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Genetic toxicity: in vivo

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Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Jan. 28, 1991 to June 19, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Version / remarks:
Adopted : 21st July 1997
Deviations:
no
GLP compliance:
yes
Remarks:
according to OECD and German principles of GLP
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: Solid
Details on test material:
- Name of test material: 2-amino-4-hydroxyethylaminoanisole sulfate
- TSIN: LGH 10183/1
- Substance type: Pure active substance
- Physical state: Grey to blue solid
- Storage condition of test material: Room temperature (protected from light)
- Stability in solvent: Oxidizes in solvent especially at pH >7.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rob 006958
- Expiration date of the lot/batch: not reported
- Purity test date: not indicated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, light protected
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: oxidizes in solvent especially at pH>7

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment (immediately before treatment), the test article was formulated in aqua bidest..
- Final dilution of a dissolved solid, stock liquid or gel: not specified
- Final preparation of a solid: formulation in aqua bidest..


OTHER SPECIFICS:

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar/WU
Details on species / strain selection:
The rat is an animal which has been used for many years as suitable experimental animal in genotoxicity investigations. There are many data available from such investigations which may be helpful in the interpretation of results from UDS test. In addition, the rat is an experimental animal in many physiological, pharmacological, and toxicological studies. Data from such experiments may also be useful for the design and the performance of the UDS test.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Male rats were obtained from SAVO-Ivanovas, med. Versuchstierzuchten GmbH D-7964 Kisslegg, F.R.G.
- Weight at study initiation: Approximately 160-180 g
- Assigned to test groups randomly: Yes, animals were randomly distributed into group and identified by cage number.
- Fasting period before study: The animals were starved overnight (4 hour treatment) or approximately 6 hours (16 hour treatment) before receiving the test substance.
- Housing: Animals were housed individually in Makrolon Type I cage, with wire mesh top and granulated soft wood bedding.
- Diet: Pelleted standard diet (ALTROMIN 1324, D-4937 Lage/Lippe, F.R.G.); ad libitum
- Water : Tap water; ad libitum
- Acclimation period: Minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3°C
- Relative humidity: 30-70%
- Air changes: Not reported
- Photoperiod: 12 hours light (artificial light 6 am - 6 pm)/12 hours dark cycle per day

Experiment start date: Jan. 28, 1991
Experiment completion date: June 19, 1991

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Aqua bidest
- Justification for choice of solvent/vehicle: The vehicle was chosen according to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: 0, 7.5 and 75 mg/mL for 0, 75 and 750 mg/kg bw, respectively
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solution was formulated in aqua bidest on the day of experiment (immediately before treatment).
Duration of treatment / exposure:
Animals were treated for 4 (750 mg/kg/ bw) and 16 hours ( 75 and 750 mg/kg bw) treatment duration.
Frequency of treatment:
Once
Post exposure period:
4 and 16 h
Doses / concentrationsopen allclose all
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
Sampling time: 4 hour after administration
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
Sampling time: 16 hour after administration
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
Sampling time : 16 hours after administration
Dose / conc.:
0 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 male rats/dose/sampling interval
Control animals:
yes, concurrent vehicle
Positive control(s):
2-acetylaminofluorene
- Justification for choice of positive control(s): The positive control used, is one of the recommended positive controls by the OECD 486 guideline.
- Route of administration: Single oral administration
- Doses / concentrations: 100 mg/kg bw
- Dose volume: 10 mL/ kg bw
- Dissolved in: Dimethylsulfoxide/polyethylene glycol 400 (1 + 9)

Examinations

Tissues and cell types examined:
Liver / Hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: For genotoxicity investigations it is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The volume to be administered should be compatible with physiological space available.

TREATMENT OF ANIMALS: The animals were starved overnight (4 hours treatment) or approximately 6 hours (16 hours treatment) before receiving the test substance, water was available continuously. Before the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the body weight of the animals. The animals received single oral administration of the test substance. 5 male animals were treated per dose group.

ISOLATION OF HEPATOCYTES: Hepatocytes were isolated at 4 and 16 hours time intervals from different groups. The rats were anesthetized with 1.5 mL/kg bw Hypnodil (intraperitoneally), the liver was perfused through the vena portae with Hanks' balanced salt solution supplemented with collagenase (0.05% w/v) adjusted to pH 7.4 and maintained at 37°C. The hepatocytes were isolated from the liver and washed twice with Hanks' balanced salt solution (HBSS) and filtered through a 94 µm stainless steel mesh to yield single cell suspension. Cell viability was determined by trypan blue dye exclusion method.

