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Genetic toxicity: in vivo

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Administrative data

in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference Type:
In vivo Comet assay - statistical analysis and power calculations of mice testicular cells
Merete Kjær Hansen, Anoop Kumar Sharma, Marianne Dybdahl, Julie Boberg, Murat Kulahci
Bibliographic source:
Mutation Research 774 (2014) 29–40

Materials and methods

Test guideline
according to guideline
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
not specified
The animal study was performed under conditions approved by The Danish Agency of Protection of Experimental Animals and the in-house Animal Welfare Committee.
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimethyl phosphate
EC Number:
EC Name:
Trimethyl phosphate
Cas Number:
Molecular formula:
trimethyl phosphate
Test material form:

Test animals

Details on test animals or test system and environmental conditions:
- Source: Taconic MB, DK-4623, Lille Skensved, Denmark
- Age at study initiation: 5 weeks old
- Weight at study initiation: mean 29.8 +/- 1.2 g
- Assigned to test groups randomly: [no/yes, under following basis: ] The mice were randomly divided into dose groups.
- Housing: individually
- Diet (e.g. ad libitum): free access to feed (Altromin 1324,Lage, Germany)
- Water (e.g. ad libitum): tap water acidified with citric acid, pH = 3.5 (to prevent growthof microorganisms)
- Acclimation period: one week

- Temperature (°C): 22 ± 1
- Humidity (%): 55 ± 5
- Air changes (per hr): 10 times/h
- Photoperiod (hrs dark / hrs light): 12 h/12h

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
Dosing suspensions were freshly prepared prior to each dosing occasion and given in a volume of 1 ml/100 g bw. All doses were placed on a magnetic stirrer until dosing. All animals were fasted overnight before the first dosing.
Duration of treatment / exposure:
2 days
Frequency of treatment:
once per day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate (EMS)
- Doses / concentrations: 300 mg/kg bw


Tissues and cell types examined:
right testicles
the DNA isolated from the testicular tissue origins from a mixture of different celltypes.
Details of tissue and slide preparation:
SAMPLING TIMES (in addition to information in specific fields): Two to four hours after the second dosing the animals were anaesthetized in CO2/O2 and decapitated. After macroscopic examination, the testicles were excised and weighed. After removing the capsule the right testicles were put in cryotubes and used later in the Comet assay. Freezing medium was not added because it was an organ that was frozen and not cell suspensions. The cryotubes were immediately frozen in Mr. Frosty for about 1 h and then transferred to −80°C freezer until analyzed in the Comet assay.

DETAILS OF SLIDE PREPARATION: The cryotubes with the testicules from each mouse were added with 1.5 ml ice cold mincing solution (Hank’s balanced salt solution (Ca2+, Mg2+free) with 20 mM EDTA and 10% dimethyl sulfoxide). The testicules were gently crushed 4–5 times with a pastil. The solution was filtered through a 100 µm nylon filter (BD Falcon, Sigma–Aldrich, Denmark). Then, the solution was centrifuged at 1200 rpm for 5 min at 4°C (Eppendorf Centrifuge 5810R, Buch & Holm, Herlev, Denmark). The supernatant was removed and the pellet was resuspended in 1.5 ml mincing solution. This solution was filtered through a 100 µm nylon filter. Three microlitres of this suspension was mixed with 150 µl of the molten CometAssay TM LM Agarose (Trevigen, Gaithersburg, Maryland, US).Thirty microlitres of this mixture was applied onto one sample area of two gels ontwo different slides (one gel on one slide) consisting of 20 gels (CometSlideTMHT,Trevigen, Gaithersburg, Maryland, US). After solidification the embedded cells werelysed werelysed in a cold alkaline lysis buffer for 60 min. For DNA unwinding the slides were placed in the alkaline electrophoresis solution (pH > 13) in the electrophoresis jarat 4°C for 40 min, and electrophoresis was run in the same buffer for 30 min at 4°C(1 V/cm and 270 mA). After neutralization, fixation in 96% ethanol and DNA staining with 10 µl SYBR Green on all gels of the slides and a drop of antifade solution was added to each gel to avoid fading. Negative (Caco-2 cells in culture medium) and positive (Caco-2 cells exposed to 200 and 400 µM ethyl methanesulfonate for 30 min) controls were included with each 20 gels slide for each electrophoresis run. Testicule samples from each mouse were analyzed in two gels on two different slides.

METHOD OF ANALYSIS: Fully automatic Comet assay scoring was performed using the PathfinderTM Cellscan Comet imaging system (IMSTAR, Paris, France). Tail intensity (% tail DNA) of each comet was used. The number of cells scored on the gels depended on the cell density of the gels. In this study 100 random cells were subsequently selected from each gel unless it was explicitly stated otherwise, hence for each mouse 200 cells were scored (100 cells per gel and two gels per mouse).
For each gel the following summary statistics were calcu-lated: the mean, median (50th), 55th, 60th, 65th, 70th, 75th, 80th, 85th, 90th and 95th percentile. The % tail DNA measured for each cell is naturally restricted to be non-negative and the distribution of the % tail DNA within each sample are strongly positively skewed. A popular endeavour to normalize the data and/or stabilize the variance of such data is to take the natural logarithm. Each of the statistics was thus calculated from the raw data as well as from data subjected to the natural logarithm. As some observations were recorded as zero, a small constant (0.001) was added todata when calculating the mean of the log-transformed data to avoid taken the logarithm of zero. Additionally, one summary statistic was calculated as the mean ofthe raw data and subsequently log-transformed. This measure will be referred to as the log(mean). In total, 23 candidate summary statistics were extracted. The summarized data was fitted using a linear mixed-effects model with dose as a fixed effect and animal as a random effect. The animals in one dose group are different from the animals in other dose groups, and this induces a nested structure in data.

Results and discussion

Test results
Key result
Vehicle controls validity:
Positive controls validity:

Applicant's summary and conclusion

Trimethyl phosphate induced a significant positive effect at 500 mg/kg bw in the present study.