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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:
07 April- 18 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to ICH guidance and GLP pinciples.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD 489 (26 september 2014)
Qualifier:
according to
Guideline:
other: - ICH S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (2012).
Principles of method if other than guideline:
The following publications were used as guiding documents: Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.-C. and Sasaki, Y.F. (2000). Single Cell Gel/Comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ mol mutagen 35, p 206 - 221; Smith CC, Adkins DJ, Martin EA, O'Donovan MR. (2008). Recommendations for design of the rat comet assay. Mutagenesis 23(3), p 233 – 240.
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane
- Substance type: Organic
- Physical state: Clear colourless liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen
- Reactivity: reactive to moisture
- Density (relative): 1.14

Test animals

Species:
rat
Strain:
other: Wistar Han
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 137 - 161g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: group housed (maximum 5 animals per sex per cage) in labelled Macrolon cages
- Diet: ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). A limited quantity of food was supplied during the night before dosing with positive control EMS (approximately 7g/rat).
- Water: ad libitum, tap-water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9– 20.8
- Humidity (%): 33 - 64
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Solvent used: corn oil
- Justification for choice of solvent: The solubility and stability of the test substance in corn oil was assessed in the present study.
- Amount of vehicle: 10 mL/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
No correction was made for the purity/composition of the test substance. Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane was dissolved in corn oil (Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands). The specific gravity of corn oil is 0.92 g/mL. The test substance was vortexed to dissolve and dosed within approximately 1 hour after preparation.

FORMULATION ANALYSIS:
Analysis were conducted on a single occasion during the main experiment, according to a validated method.
The accuracy of preparation is considered acceptable if the mean measured concentrations are 90-110% of the target concentration for solutions. Homogeneity is demonstrated if the coefficient of variation is ≤ 10%. Formulations are considered stable if the relative difference before and after storage is maximally 10%.
Duration of treatment / exposure:
Three consecutive days
Frequency of treatment:
The first dose of the test substance and vehicle was administered at t=0 h. The second and third dose were administered at approximately t=24 h and t=46 h, respectively. Positive control substance was administered was administered at t=24h and t=46 h

Post exposure period:
The animals were sacrified at approximately t=49-50 h (approximately 3-4 hours after the third treatment)
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl Methanesulfonate (EMS; CAS no. 62-50-0; Sigma Aldrich, Steinheim, Germany) at 200 mg/kg body weight dissolved in physiological saline. EMS was used within 3 hours after preparation and the route of administration was oral. The dosing volume was 10 mL/kg body weight

Examinations

Tissues and cell types examined:
Blood, liver and stomach tissue
Details of tissue and slide preparation:
ISOLATION OF BLOOD CELLS
Blood was collected under isoflurane anaesthesia from the retro-orbital sinus. At least 500 µL blood samples was taken per animal and collected into Vacuette tubes prepared with Li-heparin (Greiner Bio-one, Frickenhausen, Germany). Whole blood treated (200 µL) with heparin was added to 4.8 mL RPMI-C (RPMI 1640 medium (Invitrogen Corporation, Breda, The Netherlands), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Invitrogen Corporation) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands); Then 20 mL ice cold ery-lysis (pH 7.4) buffer containing 155 mM Ammonium chloride, 10 mM KHCO3 and 0.1 mM EDTA was added to lyse the red blood cells. The lymphocyte cultures were separated from the culture medium and lysis buffer by centrifugation (5 min, 359 g). The supernatant was removed and cells were resuspended in ice cold Hank’s Balanced Salt Solution (HBSS; Ca2+- and Mg2+-free) (Invitrogen Corporation) and kept on ice.

ISOLATION OF LIVER CELLS
The isolation method was based on the publication of Hu et al (2002). A portion of 0.6-0.7 gram from the liver was removed and minced thoroughly on aluminium foil in ice. The minced liver tissue was added to 10 mL of collagenase (20 Units/mL; Sigma Aldrich) dissolved in HBSS (Ca2+- and Mg2+-free) and incubated in a shaking waterbath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

ISOLATION OF STOMACH
The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free). The stomach was stored on ice in HBSS. The stomach was transferred to a tube containing mincing buffer. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO will be added immediately before use). The stomach was incubated in mincing buffer on ice for 15-30 minutes. After incubation, the fore-stomach was removed and discarded. The surface epithelia of the glandular epithelia was gently scraped one time softly. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis. The stomach was then minced thoroughly on aluminium foil in ice. The minced tissue was added to 10 ml of mincing buffer and incubated in a shaking waterbath at 37 °C for 15 minutes. Thereafter, a low centrifugation force was applied two times to remove undigested debris (40 g for 5 min). The supernatant was collected and filtered through a 100 µm Cell Strainer to purify the cell suspension. The purified cell suspension was centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

DETERMINATION OF CELL VIABILITY
The viability of the cells after isolation was determined by manually counting the number of viable cells using trypane blue staining. One animal per group was checked.

PREPERATION OF THE SLIDES
To 10 µL of the cell suspension, 140 µL melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added. The cells were mixed with the LMAgarose and 50 µL was layered on a precoated Comet slide (Trevigen) in duplicate. Two slides per tissue were prepared.
The slides were marked with the study identification number, animal number and group number. The slides were incubated for 15-53 minutes in the refrigerator in the dark until a clear ring appeared at the edge of the Comet slide area.