DETAILS OF SLIDE PREPARATION: The washed hepatocytes were centrifuged and transferred into Williams medium E (WME) supplemented with 2.38 mg/mL Hepes, 100 units/mL penicillin, 0.1 mg/mL streptomycin, 0.29 mg/mL glutamin, 0.5 mg/mL insulin and 100 µL/mL fetal calf serum. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (1.0 x 10(5) cells/mL) were added to 35 mm six-well cluster dishes containing one gelatinized 25 mm round plastic coverslip per well. At least five cultures were established for each animal.
After an attachment period of approximately 1.5 h in a 95% air and 5% CO2 humidified incubator at 37°C the culture medium was discarded. The cell layer was rinsed with PBS to remove non-adherent cells. Subsequently, methyl-3H-thymidine (3HTdR) (5 µCi/mL, specific activity 20 Ci/mmol) in 2.0 ml culture medium (WME, 1% FCS) was added to the cultures. After a labeling time of 4 h the cells were washed twice with WME supplemented with 1 % FCS and 0.25 mM unlabeled thymidine. Cultures were incubated overnight with the same medium. The medium was replaced by a hypotonic solution of 1% sodium citrate for 10 minutes to swell the nuclei for better grain quantification. The cells on the coverslips were then fixed by three changes of methanol: acetic acid (3+1 v/v) for 15 minutes each, rinsed with 96% ethanol and air dried. Three of the cultures from each animal were used for the UDS assay. Two cultures were used for determination of cytotoxicity and attachment efficiency with the neutral red assay.

AUTORADIOGRAPHIC PROCESSING: Cover slips were mounted the side carrying the cells up on glass slides and coated with ILFORD K-2 photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 12-14 days at 4˚C.The photographic emulsion was then developed with KODAK D-19 at room temperature, fixed in TETENAL and stained with 0.4% aceto orcein.

METHOD OF ANALYSIS (Net grain count): Evaluation was performed microscopically on coded slides using NIKON microscopes with 100 x oil immersion objectives. The number of silver grains above the nucleus was counted automatically using the ARTEK 880 counter. The number of grain counts of one nuclear-sized cytoplasm adjacent to the nucleus was counted.
- Number of hepatocyte cultures isolated: Three of the cultures from each animal were used for the UDS assay.
- Number of cells evaluated: At least two slides per animal and 50 cells per slide were evaluated. Heavily labeled S-phase cells were excluded from counting.
Evaluation criteria:
- A test substance was classified as positive if it induced either a statistically significant dose related increase in radiolabel incorporation expressed as grains per nucleus or a reproducible and statistically significant positive response for at least one of the test points.
- A test substance producing neither a statistically significant dose related increase in radiolabel incorporation expressed as grains per nucleus nor a statistically significant and reproducible positive response at any one of the test points was considered non-effective in this system.
- Statistical significance can be evaluated by means of the nonparametric Mann-Whitney test.
- However, both statistical and biological significance should be considered together.
Statistics:
A statistical evaluation of the results was not necessary to perform as the number of nuclear and net grain counts of the groups treated with the test substance were in the range of the corresponding controls.
However, if needed, the statistical significance can be evaluated by means of the nonparametric Mann-Whitney test.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
for 4 and 16 h sampling time
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY: A series of pre-experiments were performed to find dose level for the main study.
- At 1000 mg/kg bw, 2 out of 5 treated animals died after 24 h. The surviving animals showed severe toxic symptoms such as reduction of spontaneous activity, eyelid closure and pilo erection.
- At 750 mg/kg bw, all animals survived for at least 24 h and hepatocytes isolated from these animals yielded viabilities sufficient for the UDS-assay. However, dark colored kidneys, liver and urine were observed in the treated animals. In addition, the liver was relatively solid.
- At 500 mg/kg bw, the kidneys and the urine were colored, but the liver was unaffected.
Based on the results, 750 mg/kg bw was chosen as the highest dose for the UDS-assay.

RESULTS OF DEFINITIVE STUDY
- No toxic reactions were observed in animals in any dose groups.
- The viability and attachment efficiency of the hepatocytes was not substantially affected due to the in vivo treatment with the test substance at any of the treatment periods or dose groups.
- No dose level of test substance revealed UDS induction in the hepatocytes of the treated animals as compared to the vehicle control.
- The number of nuclear grains and the resulting net grains did not increase due to the in vivo treatment of the animals for 4 or 16 hours, respectively. Therefore, the net grain values obtained after treatment were consistently negative.

RESULTS OF POSITIVE CONTROL
- 2-acetylaminofluorene (2-AAF) induced significant increase in the number of nuclear and net grain counts, indicating the sensitivity of the test method.

