Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two Ames tests and an in vitro chromosome aberration study have been performed with XDI.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 12, 2008 - March 18, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
other: Chinese hamster lung fibroblasts (CHL/IU cells)
Details on mammalian cell type (if applicable):
Cells:
CHL/IU cells were supplied by Health Science Research Resources Bank, Japan Health Sciences Foundation on April 17, 2002. The modal number of chromosomes was 25 per cell. The doubling time was about 15 hours. It was confirmed in the testing facility that the cells were mycoplasma free and the spontaneous frequencies of cells with structural aberration and the numerical aberration were below 5%.

Storage of cells:
Cells were suspended in medium (Eagle's minimum essential medium, Nissui Pharmaceutical Co., Ltd.) and 10 vol% heat-inactivated newborn calf serum (NBCS, Sanko Junyaku Co., Ltd.) including 10 vol% DMSO and were frozen and stored in liquid nitrogen.

Culture condition:
Cells were cultured in a CO2 incubator (MCO-18AIC, SANYO Electric Co.), which was set at 37 degrees Celsius and 5% CO2 under humid condition

Subculture:
Cells were subcultured in a 90 mm diameter Petri dishes twice a week. Passage number of cells was at 11 for the cell growth inhibition test (dose range finder) and 17 for the chromosomal aberration test after the receipt.

Medium:
Basal medium (MEM) was prepared by adding L-Glutamine (final concentration: 0.292 g/L) and sodium hydrogen carbonate (final concentration: approximately 1.85 g/L) to Eagle's minimum essential medium (Lot no. 588803, Nissui Pharmaceutical Co., Ltd.) This medium was then supplemented with 10 vol% heat-inactivated newborn calf serum (lot. no. ASC29136, HyClone) and used for culture

Cell pre-culture:
A 60-mm diameter plastic dish (Asahi Glass Corp.) was used for cell culture. Five mL of a cell suspension of 1.5x10^4 cells/mL were seeded into a dish and were cultured continuously for 2 days in the cell growth inhibition test. Five mL of a cell suspension of 5.0x10^3 cells/mL were seeded into a dish and were cultured continuously for 3 days in the chromosomal aberration test.
Additional strain / cell type characteristics:
not applicable
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:
Dose range finding test:
Without S9 mix, 6 hr/24 hr exposure; 18 hr fixation: 7.34; 14.7; 29.4; 58.8; 118; 235; 470 and 940 µg/mL (=0.01 M)
With S9 mix, 6 hr exposure; 18 hr fixation: 7.34; 14.7; 29.4; 58.8; 118; 235; 470 and 940 µg/mL (=0.01 M)
First cytogenetic test:
Without S9-mix, 6 hr exposure; 18 hr fixation: 230; 280; 330; 380; 430 and 480 µg/mL
With S9-mix, 6 hr exposure; 18 hr fixation: 230; 280; 330; 380; 430; 480; 530 and 580 µg/mL
Second cytogenetic test: not conducted as the 1st was positive
Positive controls:
MMC, without S9 mix, 0.1 µg/mL for a 6 hr exposure period
CPA, with S9 mix, 6 µg/mL for a 6 hr exposure period
Vehicle / solvent:
- Solvent used: DMSO (purity: 100%), dehydrated with molecular sieves
- Justification for choice of solvent/vehicle: water is not possible, as the substance reacts with water. The substance dissolved at 188 mg/mL in dehydrated DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
; DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C (MMC) and Cyclophosphamide monohydrate (CPA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period:
Cell growth inhibition test (dose range finding assay): 2 days
Chromosomal aberration test: 3 days
- Exposure duration: 6 hr (with and without S9-mix) and 24 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hr

SPINDLE INHIBITOR (cytogenetic assays): demecolcine solution
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: duplicates in 1 experiment
NUMBER OF CELLS EVALUATED: 50 metaphase chromosome spreads per culture
DETERMINATION OF CYTOTOXICITY: number of cells of each culture was measured using a Microcell counter

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
The experiment was judged positive when the frequencies of cells with structural aberrations showed 10% or more with a dose-related increase or the frequencies of cells with structural aberrations showed 5% or more both in the chromosomal aberration test. The other cases were judged to be negative. No statistical analyses were performed.
Statistics:
Not applicable
Species / strain:
other: Chinese hamster lung fibroblasts (CHL/IU cells)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Precipitation: observed at the end of the treatment period at dose levels of 330 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- Little to no toxicity was observed up to and including the highest precipitating tested dose

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

- Without S9 mix, the frequencies of cells with structural aberrations at 230, 280 and 330 µg/mL were 4.0; 11.5 and 13%, respectively (more than 10% and a dose-related increase: positive). The frequencies of numerical aberration cells at 230; 280 and 330 µg/mL were 1.0; 2.0 and 1.5%, respectively (all below 5%).

