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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diethylhydroxylamine
- Analytical purity: 86.605% aqueous solution
- Purity test date: 3rd June 2002
- Lot/batch No.: HS010101
- Expiration date of the lot/batch: June 2003

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, UK.
- Age at study initiation: 8 weeks
- Weight at study initiation: 198-238 g (males)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in groups of no more than four animals of the same sex, in solid-floored cages,
cleaned
- Diet (ad libitum): Special Diets Services Ltd, RM1.(E).SQC.
- Water (ad libitum): bottled water (public
supply)
- Acclimation period: 5/6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 52-91
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
purified water
Details on exposure:
- dose volume: 10 mL/kg.
Frequency of treatment:
single administration
Post exposure period:
2-4 hour or 12-14 hour
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 and 800 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Positive control(s):
2-Acetamidofluorene (2-AAF) for the 12-14 hour experiment and dimethylnitrosamine (DMN) for the 2-4 hour experiment.

Examinations

Tissues and cell types examined:
Hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Available information indicated that the LD50 value for  Diethylhydroxylamine was greater than 2000 mg/kg (the maximum recommended  dose for the UDS assay). Accordingly, a confirmatory range-finder study  was performed at 2000 mg/kg. Additionally, both male and female animals  were tested in the range-finder study to determine any substantial  inter-sex differences in toxicity.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
In the main study groups of four male rats were treated once with the  vehicle (purified water), Diethylhydroxylamine (at 800 mg/kg or 2000  mg/kg) or the required positive control, by oral gavage, at a dose volume  of 10 mL/kg. The positive controls used were 75 mg/kg 2-acetamidofluorene  (2-AAF) suspended in corn oil (12-14 hour experiment) and 10 mg/kg  dimethylnitrosamine (DMN) dissolved in purified water (2-4 hour  experiment).

DETAILS OF SLIDE PREPARATION:
- Killing of animals and preparation of hepatocyte cultures
For Experiments 1 and 2, animals were sacrificed a nominal 2-4 hours and 12-14 hours after dosing, respectively.
Individual animals were anaesthetized with Halothane and maintained under deep anaesthesia to prevent any likelihood of recovery. The liver was surgically exposed, the hepatic portal vein and superior vena cava cannulated with suitable cannulars and the liver perfused with suitable buffers. Approximately 400 mL of calcium free Buffer 1 was pumped at a flow rate of approximately 40 mL/min to wash the liver free of blood. The liver was then perfused with Buffer 2 also at a flow rate of 40 mL/min for approximately 5 minutes. Both buffers were gassed with 5% CO2 in air (v/v) prior to use and Buffer 2 throughout perfusion.
Calcium and collagenase (approximately 50 mg of collagenase dissolved in 1 mL of 769 mM CaCl2 and 10 mL of Buffer 2) was added to the reservoir and after one to two minutes the waste line was placed in the Buffer 2 reservoir so that the perfusate recirculates and the flow rate was reduced to 20 mL/min. When the reticular pattern of the liver had begun to break up and the liver became 'spongy', the perfusion was stopped. The liver was cut free into a suitable container with Buffer 2. The liver was transferred to a sterile dish, cut open and the hepatocytes carefully teased out. The resulting hepatocyte suspension was gently washed through 150 mm nylon mesh with Williams E medium-Complete (WE-C) to a volume of approximately 100 mL. Of this suspension, approximately 50 mL was taken and centrifuged at approximately 40 x 'g' for two to three minutes. The resultant pellet was resuspended in WE-C. The centrifugation and resuspension procedure was repeated at least twice and the pellet resuspended finally in approximately 20 mL WE-C. A sample (0.5 mL) of this suspension was taken, diluted with an equal volume of 0.4% (w/v) trypan blue in phosphate buffered saline (PBS) and the proportion of viable cells (those with unstained nuclei) determined using an haemocytometer. The culture was diluted where possible to provide approximately 1.5 x 105 viable cells/mL. Three mL of hepatocyte suspension was added to each well of a six-well multiplate containing 25 mm round plastic coverslips and incubated at 37°C ± 1°C in a 5% CO2 in air (v/v) atmosphere for at least 90 minutes to allow cells to attach.
- Radiolabelling of hepatocyte cultures
Medium was removed from the cells and the monolayers washed with 2 mL Williams E medium-Incomplete (WE-I) which was then replaced with 2 mL WE-I containing 10 mCi/mL [3H] thymidine. After 4 hours incubation at 37°C in a 5% CO2 in air (v/v) atmosphere, the medium was removed and the cells washed with three changes of WE-I containing 0.25 mM thymidine. Cultures were then incubated overnight with 3 mL of the same medium.
To prepare for autoradiography, coverslips were washed with 2 mL phosphate buffered saline (PBS) and the cells fixed with three changes of 2 mL glacial acetic acid:ethanol (1:3 v/v). The coverslips were then washed four times with purified water, allowed to dry and mounted onto previously labelled microscope slides, cells side up, with DPX.
- Autoradiography
Three of the six slides from each animal were coated in Ilford K2 liquid emulsion using a dipping technique. Each slide was dipped individually into the molten emulsion, ensuring that no air-bubbles were generated. After gelling over ice, for 10 minutes face upwards, the slides were incubated in a light-tight box at room temperature for approximately 90 minutes to let the emulsion dry. The slides were then packed in light-tight boxes containing desiccant, sealed with tape and refrigerated for 14 days. At the end of this time, the emulsion was developed in Kodak D19 developer and fixed using Ilford Hypam fixer. The cell nuclei and cytoplasm were then stained with Meyers haemalum/eosin Y. Slides were then dehydrated in ethanol, cleared in xylene and mounted with coverslips for microscopic examination. The spare, duplicate sets of slides were not required.
- Grain counting
Grain counting was performed using a microscope with a video camera connected to an image analysis system (Perceptive Instruments) and a computer programmed for automatic data capture.
Each slide was examined to ensure that the culture was viable. A patch of cells was selected as a starting point and cells were scored in a regular fashion by bringing new cells into the field of view, moving only in one axis. If the desired number of cells had not been scored before coming to the edge of the slide, the stage was moved one or two fields on the other axis and counting resumed. The circular field was centred over the nucleus of a suitable cell and the grains counted. The field of view was moved and counts obtained for three separate adjacent areas of cytoplasm. Nuclear and mean cytoplasmic grain counts were then recorded, and the net grains/nucleus (NNG) determined. 100 cells were analysed per animal, where possible using two of the three slides in each case.
Evaluation criteria:
The following criteria were used for cell analysis:
1. only cells with normal morphology were scored
2. isolated nuclei with no surrounding cytoplasm were not scored
3. cells without nuclear and/or cytoplasmic graining were not scored
4. cells with unusual staining artefacts were not scored
5. heavily labelled cells in S-phase were not scored
6. all other normal cells, 100 per animal were scored
7. all slides were analysed blind (coded).

