice were also highly reproducible and were lower than for stomach cells but higher than that for liver cells in the Standard comet assay. All assays were valid according to the criteria in that the % tail DNA values for liver and stomach cells in the Standard comet assay were within the historical control range. For the duodenum, the % tail DNA was always below the pre-set limit of ≤ 20 % for a valid assay.
The different vehicles used for these studies were considered to have no effect on the % tail DNA values, with all values falling within the historical ranges. For the Standard comet assay, in all three organs, the % tail DNA was statistically significantly increased by the positive control, methyl methanesulfonate (MMS). The only exception to this was in one experiment in which the % tail DNA in stomach cells was increased; however, the increase did not reach statistical significance. In each experiment, the % tail DNA in cells from all three organs from vehicle control animals was increased by X-ray irradiation. The increases caused by irradiation were all statistically significant with the only exception being stomach cells in Experiment 4.
The increased level in stomach cells was not significantly lower than that in the other experiments; however, this finding could indicate that longer irradiation durations may be needed to ensure maximal DNA damage in this tissue. The mean % tail DNA values for all vehicle control animals (with different solvents, n = 23 animals in total) were statistically significantly increased from 1.96 ± 1.06 %, 9.97 ± 4.19 %, and 4.05 ± 2.12 % in non-irradiated cells to 12.32 ± 3.27 %; 18.18 ± 4.68 %; and 19.88 ± 7.22 % following irradiation, for liver, stomach, and duodenal cells, respectively. The increase in the % tail DNA confirmed that the X-ray irradiation had caused uniform and reproducible DNA strand breakage in the cells, such that any effects of cross-linkers may be detected as a decrease in this value.

Mitomycin C
The positive control in the Standard comet assay, MMS, caused a 10.2-, 2.0-, and 2.8-fold increase in the % tail DNA in cells isolated from the liver, stomach, and duodenum, respectively [statistically significant for liver and duodenum but not for stomach (which would invalidate the use of the stomach in this Standard comet assay but not this Modified comet assay experiment)]. In the Standard comet assay (without X-ray irradiation), there was no statistical difference in the % tail DNA in cells from duodenum or stomach for vehicle control or MMC-treated mice. MMC caused a statistically significant increase in the % tail DNA in liver cells, for which a trend was observed; however, the values were all within the historical control range for this tissue and therefore this finding was considered not to be biologically relevant.
In the modified portion of the comet assay in which the remaining cells were X-ray irradiated prior to preparing the slides, there was no statistical difference between vehicle control and MMC treated, X-ray irradiated, liver, stomach, and duodenum cells.
It was concluded that MMC was negative in liver, stomach, and duodenum cells in both the Standard and Modified comet assays.

Hexamethyl Phosphoramide (HMPA)
HMPA decreased DNA migration in the in vivo Modified comet assay. HMPA caused a significant and dose-dependent decrease in the migration of DNA from duodenal cells subjected to X-ray irradiation (17.26 ± 2.61 % in vehicle control mice and 6.49 ± 1.28 % in cells from mice treated with 2 000 mg/kg/day HMPA), indicating that HMPA is a cross-linking agent in vivo. The % tail DNA was not significantly different in X-ray irradiated cells from liver or stomach for vehicle control and HMPA-treated mice. HMPA may not be expected to affect the stomach because it is unlikely to have been metabolized to formaldehyde in the acid environment.
It was concluded that HMPA was negative in the Standard comet assay but caused a dose-dependent decrease in % tail DNA in duodenal cells in the Modified comet assay, indicating that this compound is a DNA crosslinking agent, in vivo, at least in duodenal cells. We therefore used HMPA in the Modified comet assay as a positive control for DNA-protein cross-linkers in the duodenum.

Evaluation of the test material
The test material was evaluated for its potential to elicit DNA crosslinking using MMS as a positive control for the Standard comet assay and HMPA as the positive control for the Modified assay. The positive control in the Standard comet assay, MMS, statistically increased the % tail DNA in cells isolated from the liver, stomach, and duodenum by 9.5-, 2.5-, and 6.9-fold, respectively. Although there was a statistically significant increase in the % tail DNA in cells from the stomach of mice treated with 80 mg/kg/day test material in the Standard comet assay, this value was within the historical range, and there was no dose-dependent effect. The % tail DNA in cells from liver and duodenum were the same in vehicle and test material-treated mice, indicating no DNA damage occurred under these conditions.
In the modified comet assay, the positive control, 2 000 mg/kg/day HMPA, caused a statistically significant decrease in the % tail DNA in X-ray irradiated duodenal cells compared to those from vehicle treated mice (from 27.60 ± 6.23 % in vehicle control mice to 5.76 ± 2.30% on HMPA-treated mice). This confirmed the results from the positive control identification studies and further qualifies HMPA as a positive control in the Modified comet assay. The test material also resulted in a dose-dependent and statistically significant decrease in the % tail DNA in irradiated cells from the duodenum (from 27.6 ± 6.23 % in vehicle control mice to 11.63 ± 8.9 % in cells from mice treated with 320 mg/kg/day test material). Similar decreases in % tail DNA were not observed for liver or stomach cells. The organ specific effect of the test material may be a result of a longer contact time in the small intestine (duodenum) compared to the stomach and liver. Importantly, the OECD 489 test guidance states that the duodenum and jejunum should also be considered as site-of contact tissues and that they “may be considered more relevant for humans than the rodent glandular stomach.” Thus, it was concluded that the test material is a DNA crosslinking agent in the Modified comet assay based on the statistically significant and dose-dependent decrease in the % tail DNA in irradiated cells from the duodenum.

Applicant's summary and conclusion

Although the test material was negative in the Standard comet assay, it was identified as a DNA cross-linking agent in the Modified comet assay.
Executive summary:

The test material has a structural alert for genotoxicity and was therefore tested in a combined Modified and Standard Comet Assay. To determine if the test material is a DNA cross-linker in vivo, a Modified Comet Assay was conducted using X-ray irradiation as the modification to visualize crosslinking activity. In this assay, the test material was administered orally to mice up to 320 mg/kg/day.


Results showed a statistically significant reduction in percent tail DNA in duodenal cells at 320 mg/kg/day, with a non-statistically significant but dose-related reduction in percent tail DNA also observed at the mid dose of 160 mg/kg/day. Similar decreases were not observed in cells from the liver or stomach, and no increases in percent tail DNA were noted for any tissue in the concomitantly conducted Standard Comet Assay. Taken together, the test material was identified as a cross-linking agent in the duodenum in the Modified Comet Assay.