LYSIS, ELECTROPHORESIS AND STAINING OF THE SLIDES
The cells on the slides were overnight (approximately 17-18 h) immersed in prechilled lysis solution (Trevigen) in the refrigerator. After incubation the slides were placed in freshly prepared alkaline solution for 28-49 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 1 Volt/cm. The electrophoresis was performed for 30 minutes under constant cooling (actual temperature 5.0 – 6.0°C). After electrophoresis the slides were washed twice with Milli-Q water. The slides were subsequently immersed for 6-13 minutes in 70% ethanol and allowed to dry at room temperature. The slides were stained for 5-7 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark.

COMET SCORING
To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred Comets per slide
(50 comets of each replicate LMAgarose circle) were examined. Due to the fact that the agarose was partly damaged the backup slides were scored from Rat 3A stomach slide 73and Rat 5A stomach slide 65.

The following criteria for scoring of Comets were used:
- Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
- Cells that showed overlap or were not sharp were not scored.
Evaluation criteria:
The in vivo comet is considered acceptable if it meets the following criteria:
a) The mean percentage tail intensity of the solvent control should be less than 15 in the liver and blood cells; b) The mean percentage tail intensity of the solvent control should be less than 35 in stomach cells; c) The positive control EMS should produce at least a 3-fold statistically significant increase (p<0.01) in the percentage Tail Intensity compared to the vehicle treated animals in liver and blood cells; d) The positive control EMS should produce at least a 2-fold statistically significant increase (p<0.01) in the percentage Tail Intensity compared to the vehicle treated animals in stomach tissue.

A test compound is considered positive in the Comet assay (in a tissue) if the following criteria are met:
It induces a biologically as well as a statistically significant (Students t test, one sided, p < 0.01) dose-dependent increase in percentage Tail Intensity. In case of other non-dose-dependent significant increases the data interpretation will be on a case by case base.

A test compound is considered as negative in the Comet assay (in a tissue) if the following criteria are met:
None of the tested concentrations show a statistically significant (Students t test, one sided, p < 0.01) dose-dependent increase in percentage Tail Intensity.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.
Statistics:
Significance is assessed by using the Students t test.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
1000 and 2000 mg/kg: clinical signs (lethargy and diarrhea, rough coat, hunched posture) and body weight loss; 500 mg/kg: clinical signs (lethargy and hunched posture)
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw (3 males)
- Clinical signs of toxicity in test animals: No mortality was observed in the 2000 mg/kg body weight treatment group. The following clinical signs were observed during the duration of the study (days 1-3): lethargy, hunched posture, rough coat, diarrhea (1/3 animals). The body weight of the three animals decreased from 151, 221 and 202 g just before the start of the first treatment to 140, 204 and 188 g just before the third treatment.

RESULTS OF DEFINITIVE STUDY
The data are attached as background material.

- No statistically significant increase in the mean Tail Intensity (%) was observed in liver, blood and stomach cells of treated males at any of the dose levels tested compared to the vehicle treated animals.

- Positive control: EMS induced a statistically significant increase in the mean Tail Intensity (%) in cells of males when compared to the vehicle (12.6-fold in liver cells, 8.4-fold in blood cells, 1.9-fold in stomach). Hence, the acceptability criteria of the test were met.

-Vehicle control: The tail intensity of liver, blood and stomach cells of male rats were resp. 7.51%, 11.3% and 47.2%.

- Cell viability: The viability of all single suspension was high and in the range of 80 – 100%

- The animals of the groups treated with the negative and positive controls showed no treatment related clinical signs of toxicity or mortality. The following clinical observations were made in the groups treated with 500, 1000 and 2000 mg Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane /kg body weight:
- within approximately 1 hour after the first treatment none of the animals showed treatment related clinical signs;
- within approximately 25 and 49 hours after treatment, all animals treated with 1000 and 2000 mg/kg were lethargic and had diarrhea, a rough coat, and a hunched posture;
- within approximately 25 hours after treatment with 500 mg/kg, three animals were lethargic and showed a hunched posture. Within 48 hours after treatment one animals was lethargic and showed a hunched posture.

Any other information on results incl. tables

The concentrations of test substance solutions were in agreement with target concentrations (i.e. mean accuracies of respectively 90, 95 and 99%). No test substance was detected in control group (vehicle) formulation.

 The formulations of the highest and the lowest test concentrations were homogenous (i.e. coefficient of variation of respectively 3.4 and 2.4%).

The formulations at the entire range were stable at room temperature under normal laboratory light conditions for at least 4 hours (relative difference of the analysed concentration in the highest and the lowest test concentration before and after storage was -4.5% and 0.3%, respectively).



Applicant's summary and conclusion

Conclusions:
In an alkaline Comet assay, performed according to GLP principles, Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane did not induce DNA damage in blood, liver and stomach cells when tested up to a maximum single dose of 2000 mg/kg bw orally in rats.
Executive summary:

An alkaline Comet assay was performed with Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane according to GLP principles with 5 male rats dosed at 500, 1000 and 2000 mg/kg bw by gavage. Clinical signs including lethargy and diarrhea, rough coat, hunched posture were noted at 1000 and 2000 mg/kg bw, lethargy and hunched posture were seen at 500 mg/kg bw. No statistically significant increase in the mean Tail Intensity was observed in the blood, liver and stomach of Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane-treated male animals at any of the dose levels tested compared to the vehicle treated animals. Based on these results it is concluded that the vehicle control and the positive control showed adequate results and the test substance did not induce DNA damage in blood, liver and stomach cells when tested up to a maximum dose of 2000 mg/kg bw.