Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro tests were performed with the registered substance: an Ames test, and an in vitro mammalian chromosome aberration test and a mammalian cell gene mutation assay. The results of the mammalian cell gene mutation assay triggered the need to perform further in vivo testing.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 7, 1991 - June 26, 1991
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
The preliminary test was carried out at 8 doses of 39 to 5000 µg/plate
Main study:
With metabolic activation: 39; 78; 156; 313; 625, 1250 and 2500 µg/plate
Without metabolic activation: 20; 39; 78; 156; 313, 625 and 1250 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
(see below)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 hour
NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if the total number of revertants in the tester strains is not greater than two (2) times the concurrent negative controls.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(in all strains +/- S9 but starting at different concentration)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 2500 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative (non-treatment and solvent) control values were within the laboratory historical control data ranges indicating that the test conditions were adequate
ADDITIONAL INFORMATION ON CYTOTOXICITY:
-Toxicity was observed in all strains tested in both experiments
Conclusions:
A bacterial reverse mutation assay was conducted equivalent to OECD 471 (Ames test) and GLP principles. All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 22, 1990 - June 26, 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Conducted according to the EC-method and GLP principles. Deviations: - The positive control in the 6h treatment time (without S9-mix) was not valid as no significantly increase in chromosome aberrations was observed when compared with the negative control. - No data about precipitation and no cytotoxicity was observed in the main study in all treatment times. The data has been approved by the German Competent Authority in 1993.
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese Hamster Lung (CHL/IU)
Details on mammalian cell type (if applicable):
Details: Established CHL/IU cells (purchased from Dai Nippon Pharmaceutical company on June 20, 1989) derived from lungs of female Chinese Hamster were used. Cell suspension supplemented with 10% of DMSO was frozen and stored in liquiid nitrogen. These cells were cultured in growth medium prior to use; out of the cultured cells, cells within 5 passages after origination from the cryopreserved state were used for the test.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix: 25, 50, 75, 100, 125 and 150 µg/mL
Main test:
Without S9-mix, 24 hr exposure: 20, 40 and 80 µg/mL
Without S9-mix, 48 hr exposure: 17.5, 35 and 70 µg/ mL
With S9-mix and without S9-mix, 6 hr exposure: 15, 30 and 60 µg/ mL
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix: dose 0.03 µg/mL (24h and 48h exposure)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with and without S9-mix: dose 20 µg/mL (6h exposure)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 3 days
- Exposure duration: 6 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
NUMBER OF REPLICATIONS: 2
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
A test substance is considered positive (clastogenic) in the chromosome aberration test if it induces a (dose-related statistically) significant increase in the number of cells with chromosome aberrations.
Species / strain:
other: Chinese Hamster Lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the dose range finder
Vehicle controls validity:
valid
Positive controls validity:
other: Positive controls valid in the 6h (with S9-mix), and 24h and 48h treatment time (without S9-mix). The positive control in the 6h treatment time (without S9-mix) was not valid as no significantly increase in chromosome aberrations was observed.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no data

COMPARISON WITH HISTORICAL CONTROL DATA:
The findings are within the laboratory historical control data, for the solvent and for the positive control.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was observed in the dose range finding study:
Survival:
-50% at 75 µg/mL after 24 hours of treatment (-S9 mix)
-31% at 75 µg/mL after 48 hours of treatment (-S9 mix)
-14% at 75 µg/mL after 6 hours of treatment (+S9 mix)
Conclusions:
The substance did not induce structural chromosomal aberrations and polyploid cells, but no data about precipitation and no cytotoxicity was observed in the main study in all treatment times.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19-Mar-2010 to 17-May-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
-Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Exp 1: Without and with S9-mix, 3 hours treatment: 33, 100, 333, 1000 and 2062 µg/mL
Exp 2: Without and with S9-mix, 3 hours treatment: 0.003, 0.03, 0.3, 3 and 33 µg/mL
Mutation Experiment:
Without S9-mix, 3 hours treatment: 0.03, 0.1, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 µg/mL
With S9-mix, 3 hours treatment: 0.3, 3, 5, 7.5, 10, 13 and 16 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 15 µg/mL
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more then MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 333 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 3 and 33 µg/mL in the absence and presence of S9-mix, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The relative total growth of the highest test substance concentrations was reduced by 88 and 89% compared to the total growth of the solvent controls in the absence and presence of S9-mix, respectively.


