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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Version / remarks:
Principles of method if other than guideline:
The inhalation part of this study was carried out in accordance to OECD TG 403 (exposure technology).
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: unscheduled DNA synthesis (sacrifice time 16 hours)

Test material

Constituent 1
Chemical structure
Reference substance name:
N-[6-(3-{6-[3-(6-formamidohexyl)-2,4-dioxo-1,3-diazetidin-1-yl]hexyl}-2,4-dioxo-1,3-diazetidin-1-yl)hexyl]formamide; N-{6-[3-(6-formamidohexyl)-2,4-dioxo-1,3-diazetidin-1-yl]hexyl}formamide
EC Number:
Molecular formula:
N-[6-(3-{6-[3-(6-formamidohexyl)-2,4-dioxo-1,3-diazetidin-1-yl]hexyl}-2,4-dioxo-1,3-diazetidin-1-yl)hexyl]formamide; N-{6-[3-(6-formamidohexyl)-2,4-dioxo-1,3-diazetidin-1-yl]hexyl}formamide
Constituent 2
Reference substance name:
Cas Number:

Test animals

Details on test animals or test system and environmental conditions:
- Strain: Wistar rats Crl:(WI)BR (SPF)
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 7 weeks of age
- Weight at study initiation: 176 g-199 g
- Assigned to test groups randomly: yes
- Housing: animals were kept singly in type III h cages
- Diet and water: ad libitum
- Acclimation period: at least five days; The rats were adapted to the exposure tubes on 3 days prior to exposure.

- Temperature (°C): 22.5-23°C
- Humidity (%): 25-30% mean relative humidity
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
conditioned air
Details on exposure:

dynamic directed-flow nose-only exposure conditions
- Exposure apparatus: The chambers used are commercially available (TSE, 61348 Bad Homburg) and the performance as well as their validation has been published (Pauluhn, Journal of Applied Toxicology 13, 55-62, 1994). The aluminum inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 I).
- Method of holding animals in test chamber: Animals were in Plexiglas exposure restrainers. Restrainers were chosen that accommodated the animal's size.
- Source of air/ Method of conditioning air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer.
- System of generating particulates/aerosols: Under dynamic conditions the test substance is nebulized neat into a baffle (pre-separator) from which the substance was conveyed into the mixing unit and air dilution cascade, which is connected with the intake of the cylindrical inhalation chamber. The test substance atmosphere is generated using a Schlick binary nozzle, which is heated to 40 °C, with conditioned compressed air (15 I/min); dispersion pressure approximately 600 kPa. The liquid test substance is fed into the nozzle by a digitally controlled Harvard PHD 2000 infusion pump with an injection of 40 µl test substance/min. The generated atmosphere had to be diluted into the range targeted.
- Temperature, humidity, pressure in air chamber: Temperature and humidity measurements were recorded using a computerized system (Hydra, Fluke-Philips) at intervals of 5 min.
- Air flow rate: At each exposure port a minimal air flow rate of 0.75 I/min was provided.
- Air change rate: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (> 200 x, continuous generation of test atmosphere).
- Method of particle size determination: The particle-size distribution was analyzed using a Berner type impactor.
- Treatment of exhaust air: The exhaust air was purified via cotton-wool/HEPA filters.

The integrity and stability of the aerosol generation and exposure system was measured by using a RAS-2 real-time aerosol photometer (MIE, Bedford, Massachusetts, USA).
- Brief description of analytical method used: gravimetric determination (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
- Samples taken from breathing zone: yes
- Particle characterisation: Aerosol mass < 3 %: at 50 mg/m³ 93.0 %, at 140 mg/m³ 90.8 %.
MMAD (µm)/GSD: approx. 1.6/ approx. 1.5
Duration of treatment / exposure:
3 hours
Frequency of treatment:
Post exposure period:
Sacrifice time: 16 hours
Liver cells of the animals of the negative controls and treated with the test substance were prepared 16 hours after the mid of the inhalation exposure.
Doses / concentrationsopen allclose all
Doses / Concentrations:
50 and 140 mg/m³
other: target conc.
Doses / Concentrations:
52.7 and 144 mg/m³
analytical conc.
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):
Liver cells of the positive control 2-acetylaminofluorene treated animals were prepared separately 16 hours after the oral treatment.

- Route of administration: oral; For that purpose 2-acetylaminofluorene was dissolved in corn oil. The volume administered was 10 ml/kg. For the positive control, approximately 6 hours before treatment food was withdrawn for animals and approximately 30 min after treatment animals received food again.
- Doses / concentrations: 100 mg/kg


Tissues and cell types examined:
Liver cells (primary hepatocytes)
Details of tissue and slide preparation:
In a micronucleus-test in male mice with 6 hours inhalation (report AT04888, Bayer AG, 2008) for which 7, 25, 50 and 70 mg/m³ were planned, one mouse of the 70 mg/m³ group died 24 hours after start of treatment. It has also to be considered, that rats have a much lower minute volume. Therefore, 140 mg/m³ using an inhalation period of 3 hours are considered to be the MTD for the present study with rats.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Anesthesia (Narcoren i.p.) was performed prior to liver perfusion procedure. Perfusion was performed with two consecutive solutions (EGTA-solution, Collagenase solution) kept at 37°C using two peristaltic pumps (Infusomat Secura, Braun Melsungen AG). Then the liver was removed, rinsed with sterile saline or medium and placed in a sterile Petri dish containing 30 ml of collagenase-solution and transferred to a sterile hood for cell isolation. After perfusion, primary hepatocytes were prepared under sterile conditions according to the protocol of Butterworth et al. (Mutation Res. 189, 113-121, 1987). The cell viability of freshly isolated hepatocytes was determinated. For treated animals the relative viability in comparison to the viability of the negative control, which represents a survival of 100%, is an indicator for a substance induced cytotoxicity during the in vivo exposure.