Treatment of data
The following were calculated for each slide, animal and dose point:
1. the population average NNG and standard deviation (SD)
2. the percent of cells responding or in repair (ie ³ 5 NNG)
3. the population average cytoplasmic and nuclear grain count.

Acceptance criteria
The study would be considered valid if the negative control animals had a group mean NNG value that did not exceed the upper limit of the historical range. The positive control treatments should have group mean values of five or more NNG with 50% or more cells having NNG counts of five or greater.
Evaluation criteria
The test article would be considered as positive in this assay if, at any dose and at either time point:
1. the test article yielded group mean NNG values greater than 0 NNG and 20% or more of cells responding (mean NNG values >= 5)
2. an increase above solvent control levels was seen in both NNG and the percentage of cells in repair.
Cytoplasmic and nuclear grain count values as well as the concurrent negative control data would be considered in relation to the overall NNG values of cultures from treated animals.
If the test article failed to induce UDS at any dose tested after both 2-4 and 12-14 hours exposure, it would be considered clearly negative in this system.
Statistics:
None

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the range-finder study, groups of three male and three female out bred  Han Wistar (Crl:WI (Glx/BRL/Han) BR) rats were dosed once with up to 2000  mg/kg Diethylhydroxylamine. During a 2 day post-dose observation period  male animals dosed at 2000 mg/kg showed clinical signs of eye closure,  piloerection, lethargy and weight loss. Female animals dosed at 2000  mg/kg showed clinical signs of eye closure, piloerection, lethargy,  abnormal gait and being cold to the touch. However, due to the severity  of these signs the female animals were killed in extremis. Therefore, a  further three female animals were dosed at 1400 mg/kg and acceptable  clinical signs including piloerection, lethargy, abnormal gait, being  cold to the touch and weight loss were observed. Accordingly, as no  substantial inter-sex differences in toxicity were observed, male animals  only were tested in the main study at a maximum dose of 2000 mg/kg. A  lower dose level of 800 mg/kg (40% of the maximum dose) was also selected.