Conclusions:
A mammalian cell gene mutation assay was performed according to OECD and/or EC guidelines and GLP principles. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. It is concluded that 2,5(or 2,6)-bis-isocyanatomethyl-bicyclo[2.2.1]heptane is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.
Executive summary:

2,5(or 2,6)-bis-isocyanatomethyl-bicyclo[2.2.1]heptane induced 5.3- and 6.2-fold increases in the mutation frequency in the absence and presence of S9-mix, respectively. The mutation frequencies were above the GEF + MF(controls)(203per 106survivorsin the absence and presence of S9-mix). Since the increases observed were above the GEF, more than three-fold, outside the historical control data range and in a dose dependent manner, these increases are considered biologically relevant and 2,5(or 2,6)-bis-isocyanatomethyl-bicyclo[2.2.1]heptane is considered mutagenic.

  

Both in the absence and presence of S9-mix, 2,5(or 2,6)-bis-isocyanatomethyl-bicyclo[2.2.1] heptane induced significant increases in the mutation frequency of both the small and large colonies, as compared to the mean mutation frequency of the small and large colonies of the solvent controls. This indicates increases in both chromosome aberrations and gene mutations.

 

Since clear, dose dependent, positive responses were observed in the mutation frequency at the TK locus after treatment of the L5178Y cells with 2,5(or 2,6)-bis-isocyanatomethyl-bicyclo[2.2.1] heptane, no repeat assay was performed.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo Micronucleus assay combined with an alkaline Comet assay was performed. As the results of the Comet assay in stomach cells was not considered reliable, a second alkaline Comet assay with stomach tissue was performed. The data show that the registered substance is not genotoxic in vivo.

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Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 February 2018 - 22 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The test was initiated upon request of ECHA (decision number TPE-D-2114376203-54-01/F).
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
Rat was selected as recommended in the guideline. The Crl:CD(SD) strain was selected, because BSRC has abundant usage experience and a large historical database on this strain.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 283 to 309 g
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: 2 rats/cage
- Diet: free access to commercial diet (CRF-1, lot No. 171024, Oriental Yeast)
- Water: free access to tap water from water bottles
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8-23.2
- Humidity (%): 47-62
- Air changes (per hr): 12 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 7 March 2018 - 8 March 2018
Route of administration:
oral: gavage
Vehicle:
- Solvent used: corn oil
- Concentration of test material in vehicle: 25, 50 and 100 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot no.: SAG4916 (Wako Pure Chemical Industries, Ltd.)
- The test substance is reported to be stable in the vehicle (corn oil) at room temperature under normal laboratory light conditions for at least 4 hours.
Details on exposure:
The individual dosing volume (mL) were calculated on the basis of the body weight measured at the time of assignment to dose groups.
Duration of treatment / exposure:
Two consecutive days
Frequency of treatment:
Once daily
Post exposure period:
3 hours
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 males were dosed, 5 males analysed
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate (500 mg) was dissolved in 25 mL of water for injection to prepare a 20 mg/mL solution (dose of 200 mg/kg bw). This positive control solution was prepared just before administration.
Tissues and cell types examined:
Stomach tissue
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In dose range-finding study in rats treated with 0, 222, 667 or 2000 mg/kg of test substance in corn oil, decrease in locomotor activity, salivation and diarrhea were observed in the 667 mg/kg group, and decrease in locomotor activity, salivation, loose stool, diarrhea and soiled fur were observed in the 2000 mg/kg bw group. No clinical signs of toxicity were observed in the 222 mg/kg bw group. The body weight of 667 and 2000 mg/kg bw groups decreased. In the gross observation, edema of the forestomach was observed in the 2000 mg/kg bw group. Therefore, 1000 mg/kg, which was considered to be the maximum tolerated dose, was selected as the highest dosage level and a total of 3 dosage levels, 250, 500 and 1000 mg/kg bw, were selected in the present study.