- With S9 mix, the frequencies of cells with structural aberrations at 230, 280; 330 and 380 µg/mL were 5.0; 16.0; 23.5 and 38.5%, respectively (more than 10% and a dose-related increase: positive). The frequencies of numerical aberration cells at 230; 280; 330 and 380 µg/mL were 3.0; 3.5; 3.0 and 2.5%, respectively (all below 5%).

- No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the substance does not disturb mitotic processes and cell cycle progression.

Conclusions:
The substance did induce a relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix. It is concluded that this test is valid and that the substance is clastogenic in Chinese hamster lung fibroblasts.
Executive summary:

The potential of the substance to induce structural chromosome aberrations in cultured mammalian somatic cells has been investigated according to OECD 473 and GLP. Chinese hamster lung fibroblasts cells were treated for 6 hr (with and without S9-mix) and for 24 hr (without S9-mix) with the substance at doses ranging from 7.34 to 940 µg/mL (=0.01 M). Precipitation was observed (at the end of the treatment period) at dose levels of 330 µg/mL and above. The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

Without S9 mix, the frequencies of cells with structural aberrations at 280 and 330 µg/mL were 11.5 and 13%, respectively and with S9 mix, the frequencies of cells with structural aberrations at 280; 330 and 380 µg/mL were 16.0; 23.5 and 38.5%, respectively. As the frequency is more than 10% and increases in a dose-related manner, it is concluded that the substance is clastogenic in Chinese hamster lung fibroblasts.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 28, 1990 - April 3, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Performed in accordance with OECD 471 and GLP principles. But the report does not contain the laboratory historical control data of the vehicle and of the strain-specific positive controls. Also the doses of the test substance were tested in duplicate in each strain in the preliminary study, without a justification.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium, other: TA100, TA1535, TA98, TA1537 and TA1538
Species / strain / cell type:
E. coli WP2 uvr A
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:
Preliminary test (without and with S9-mix):
S. typhimurium TA100, TA1535, TA98, TA1537, TA1538 and E. coli WP2 uvr A: 50, 100, 500, 1000 and 5000 µg/plate

Main study:
Without and with S9-mix:
S. typhimurium TA100, TA1535 and E. coli WP2 uvr A: 125, 250, 500, 1000, 2000 and 4000 µg/plate
Without S9-mix:
S. typhimurium TA98, TA1537 and TA1538: 63, 125, 250, 500 and 1000 µg/plate
With S9-mix:
S. typhimurium TA98, TA1537 and TA1538: 125, 250, 500, 1000, 2000 and 4000 µg/plate

Experiment 2:
Without and with S9-mix:
S. typhimurium TA100, TA1535 and E. coli WP2 uvr A: 125, 250, 500, 1000, 2000 and 4000 µg/plate
Without S9-mix:
S. typhimurium TA98, TA1537 and TA1538: 63, 125, 250, 500 and 1000 µg/plate
With S9-mix:
S. typhimurium TA98, TA1537 and TA1538: 125, 250, 500, 1000, 2000 and 4000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods inc. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Preliminary study: doses of the test substance were tested in duplicate in each strain
- Main study: doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

Evaluation criteria:
Non parametric multiple range test (Ryan) was employed for analyzing the data. A reproducible and statistical significant dose related increase in revertant colonies for at least 1 strain is considered positive.
Species / strain:
S. typhimurium, other: TA100, TA1535, TA98, TA1537 and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(starting at 2000 µg/plate)
Vehicle 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:
(starting at 2000 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: No data

Mutagenic response of XDI in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay: see the attached background information.

