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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
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-01-19 To: 2005-02-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Methods and results were well documented and scientifically defensible; GLP.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Conduct and evaluation of the study was performed according to an internationally accepted protocol for the Comet Assay
in vivo (A.Hartmann et.al.,2003).
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
481-170-7
EC Name:
-
Cas Number:
502453-61-4
Molecular formula:
Hill formula: C23H30BrN3O2 CAS formula: C23H30N3O2.Br
IUPAC Name:
dimethyl(3-{[4-(methylamino)-9,10-dioxo-9,10-dihydroanthracen-1-yl]amino}propyl)propylazanium bromide
Constituent 2
Reference substance name:
Dimethyl-(3-(4-methylamino-9,10-dioxo-9,10-dihydro-anthracen -1-ylamino)propyl)propylammonium bromide
IUPAC Name:
Dimethyl-(3-(4-methylamino-9,10-dioxo-9,10-dihydro-anthracen -1-ylamino)propyl)propylammonium bromide
Details on test material:
- Name of test material (as cited in study report): B119 HC Blue 16
- Molecular formula (if other than submission substance): C23H30N3O2Br
- Molecular weight (if other than submission substance): 460.42
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): N/A
- Substance type: Active
- Physical state: Solid, dark blue powder

Test animals

Species:
rat
Strain:
other: Wistar Crl:WI BR
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-8 weeks of age
- Weight at study initiation: 152-203 g
- Assigned to test groups randomly: Yes; Animals were randomized according to randomization numbers generated by a computer program created and validated in house.
- Fasting period before study: Food withdrawn prior to weighing at the start of the pilot and main studies, 4-5 hours before the first treatment and were given food again 1 hour after treatment
- Housing: Individually in Makrolon type IIA cages
- Diet (e.g. ad libitum): Fixed- formula feed "Maus/Ratte Haltung (NAFAG 9441), 3883.0.15" for mice and rats, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-23.0 °C
- Humidity (%): 41-52%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: N/A To: N/A

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Deionized water
- Justification for choice of solvent/vehicle: Test material solubility in water is > 20 weight% (pH 6.1)
- Concentration of test material in vehicle: N/A
- Amount of vehicle (if gavage or dermal): 20 ml/kg bw
- Type and concentration of dispersant aid (if powder): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was dissolved in deionized water.

DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A
Duration of treatment / exposure:
single administration (two treatments)
Frequency of treatment:
Test material and vehicle exposed groups received two administered treatments. Positive control only administered once.
Post exposure period:
Animals in treatment and vehicle control groups were sacrificed 23 hours after first treatment, positive control groups were sacrificed 3 hours after treatment.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
150 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
600 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Five animals were treated per day including at least one control animal (positive and/or negative control). Each treatmeant group consisted of 5-6 animals.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: ethylmethanesulphonate (EMS)
- Justification for choice of positive control(s): Known DNA-damaging agent
- Route of administration: oral gavage
- Doses / concentrations: 400 mg/kg bw (volume: 10 ml/kg bw); single treatment
- Other: EMS was suspended in corn oil. The EMS treated animals did not seem to drink water after the compound adminstration, which could have influenced the compound distribution in the body so the animals also received 10 ml/kg be deionized water about 1 hour after the EMS administration.

Examinations

Tissues and cell types examined:
Liver, stomach and urinary bladder epithelium
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The selection of the B119 HC Blue 16 doses were based on two pilot studies.


TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 5 animals per treatment group were available for the liver and for the positive control groups of stomach and urinary bladder epithelium. All other treatment groups comprised of 6 evaluated animals for stomach and urinary bladder epithelium. 23 hours after the first application and 3 hours after the second application, rats were anaesthetized by i.p. injection of Narcoren (Pentaobarbital-sodium). Anaesthesia was performed about 10 minutes prior to the perfusion procedure.