TREATMENT AND SAMPLING TIMES:
The oral route that was assumed to be one of the exposure routes for the human was selected as an administration route of the vehicle and the test substance formulations.

DETAILS OF SLIDE PREPARATION:
The stomachs were macroscopically examined at necropsy, and gross findings were recorded including the affected portion, color, size. Any fouling (blood, etc.) was rinsed off using homogenizing buffer. The forestomach was removed and discarded. The glandular section of the stomach was cut open and rinsed with the homogenizing buffer. After collecting a part of the glandular stomach for pathological specimen, the remaining glandular stomach was incubated in cooled homogenizing buffer for 15 to 30 minutes. After incubation, the surface epithelium was gently scraped several times using a blade and discarded. In a dish containing homogenizing buffer, the stomach epithelium was scraped several times with the blade to release the cells. The cell suspension was put into a tube. After the appropriate amount of the supernatant is discarded by centrifugation (800 rpm, 5 minutes), the cells were re-suspended in the remaining supernatant and 10 μL of the cell suspension was put into a microtube.
A superfrosted glass slide was pre-coated with 1.0% agarose gel. Ninety microliters of 0.5% low-melting agarose gel was added to a microtube. After mixing, 90 μL of the cell-agarose mixture was placed on the pre-coated slide and covered with a non-coated superfrosted glass slide. After the agarose solidified, the covering glass slide was removed. Another layer of 0.5% low-melting agarose (90 μL) was added in the same manner.
Each slide was placed in lysing solution and left overnight under the refrigerated and light-protected conditions. Three slides (2 slides for evaluation, 1 for spare) per organ were prepared.

METHOD OF ANALYSIS:
One hundred fifty cells (75 cells per slide), i.e. 750 cells per group (5 animals), were analyzed. Hedgehogs were excluded from comet image analysis. Separately, 150 cells per animal (75 cells per slide) were analyzed using the fluorescence microscope (×200), and the number of hedgehogs was counted.
The percentage of DNA in the tail relative to the total (% tail DNA: Tail % intensity) was used as an indicator for DNA damage. The median % tail DNA for each slide (slide value) was determined, and the mean of the slide values for each animal (animal value) was calculated.
Evaluation criteria:
Interpretation of the results:
a) At least one of the test substance-treated groups exhibits a statistically significant increase of % tail DNA compared with the negative control.
b) A significant dose dependence in the % tail DNA in the test substance-treated groups is present.
c) Any of the % tail DNA (mean of animal values) in the test substance-treated group is outside the distribution (mean±3SD) of the test facility’s historical data of the negative control group.
If all of the above criteria were met, the test result was considered to be positive. In addition, the biological relevance of the results was taken into consideration for the final judgment.

Validity of the study:
a) The mean % tail DNA in the negative control group should be within the acceptable ranges (mean±3SD) calculated from the test facility’s historical data.
b) The mean % tail DNA in the positive control group should be increased with a statistically significant difference compared with that in the negative control group. The study was considered to be successfully performed because the above criteria were met.
Statistics:
Each animal value of % tail DNA was logarithmically transformed prior to statistical analysis. The % tail DNA (mean of animal values) was analyzed by Dunnett’s multiple comparison test (two-sided, significant level of 0.05) between the negative control group and each test substance-treated group. Since no significant difference was observed, the dose dependence (trend) was not analyzed. For the comparison between the negative control group and positive control group, the % tail DNA (mean of animal values) was analyzed by Aspin-Welch’s t test (one-sided, significant level of 0.025).
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Decreased body weights, clinical signs and local effects on forestomach were seen at 500 and 1000 mg/kg bw.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mortality
No mortality occurred.

Gross necropsy
In the 500 and 1000 mg/kg bw treatment groups, edema in the forestomach was observed in 2 and 3 rats, respectively. There were no macroscopic findings in the 250 mg/kg bw group.