Conclusions:
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. It is concluded that this test is valid and that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The genetic toxicity of the substance was assessed at concentrations of 4000 µg/plate using S. typhimurium TA100, TA1535, TA98, TA1537, TA1538 and E. coli WP2 uvr A strains. All bacterial strains showed negative responses with and without metabolic activation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus test was performed with XDI. Two COMET assays are available, perforned with substance analogue NBDI. The rationale to read across this data is attached in Section 13.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The rationale to read across the data is attached in Section 13.
Reason / purpose for cross-reference:
read-across source
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. This result is read across to the registered substance.
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. This result is read across to the registered substance.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The read across rationale is attached in Section 13.
Reason / purpose for cross-reference:
read-across source
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. This result is read across to the registered substance.
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. This result is read across to the registered substance.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 7 - March 19, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc. (Hino Breeding Center)
- Age at study initiation: 7 weeks old
- Weight at study initiation: 217-266.2 g, average 237.4 g
- Assigned to test groups randomly: yes
- Housing: 2 animals housed per cage (3 cages/group). Housed in hanging steel cages with wire-mesh floors.
- Diet: ad libitum to a diet of pellets (MF, Oriental Yeast Co., Ltd.)
- Water: ad libitum Hita City Water Supply (chlorine addition water) via an automatic water supply device.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8-23.9
- Humidity (%): 54.9-64.8
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Olive oil
- Justification for choice of solvent/vehicle: The test substance was dissolved at 100 mg/ml in olive oil. The test substance solution at 100 mg/ml in olive oil showed no change in color, exothermic reaction nor gas generation at room temperature within 4 hours after preparation. Therefore olive oil was preferably selected as vehicle.
- Concentration of test material in vehicle: 100 mg/ml was further diluted.
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no.: 042OMS
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulation was prepared on each administration day, stored at room temperature and used within 4 hours of preparation.
5.0 gram of test substance was weighed, vehicle was added and the test substance was dissolved by stirring to make 50 ml of 100 mg/ml tes substance solution. A portion of 50.0, 25.0, 12.5 and 6.25 mg/ml test substance formulation was made.
10 ml/kg was administered.
Duration of treatment / exposure:
Twice
Frequency of treatment:
24 hour interval
Post exposure period:
24 hours
Remarks:
Doses / Concentrations:
0, 62.5, 125, 250, 500, 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control: mitomycin C
- Justification for choice of positive control(s): MMC was described as the positive control substance in OECD guideline and the testing facility has background data.
- Route of administration: Intraperitoneal
- Doses / concentrations: 2 mg/kg/day
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the results of the acute oral toxicity study and fourteen day repeated dose oral toxicity study, it was judged that there was no sex difference in toxicity of the test substance. Dead animals were observed on the fourth day of the administration of 1000 mg/kg/day in the 14-d study. Therefore, male rats were used and the highest dose of test substance was 1000 mg/kg/day.

DOSES for observation:
The specimens of the negative and the positive control group and the top three doses were selected for observation. Specimens of 5 animals were taken for analysis.

TREATMENT AND SAMPLING TIMES:
24 hours after second administration animals were euthanized by cervical dislocation. The femur was removed and bone marrow cells were collected with approximately 3 ml of heat inactivated fetal bovine serum into a centrifuge tube, and the tube was centrifuged at 1000 rpm for 5 minutes.

DETAILS OF SLIDE PREPARATION:
A small amount of cell suspension was smeared on a slide glass. The smear specimens were fully air dried and fixed with methanol. In all animals, 2 specimens were prepared per animal. A drop of 40 microg/ml acridine orange solution was dropped onto the specimens, which were then covered with cover glass. Staining was carried out immediately before observation and specimens were observed under a fluorescence microscope.

METHOD OF ANALYSIS:
Two thousand immature erythrocytes (IE) per animal (1000 IE per specimen) were observed and the incidence of the IE with micronuclei (MNIE) per IE, abbreviated as MNIE%, was calculated.
Two hundred total erythrocytes per animal (100 TE per specimen) were observed and the ratio of IE per TE, abbreviated as IE%, was also calculated.
Evaluation criteria:
Study was valid, when the means of the MNIE% in the negative and the positive control groups were within the range of the background data in the testing facility and IE% of each dose of the test substance were not under 20% of negative control.
When MNIE% of the test substance treatment groups increased significantly compared with the negative control group and dose dependency was exhibited, the results for the test substance are judged to be positive.
Statistics:
Judgment of MNIE%: Conditional Binomial test (Kastenbaum and Bowman) was performed. When a significant increase was obtained at a dose, the Cochran-Armitage trend test would be expected to conduct to confirm whether there was a dose dependency.
Judgment of IE%: Bartlett's test for homogeneity of variance (all data except positive control). Because the variances were homogeneous, Williams' test was performed. No significant differences were found, Dunnett's test was performed to compare the mean in the negative control group with that in each dose group. Data from the negative and positive control groups were tested by the F test for homogeneity of vaiance between the groups. Since the result showed homogeneity, Student's t test was performed in order to compare the mean in the negative control group with that in the positive control group.
Statistical tool StatLight was used for mentioned tests.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No animals died before the end of the study. Clinical signs were seen in 1 animal in the 62.5 and 125 mg/kg/day dose group and in 4 animals in the 250 mg/kg/day dose group and in all animals in 500 and 1000 mg/kg/day group, until 1 day after the second administration. Clinical signs included decreased spontaneous locomotion, salivation, aperture respiration and incomplete eyelid opening.
Bodyweight of 2 animals in 250 mg/kg/day group and 1 animal in 1000 mg/kg/day group decreased by 11, 17 and 14%, respectively at 1 day after second administration. Other animals showed body weight within 10 % of starting body weight.