DETAILS OF SLIDE PREPARATION: Aliquots of the liver, stomach and urinary bladder epithelium cell suspensions were taken to reach an approximate viable cell number of 4-5E4 cells, respectively. Cells were centrifuged at about 100xg for 5 min. The resulting pellet was mixed with 50 µl low melting agarose (LMA) (0.7%). The cell/LMA suspension was carefully pipetted onto slides (76 X 26 mm) already covered with two layers of 0.5% normal melting agarose (NMA; 50 µl each). Afterwards, a second LMA layer (0.7%, 50 µl) was placed on top (agarose from Biozym). Starting with the lysis all subsequent steps were performed under red light. After lysis (overnight; lysis buffer containing Na2EDTA, sarcosinate, and freshly added Triton X-100 and DMSO), alkaline treatment (20 min, NaOH and Na2EDTA, pH >= 13, in an ice bath) was performed followed by electrophoresis in an ice bath and neutralisation (Tris base, pH 7.5). Electrophoresis conditions were 40 min, 25 V, 300 mA, for the liver and urinary bladder epithelium cells. Electrophoresis of stomach cells was performed under the same conditions, but for 30 min. Subsequently, slides were stained with ethidium bromide.


METHOD OF ANALYSIS: Evaluation of comets was performed using image analysis and a validated evaluation program (Comet III, Perceptive Instruments, Haverhill, UK). Tail length, defined as distance between the middle of the head and the end of the tail, was used as assessment parameter. 50 cells per slide and two slides per animal were scored (100 cells total).


OTHER: PERFUSION PROCEDURE: The abdomen was shaven and thoroughly wetted with 70% ethanol. An incision was made through both skin and muscle from the centre of the lower abdomen to the lateral aspects of the rib cage. The intestines were gently moved out to reveal the portal vein. A suture was put in place (but not tightened) around the vena cava just below the left renal branch. Additionally, the vena cava was pinched off between diaphragm and liver by a clamp. The vena cava was then cannulated with an appropriate syringe about 3 mm caudally of the suture. The inner needle was removed, and the plastic catheter was further inserted into the vein and tied in place by the suture, which was tightened at that point of the perfusion procedure.
Perfusion was performed with two peristaltic pumps. Solutions were kept at 37°C with a minimal distance to the animal and the shortest possible tube length to minimize cooling in the line.
The tube with the flowing perfusion solution I was then inserted in the catheter and taped in place. The portal vein was immediately cut off and the liver was drained of blood. The flow rate of perfusion solution I was 5 ml per minute for 1.5 minutes and then the flow rate was increased to 10 ml per minute for approximately 2 minutes. Subsequently, a switch was performed to the perfusion solution II (15 ml/ min until approximately 200 ml were used). After the perfusion was over, the liver was carefully removed and placed in a sterile Petri dish containing 30 ml of perfusion solution II and taken to a sterile hood to prepare the cells.

Perfusion solution I (sterile): 0.5 ml EGTA (190 mg dissolved in 1 ml 2N NaOH, 2.5 ml 2M HEPES, 0.5 mi of gentamycin sulfate (stock: 50 mg/ml), filled up to 500 ml with Hanks balanced salt solution without Ca2+/Mg2+.

Perfusion solution II (sterile): 497 ml Williams medium E, 2.5 ml 2M HEPES, 0.5 ml of gentamycin sulfate (stock: 50 mg/ml) 0.1 ml 2N NaOH, 25,000 - 50,000 units collagenase (Type CLS II).
Evaluation criteria:
The viability of liver, stomach and urinary bladder epithelium cells of the vehicle control animals collected by this process should normally exceed 70%. For single animals values between 50% and 70% viability were accepted, if the cell preparation demonstrates adequate quality. Vehicle control animals with organ cell preparations below 50% viability were considered unacceptable. No such limits are set for treated animals.
An assay is normally considered acceptable if the following criteria are met: Tail length data obtained for a giventreatment are acceptable as part of the evaluation if obtained from at least two slides and 100 cells per animal. The positive control EMS should induce a mean tail length increase of at least 30% compared to the mean tail length value of the vehicle control group.
The following criteria are used for assessment of the data: A response is considered positive, if a chemical induces a dose dependent mean increase of the tail length per dose group of more than 25% above the vehicle control group mean. Mean tail length increases in test compound treated groups between 15% and 25% compared to the vehicle control group have to be considered case by case. Increases below 15% compared to the vehicle control group are considered negative.
Statistics:
Descriptive statistical methods were used to calculate means and standard deviations using the standard Excel program. The means and standard deviations of the tail length were calculated from the means calculated individually for each of the two slides per animal. The means and standard deviations of the tail length per dose group were calculated from the means calculated individually for each of the evaluated animals.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Initially 2000 mg/kg bw was administered which caused the rats to die shortly after administration so another range finding study was conducted using doses of 100 and 200 mg/kg bw. Since the maximum tolerated dose was not reached another range finding study was conducted at levels of 400 and 600 mg/kg bw.
- Solubility: N/A
- Clinical signs of toxicity in test animals: Animals died at the 1000 mg/kg bw level. Roughened fur, rapid breathing, twitching at the 100 and 200 dose levels. At the 400 mg/kg dose level, rats were observed with roughened fur, rapid breathing, and discolored feces. At the 600 mg/kg bw dose level, rats were observed with roughened fur, rapid breathing, palmospasms, discolored feces and langour. Based on these results 600 mg/kg B119 HC Blue was selected as maximum tolerated dose for this test in male rats for both consecutive treatments.
- Evidence of cytotoxicity in tissue analyzed: N/A
- Rationale for exposure: Range finding study to determine maximum tolerated dose levels for the main study.
- Harvest times: N/A
- High dose with and without activation: N/A
- Other: N/A