Body weight and general condition
The body weights of animals in 500 and 1000 mg/kg bw groups were decreased compared to control animals (mean weight loss of -7g, -17g, -32g for the groups dosed with 250, 500 and 1000 mg/kg bw versus a mean weight loss of -5g for the controls). No deaths were observed in any groups. In the 250 mg/kg group, loose stool was observed in two animals. In the 500 mg/kg bw and 1000 mg/kg bw groups, loose stool, soiled fur, decrease in locomotor activity, or diarrhea were observed in several animals.

DNA damage
In the negative control group, the mean % tail DNA was 3.87, which was within the acceptable ranges calculated from the test facility’s historical data.
The mean % tail DNA was 3.42, 3.63 and 3.83 for the NBDI-treated groups at 250, 500 and 1000 mg/kg bw, respectively. No statistically significant increase was observed as compared with the negative control group.
The frequencies of hedgehogs were 0.3, 0.7 and 0.4% for the NBDI-treated groups at 250, 500 and 1000 mg/kg bw, respectively, and no apparent increase was observed in any of the treatment groups as compared with the negative control group (1.6%).
The mean % tail DNA in the positive control group was 27.86 with a significant increase, indicative of a valid response.

Conclusions:
In an alkaline Comet assay performed according to OECD guideline 489 and GLP principles, NBDI did not induce DNA damage in stomach cells when tested up to a maximum oral dose of 1000 mg/kg bw in rats.
Executive summary:

An in vivo alkaline comet assay was conducted according to OECD guideline 489 and GLP principles using Crl:CD(SD) male rats to assess the potential of NBDI to induce DNA damage. Based on a dose range finding study, the maximum tolerated dose was concluded to be 1000 mg/kg bw/day, therefore the assay was performed with three dose levels 250, 500 and 1000 mg/kg bw.

In the main study, decreased body weights, clinical signs and local effects on forestomach were seen at 500 and 1000 mg/kg bw. No statistically significant increase in the % tail DNA was observed in the NBDI-treated groups as compared with the negative control group. In addition, the % tail DNA were within the acceptable ranges (mean±3SD) calculated from this test facility’s historical data of the negative control group.

The % tail DNA in the negative control group were within the acceptable ranges calculated from this test facility’s historical data, and the % tail DNA in the positive control group was increased with a statistically significant difference compared with that in the negative control group. These results supported the validity of this study. It is therefore concluded that NBDI did not induce DNA damage in the stomach of rats under the conditions employed in this study.

Endpoint:
in vivo mammalian cell study: 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.
Reason / purpose:
reference to same study
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
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
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)
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
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.
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.

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).



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.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
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 the OECD guideline and GLP pinciples.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(21 July 1997)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(31 May 2008)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
(August 1998)
Qualifier:
according to
Guideline:
other: - ICH S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (2012).
GLP compliance:
yes
Type of assay:
micronucleus assay
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
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 once at t=0 h.

Post exposure period:
The animals were sacrified at approximately t=49-50 h (approximately 3-4 hours after the third treatment)
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):
Cyclophosphamide (CP; CAS no. 50-18-0; Baxter B.V., Utrecht, The Netherlands) at 20 mg/kg body weight dissolved in physiological saline. The stock solutions of CP were stored in aliquots at ≤-15°C in the dark and one sample was thawed immediately before use. The route of administration was consistent with those of the test substance. The dosing volume was 10 mL/kg body weight.
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CELL ISOLATION
One femur was removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 4 mL of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 216 g for 5 min.

PREPARATION OF BONE MARROW SMEARS
The supernatant was removed with a Pasteur pipette. Approximately 500 µl serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. Two slides were prepared per animal.

STAINING OF BONE MARROW SMEARS
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

ANALYSIS FOR MICRONUCLEI
To prevent bias, all slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 2000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated. Parts on the slides that contained mast cells that might interfere with the scoring of micronucleated polychromatic erythrocytes were not used for scoring.
Evaluation criteria:
The micronucleus test is considered acceptable if it meets the following criteria:
a) The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the historical control data range; b) The positive control substance induces a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes; c) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range. If (one of) the acceptability criteria are not met and the Study Director decides that this has a critical effect on the study, the test will be rejected and repeated.