RESULTS OF DEFINITIVE STUDY
Clinical signs observed in the main study included piloerection and  lethargy (2-4 hour experiment, 2000 mg/kg dose group). In the 12-14 hour  experiment slight weight loss was observed (2000 mg/kg dose group). Approximately 2-4 hours (Experiment 1) or 12-14 hours (Experiment 2)  after dosing, animals were sacrificed and their livers perfused with  collagenase to provide a primary culture of hepatocytes. Cultures were  made from three animals in each dose group and were treated with [3H]  thymidine. Six slides from each animal were prepared with fixed  hepatocytes and of these, three were dipped in photographic emulsion to  prepare autoradiograms. Slides were examined microscopically after  development of the emulsion and staining, and the net grain count (NNG),  the number of grains present in the nucleus minus the mean number of  grains in three equivalent areas of cytoplasm, was determined for each of  two of the three slides, each animal and dose group. Negative (vehicle) control animals gave a group mean NNG value of less  than 0.2 with only 0 to 1% cells in repair. Group mean NNG values were  increased by 2-AAF and DMN treatment to 2.9 or more with more than 20%  cells found to be in repair. It may be noted that the Experiment 1  vehicle control NNG value was slightly higher than normal (0.2), however  this value is within the negative historical control range and therefore  acceptable. Additionally, although the positive control response for DMN  was lower than normal (falling outside of historical range), the response  was considered to be clearly indicative of UDS. Accordingly, in this  study the vehicle control NNG value was consistent with both published  and historical control data, and the system was shown to be sensitive to  two known DNA damaging agents requiring metabolism for their action. The  assay was therefore accepted as valid.  Treatment with 800 or 2000 mg/kg Diethylhydroxylamine did not produce a  group mean NNG value greater than zero nor were any cells found in repair  at either dose.

Applicant's summary and conclusion

Conclusions:
When treated once via oral gavage with Diethylhydroxylamine at doses up to 2000 mg/kg male Han Wistar rats showed no induction of UDS in hepatocytes isolated ex vivo approximately 2-4 or 12-14 hours after dosing. It is concluded that Diethylhydroxylamine had no genotoxic activity detectable in this test system under the experimental conditions employed.
Executive summary:

Diethylhydroxylamine was tested for its ability to induce unscheduled DNA synthesis (UDS) in the livers of orally dosed male rats using anin vivo/in vitroprocedure. Information available indicated that the LD50value for Diethylhydroxylamine was greater than 2000 mg/kg (the maximum recommended dose for the UDS assay). Accordingly, a confirmatory range-finder study was performed at 2000 mg/kg. Additionally, both male and female animals were tested in the range-finder study to determine any substantial inter-sex differences in toxicity. In the range-finder study, groups of three male and three female out bred Han Wistar (Crl:WI (Glx/BRL/Han) BR) rats were dosed once with up to 2000 mg/kg Diethylhydroxylamine. During a 2 day post-dose observation period male animals dosed at 2000 mg/kg showed clinical signs of eye closure, piloerection, lethargy and weight loss. Female animals dosed at 2000 mg/kg showed clinical signs of eye closure, piloerection, lethargy, abnormal gait and being cold to the touch. However, due to the severity of these signs the female animals were killedin extremis. Therefore, a further three female animals were dosed at 1400 mg/kg and acceptable clinical signs including piloerection, lethargy, abnormal gait, being cold to the touch and weight loss were observed. Accordingly, as no substantial inter-sex differences in toxicity were observed, male animals only were tested in the main study at a maximum dose of 2000 mg/kg. A lower dose level of 800 mg/kg (40% of the maximum dose) was also selected.

In the main study groups of four male rats were treated once with the vehicle (purified water), Diethylhydroxylamine (at 800 mg/kg or 2000 mg/kg) or the required positive control, by oral gavage, at a dose volume of 10 mL/kg. The positive controls used were 75 mg/kg 2-acetamidofluorene (2-AAF) suspended in corn oil (12-14 hour experiment) and 10 mg/kg dimethylnitrosamine (DMN) dissolved in purified water (2-4 hour experiment). Clinical signs observed in the main study included piloerection and lethargy (2-4 hour experiment, 2000 mg/kg dose group). In the 12-14 hour experiment slight weight loss was observed (2000 mg/kg dose group). Approximately 2-4 hours (Experiment 1) or 12-14 hours (Experiment 2) after dosing, animals were sacrificed and their livers perfused with collagenase to provide a primary culture of hepatocytes. Cultures were made from three animals in each dose group and were treated with [3H] thymidine. Six slides from each animal were prepared with fixed hepatocytes and of these, three were dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically after development of the emulsion and staining, and the net grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for each of two of the three slides, each animal and dose group.

Negative (vehicle) control animals gave a group mean NNG value of less than 0.2 with only 0 to 1% cells in repair. Group mean NNG values were increased by 2-AAF and DMN treatment to 2.9 or more with more than 20% cells found to be in repair. It may be noted that the Experiment 1 vehicle control NNG value was slightly higher than normal (0.2), however this value is within the negative historical control range and therefore acceptable. Additionally, although the positive control response for DMN was lower than normal (falling outside of historical range), the response was considered to be clearly indicative of UDS. Accordingly, in this study the vehicle control NNG value was consistent with both published and historical control data, and the system was shown to be sensitive to two known DNA damaging agents requiring metabolism for their action. The assay was therefore accepted as valid. Treatment with 800 or 2000 mg/kg Diethylhydroxylamine did not produce a group mean NNG value greater than zero nor were any cells found in repair at either dose.

It was concluded that Diethylhydroxylamine did not induce UDS detectable under the experimental conditions employed.