The average of MNIE% in the negative control and positive control groups were 0.17 and 3.47% respectively. MNIE% in te test substance groups was 0.13% at 250 mg/kg/day, 0.11% at 500 mg/kg/day and 0.10% at 1000 mg/kg/day. Thus, no significant increase was noted at any dose of the test substance treatment groups when compared with the negative control group. A significant increase was observed in the positive control group.

IE% in the negative control and positive control groups were 58.0 and 54.4% respectively. IE% at 250 mg/kg/day was 58.7%, at 500 mg/kg/day was 58.3% and at 1000 mg/kg/day was 55.6%. Thus, no significant difference was noted at any doses of the treatment groups and the positive control group compared with the negative control group.

Conclusions:
The frequency of the micronuclei did not increase by administering the test substance to rats. The positive control showed a significant increase compared to the negative control, it was confirmed that the test was valid.
The exposure of substance to bone marrow cells could not be proved as the IE% did not show significant differences in substance treated compared to negative control. However, doses were set based on dead animals found after 4 days of administration in 14-d repeated dose toxicity study, and toxic symptoms were observed in this study. The potential to induce micronuclei could be evaluated adequately in this study.
Executive summary:

In an in vivo micronucleus study performed according to OECD test guideline 474, XDI was administered orally twice up to 1000 mg/kg bw/day to rats. The frequency of the micronuclei did not increase by administering the test substance to rats. The positive control showed a significant increase compared to the negative control, it was confirmed that the test was valid. The exposure of substance to bone marrow cells could not be proved as the IE% did not show significant differences in substance treated compared to negative control. However, doses were set based on dead animals found after 4 days of administration in 14-d repeated dose toxicity study, and toxic symptoms indicative of systemic exposure were observed in this study. The potential to induce micronuclei could be evaluated adequately in this study. Therefore, it was concluded that XDI had no potential to induce the micronuclei under the present test conditions.

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

Additional information

In vitro

The genetic toxicity of the substance was assessed at concentrations of 4000 µg/plate using S. typhimurium TA100, TA1535, TA98, TA1537, TA1538 and E. coliWP2 uvr A strains. All bacterial strains showed negative responses with and without metabolic activation. In an old Ames test, performed with less strains, this result was confirmed.

The potential of the substance to induce structural chromosome aberrations in cultured mammalian somatic cells has been investigated according to OECD 473 and GLP. Chinese hamster lung fibroblasts cells were treated for 6 hr (with and without S9-mix) and for 24 hr (without S9-mix) with the substance at doses ranging from 7.34 to 940 µg/mL (=0.01 M). Precipitation was observed (at the end of the treatment period) at dose levels of 330 µg/mL and above. The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Without S9 mix, the frequencies of cells with structural aberrations at 280 and 330 µg/mL were 11.5 and 13%, respectively and with S9 mix, the frequencies of cells with structural aberrations at 280; 330 and 380 µg/mL were 16.0; 23.5 and 38.5%, respectively. As the frequency is more than 10% and increases in a dose-related manner, it is concluded that the substance is clastogenic in Chinese hamster lung fibroblasts.

In accordance with REACH column 2, Annex VIII and IX, no further in vitro studies have been performed, as the chromosome aberration study is positive. Therefore, in vivo testing has been performed.

In vivo

In an in vivo micronucleus study performed according to OECD test guideline 474, XDI was administered orally twice up to 1000 mg/kg bw/day to rats. The frequency of the micronuclei did not increase by administering the test substance to rats. The positive control showed a significant increase compared to the negative control, it was confirmed that the test was valid. The exposure of substance to bone marrow cells could not be proved as the IE% did not show significant differences in substance treated compared to negative control. However, doses were set based on dead animals found after 4 days of administration in 14-d repeated dose toxicity study, and toxic symptoms indicative of systemic exposure were observed in this study. The potential to induce micronuclei could be evaluated adequately in this study. Therefore, it was concluded that XDI had no potential to induce the micronuclei under the present test conditions.

In an alkaline Comet assay, performed according to GLP principles, substance analogue 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. This result is read across to XDI


Justification for classification or non-classification

Based on the available data, XDI does not have to be classified according to the CLP Regulation (EC) No. 1272/2008 for mutagenicity.