A test substance is considered positive in the micronucleus test if:
It induces a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals should be above the historical control data range.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations show a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals should be within the historical control data range.
Statistics:
Wilcoxon Rank Sum Test, one-sided is used to test for significant increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes.
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
- Induction of micronuclei (for Micronucleus assay): No statistically significant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of test substance treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio showed a dose dependent decrease from 0.94 ± 0.05 in the vehicle treated rats to 0.58 ± 0.22 in the 2000 mg/kg group (demonstrating toxic effects on erythropoiesis).
- 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.

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).



Conclusions:
In an in vivo Micronucleus assay, performed according to OECD guideline and GLP principles, Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane is not clastogenic or aneugenic in the bone marrow when tested up to a maximum single oral dose of 2000 mg/kg bw in rats.
Executive summary:

An in vivo Micronucleus 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 OECD guideline and 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. The PCE/NCE ratio showed a dose dependent decrease from 0.94 ± 0.05 in the vehicle treated rats to 0.58 ± 0.22 in the 2000 mg/kg group (demonstrating toxic effects on erythropoiesis). Analysis of the bone marrow smears showed no statistically significant increase in the mean frequency of micronucleated polychromatic erythrocytes in the bone marrow of test substance treated animals compared to the vehicle treated animals. Based on these results it is concluded that the test is valid and that the test substance is not clastogenic or aneugenic in the bone marrow when tested up to a maximum dose of 2000 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro data

In a bacterial reverse mutation assay conducted equivalent to OECD 471 (Ames test) and GLP principles, all strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

In an in vitro mammalian chromosome aberration test, the substance did not induce structural chromosomal aberrations and polyploid cells.

In a mammalian cell gene mutation assay, performed according to OECD and/or EC guidelines and according to GLP principles, dose dependent, positive responses were observed in the mutation frequency at the TK locus after treatment of L5178Y cells with the substance. Both in the absence and presence of S9-mix, the substance induced significant increases in the mutation frequency of both the small and large colonies, as compared to the mean mutation frequency of the small and large colonies of the solvent controls.

Based on the results obtained in the mammalian cell gene mutation assay, further in vivo testing was performed.

 

In vivo data

An in vivo Micronucleus 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 OECD guideline and 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. The PCE/NCE ratio showed a dose dependent decrease from 0.94 ± 0.05 in the vehicle treated rats to 0.58 ± 0.22 in the 2000 mg/kg group (demonstrating toxic effects on erythropoiesis). Analysis of the bone marrow smears showed no statistically significant increase in the mean frequency of micronucleated polychromatic erythrocytes in the bone marrow of test substance treated animals compared to the vehicle treated animals. Based on these results it is concluded that the test is valid and that the test substance is not clastogenic or aneugenic in the bone marrow when tested up to a maximum dose of 2000 mg/kg bw.

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.  Short description of key information: Negative in the Ames test, negative in the in vitro chromosome aberration test, but positive in the mouse lymphoma test. In an in vivo Micronucleus assay, performed according to OECD guideline and GLP principles, Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane is not clastogenic or aneugenic in the bone marrow when tested up to a maximum single oral dose of 2000 mg/kg bw in rats. 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. 

In order to verify the results in stomach, a second in vivo Comet assay was performed according to OECD guideline 489 and GLP principles using Crl:CD(SD) male rats. In the main study, decreased body weights, clinical signs and local effects on forestomach were seen at 500 and 1000 mg/kg bw. No statistically significant increase in the percentage tail DNA was observed in the NBDI-treated groups as compared with the negative control group. In addition, the percentage tail DNA were within the acceptable ranges (mean±3SD) calculated from this test facility’s historical data of the negative control group. The percentage tail DNA in the negative control group were within the acceptable ranges calculated from this test facility’s historical data, and the percentage tail DNA in the positive control group was increased with a statistically significant difference compared with that in the negative control group. These results supported the validity of this study. It is therefore concluded that NBDI did not induce DNA damage in the stomach of rats under the conditions employed in this study.

Justification for classification or non-classification

Based on the available data, the substance is not classified for genotoxic properties in accordance with the CLP Regulation.