For slide preparation a Thermanox round plastic coverslip (diameter 25 mm) pre-coated with collagen was placed into each of 4 wells of a 6 well culture dish. Approximately 3.75x10exp5 viable cells were seeded per well in culture medium at a total volume of 2.5 ml. Cultures were incubated for attachment for about 90 minutes at 37 °C in a humidified atmosphere containing approximately 5% CO2. After the attachment period, cells in the 6-well dishes were washed and labelled (10 µCi/ml 3H-thymidine added to each culture, incubation 4 hours at 37°C, humidified atmosphere with 5% CO2). After washing steps and incubation with unlabelled thymidine 1% sodium citrate solution was added for 5-10 min. The cells were then fixed (acetic acid/ethanol 1/4), washed with deionized water and air dried. An autoradiography-procedure was performed follpwed by a hematoxylin and eosin staining.

Slides were put onto a light microscope using an oil immersion objective with a magnification of 100. Nuclear grains and cytoplasma grains were counted on this TV color screen by the naked eye. Slides were read blind and meanderwise. Generally, three slides and 50 cells per slide were examined per animal. Per cell the nuclear grain count (NG) was determined once. In addition, the number of grains was determined in 3 cytoplasmic areas
of this cell. These areas had the same size as the corresponding nucleus. The NG was reduced by the mean of the grains in the cytoplasmic areas (CG). This value was referred to as the nuclear net grain (NNG) count of the cell. The mean NNG value per dose group was calculated from the mean NNG value of each animal of the respective dose group. The number of cells in repair (nuclei with 5 or more net grains) was also determined.
Evaluation criteria:
A test was considered positive if there was a biologically relevant and statistically significant increase of nuclear net grains (NNG) in hepatocytes of treated animals in comparison to the negative control. A test will be considered also positive, if a chemical yields dose-dependently a group mean of at least +2 NNG. Cells in repair have to be considered in addition.
A test was considered negative if there was no biologically relevant and statistically significant increase of NNG. A test was also considered negative if there was a significant increase of NNG, which according to the laboratory's experience was within the range of historical negative controls. A group mean of 0 NNG or lower will also be indicative for a negative response.
In addition, a test was considered equivocal if there was an increase of mean NNG above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. In this case, normally a second test will be performed.
However, these criteria may be overruled by good scientific judgement.
Descriptive statistical methods were used for nuclear grains (NG), cytoplasmic grains (CG) and nuclear net grains (NNG). Means and standard deviations were calculated for each animal and for each group. The standard deviations for an animal were calculated from the respective means calculated individually for each of its three coverslips. The standard deviations for a group were calculated on the basis of these animal means. In addition, numbers of NNG in the test substance groups and in the positive controls (provided this exceeded 0) were checked by Wilcoxon's non-parametric rank sum test.
For statistical evaluations of body weights and rectal temperatures a one-way ANOVA (vide infra) was used.

Results and discussion

Test results
starting at 50 mg/m³
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
After single inhalation exposure with 50 mg/m³ and 140 mg/m³ of the aerosolised substance, for the sacrifice time of 16 hours, no biologically relevant increases were found, neither concerning nuclear net grains nor concerning cells in repair.

Male rats treated with doses up to 140 mg/m³ showed symptoms of toxicity after administration, starting at 50 mg/m³. These symptoms demonstrate relevant systemic exposure to the substance. However, all animals survived until the end of the test.
After cell isolation, no relevant cytotoxic effects could be observed in hepatocytes of male rats exposed to the test substance. The same was true for cells of the positive control animals.

The positive control 2-Acetylaminofluorene induced relevant genotoxic effects and thus demonstrated the sensitivity of the method for the detection of induced DNA repair synthesis.

Any other information on results incl. tables

Exposure chamber temperature and humidity (mean): 22.4-23.2 °C, < 5.0-5.6 %

The liquid aerosol was respirable to the rats (MMAD approx. 1.6 µm, GSD approx. 1.5, aerosol mass < 3 µm 93.0-90.8%).

Toxicological results obtained during and after single exposures of male rats: All rats survived the exposure period. The rats showed following clinical signs: bradypnea, labored breathing patterns, breathing irregular, piloerection, motility reduced, limp, nose with serous discharge and hypothermia. At the UDS-part, symptoms of the animals before narcosis were: slitted eyes, rapid breathing and roughened fur.

Applicant's summary and conclusion

Executive summary:

An Unsheduled DNA Synthesis test according to OECD TG 486 was conducted on male rats to investigate a possible genotoxic effect on the DNA of liver cells following a single aerosol inhalation exposure (3h, nose-only). Concentration were 0 (air control), 52.7 or 144 mg/m³. Liver cells of the animals were prepared 16 hours after the mid of the inhalation exposure.

Male rats treated with the test substance showed symptoms of toxicity after exposure, starting at 52.7 mg/m³. However, no mortalities occurred.

No biologically relevant increases were found in the prepared liver cells after evaluation, neither concerning nuclear net grains nor concerning cells in repair. The positive control 2-Acetylaminofluorene induced relevant genotoxic effects and thus demonstrated the sensitivity of the method for the detection of induced DNA repair synthesis.

Based on the study result the test substance is not considered to be genotoxic in vivo.