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): Cells of vehicle controls used for assessment showed good cell viabilities fulfilling our cell quality criteria. No relevant cytotoxic effects could be observed in liver, stomach and urinary bladder epithelium cells of rats exposed to B119 HC Blue 16. The same was true for cells of all evaluated organs of the positive control animals.
- Induction of micronuclei (for Micronucleus assay): N/A
- Ratio of PCE/NCE (for Micronucleus assay): N/A
- Appropriateness of dose levels and route: N/A
- Statistical evaluation: No biological relevant increases in tail length values were observed in cells of the liver, stomach, and the urinary bladder epithelium after oral treatment of rats with the test material. The positive control induced biologically significant increases of the tail length in liver, stomach and urinary bladder epithelium.

Any other information on results incl. tables

N/A

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
B119 HC Blue 16 did neither induce a biologically relevant increase in the level of DNA migration (tail length as criterion) nor any relevant cytotoxic effect in cells of the liver, stomach and urinary bladder epithelium sampled from treated animals in the in vitro Comet Assay.
Executive summary:

B119 HC Blue 16 was tested in a comet assay in vivo with liver, stomach and urinary bladder epithelium cells of male Wistar rats. Ethyl­methanesulfonate (EMS) served as a positive control. The vehicle control group was treated with deionized water.

Based on the results of two pilot studies 600 mg/kg body weight B119 HC Blue 16 was selected as the maximum tolerated dose (MTD) for this test for both consecutive treatments. In a first treatment, rats received B119 HC Blue 16 at a single oral dose of 600 mg/kg, 300 mg/kg and 150 mg/kg, respectively, by gavage. 20 hours after the first treatment a second treatment was performed with the same dose as at the first treatment. Animals were sacrificed 3 hours after the second treatment. Intact liver cells were prepared via in situ perfusion.

Intact stomach cells from the stomach epithelium and intact urinary bladder cells were isolated via incubation with trypsin. Subsequently, organ cells were subjected to the comet assay procedure.

In all cases 400 mg/kg EMS was used as the positive control. Cells of the liver, stomach and urinary bladder epithelium of positive control animals were prepared 3 hours after administration.

Animals showed symptoms of toxicity at all doses of B119 HC Blue 16. As a further evidence for the systemic availability of the test substance in animals of the high and mid dose groups discolored feces and/or urine were observed. No compound-related cytotoxicity was observed in cells of liver, stomach and urinary bladder epithelium after isolation.

After oral treatment of male rats with B119 HC Blue 16 according to the treatment regimen no biologically relevant increase of the mean tail length value was observed in cells of the liver, the stomach and the urinary bladder epithelium at all dose groups compared to the vehicle control group.

The positive control EMS had clear genotoxic effects in cells of liver, stomach and urinary bladder epithelium as shown by the biologically relevant increase of the tail length, demonstrating the sensitivity of the method used for the detection of induced DNA damage.

Based on these results and under the conditions described, B119 HC Blue 16 was considered to be non-genotoxic in the comet assay in vivo using rat liver, stomach and urinary bladder epithelium cells.

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