COMPARISON WITH HISTORICAL CONTROL DATA
- The inter-individual variations obtained for the numbers of isolated hepatocytes as well as for the attachment-efficiency (NR-assay) were in the range of historical laboratory control.

Any other information on results incl. tables

Table 1: UDS in primary rat hepatocytes after in vivo treatment after 4 and 16 h treatment with 2-amino-hydroxyethylamino-anisole sulfate (study # 83560)

Treatment

Grains per nucleus (Mean*± SD)

Grains per cytoplasmic area (Mean* ± SD)

Net grains per nucleus (Mean* ± SD)

Vehicle control (Aqua bidest/16h)

5.03 ± 3.54

8.00 ± 4.27

-2.97 ± 3.76

2AAF(100 mg/kg/16h)

21.56 ± 10.80

5.35 ± 2.75

16.22 ± 10.76

75 mg/kg/16h

3.65 ± 2.22

6.14 ± 2.56

-2.49 ± 2.83

750 mg/kg/ 4h

4.08 ± 2.19

7.38 ± 2.86

-3.31 ± 2.64

750 mg/kg/16h

3.98 ± 2.49

6.67 ± 2.94

-2.69 ± 2.99

* Mean of 3 animals, 100 cells each

Applicant's summary and conclusion

Conclusions:
Under the test conditions of in vivo unscheduled DNA synthesis assay, 2-aminohydroxyethylamino- anisole sulfate when administered orally at doses of 75 mg/kg bw (16 h sampling time) and 750 mg/kg bw (4 h and 16 h sampling time) did not induce unscheduled DNA synthesis in primary rat hepatocytes in an autoradiographic in vivo unscheduled DNA synthesis assay.
Executive summary:

The in-vivo DNA damage and repair assay of 2-aminohydroxyethylamino-anisole sulfate was determined following method comparable to OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo).

Male Wistar/WU rats weighing approximately 160-180 g were obtained from SAVO-Ivanovas, med. GmbH Kisslegg, F.R.G. The animals were housed individually in Makrolon Type I cages with wire mesh tops and granulated soft wood bedding and maintained under standard laboratory conditions (temperature: 21 ± 3°C, humidity: 30-70%; artificial light: 12 hours light /12 hours dark cycle per day).  

The induction of UDS was evaluated using the definitive autoradiographic method. Test solutions were prepared in aqua bidest, which was also used as the vehicle control, and were administered once by oral gavage (volume: 10 mL/ kg bw). 2-Acetyl aminofluorene (2-AAF) served as a positive control in the study.

Based on the result of pre-experiments, 750 mg/kg bw was selected as the highest dose for main study. Two independent experiments with two sampling times (4 and 16 h) were performed in the main study.

The following dose levels with 5 males per dose group were tested:

 Experiment 1 (4 h sampling time): 750 mg/ kg bw

 Experiment 2 (16 h sampling time): 75 and 750 mg/ kg bw

The hepatocytes were collected at 4 h (Experiment 1) and 16 h (Experiment 2). Primary rat hepatocytes were prepared by perfusing rat livers in situ. Viability of hepatocytes was determined by trypan blue exclusion assay. Hepatocytes were processed and treated with radiolabelled thymidine (3H-Thymidine) for autoradiographic measurement of UDS.

At least 5 cultures were established for each animal. Three of the cultures from each animal were used for the UDS assay. Two cultures were used for determination of cytotoxicity and attachment efficiency with the neutral red assay. At least two slides/animal and 50 cells/slide were evaluated. The slides from both experiments were analyzed for nuclear and cytoplasmic grain counts and the net grain counts.

No toxic reactions were observed in animals in anydose group. The viability and attachment efficiency of the hepatocytes were not substantially affected due to thein vivotreatment with the test substance at any of the treatment periods or dose group.

No dose level of test substance revealed UDS induction in the hepatocytes of the treated animals as compared to the vehicle control. The number of nuclear grains and the resulting net grains did not increase due to the in vivo treatment of the animals for 4 or 16 hours. Therefore, the net grain values obtained after treatment were consistently negative.

2-acetylaminofluorene (2-AAF) induced distinct increases in the number of nuclear and net grain counts, indicating the sensitivity of the test method.

In conclusion, it can be stated that under the experimental conditions reported, 2-Aminohydroxyethylamino-anisole sulfate did not induce DNA damage that is detectable with the UDS test.

This in-vivo DNA damage and repair assay is classified as acceptable, and satisfies the guideline requirements of the OECD 486 method.