Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For 1,4-H6XDI, an analogue of 1,3-H6XDI, an AMES test is available in which the substance was found to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation. This result is read across to 1,3-H6XDI. The result is supported by the results of an AMES study performed with H6XDI, which is concluded to be 1,3-H6XDI (supporting study). As in vitro testing with analogues (isocyanates) resulted in false positive outcome, the validity of in vitro testing with 1,4-H6XDI is questionable. Further in vitro testing was therefore omitted.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
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:
read-across source
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 4.88 µg/plate without S9-mix and at 19.5 µg/plate with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1.22 µg/plate without S9-mix and at 4.88 µg/plate with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4.88 µg/plate without S9-mix and at 78.1 µg/plate with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 9.77 µg/plate with and without S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 39.1 µg/plate without S9-mix and at 78.1 µg/plate with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Precipitation: in all tester strains, at 5000 µg/plate without metabolic activation and at and above 1250 µg/plate with metabolic activation.
Cytotoxicity: in tester strain TA1537 at and above 1.22 µg/plate without S9-mix and at 4.88 µg/plate with S9-mix; in tester strain TA98 at 4.88 µg/plate without S9-mix and at 78.1 µg/plate with S9-mix; in tester strain TA100 at and above 9.77 µg/plate with and without S9-mix; in tester strain TA1535 at 4.88 µg/plate without S9-mix and at 19.5 µg/plate with S9-mix; in tester strain WP2uvrA at and above 39.1 µg/plate without S9-mix and at 78.1 µg/plate with S9-mix.
Mutagenicity: in all strains, in the absence and in the presence of S9-mix, no increase in the number of revertants was observed.

ACCEPTABILITY
- in the positive control, the increase in the number of revertant colonies was more than two-fold, compared to the solvent control.
- the number of revertant colonies in the plates in the solvent control group and in the positive control group were within the historical data range.
- no contaminants such as other bacteria were seen in this test system.

See attached illustration for more details.

Conclusions:
In an AMES test, 1,4-bis(isocyanatomethyl)cyclohexane was found to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation. This result is read across to 1,3-H6XDI.
Executive summary:

The mutagenic potential of 1,4-bis(isocyanatomethyl)cyclohexane was assessed in an Ames test, performed under GLP principles. The test item was tested in several strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and in one strain of Escherichia coli (WP2uvrA) in the absence and in the presence of a metabolic activation system (S9 -mix). A dose-range finding study and two independent main studies were conducted.Concentrations up to and including 5000 µg/plate were used in the dose-range finding study. Based on the results of the dose-range finding study, the concentrations were determined for the main studies: the top dose was the minimum dose at which growth inhibition was observed in the dose-range finding study.

The test item precipitated on the plates at the top dose of 5000 μg/plate in the absence of S9 -mix and at a dose of 1250 µg/plate in the presence of S9 -mix. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all tester strains at different dose levels, with and without S9 -mix. In all strains, in the absence and in the presence of S9-mix, no increase in the number of revertants was observed. The results of the solvent control and the positive controls were within the historical range of the test facility.

In conclusion, 1,4 -bis(isocyanamethyl)cyclohexane is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, with and without metabolic activation. This result is read across to 1,3-H6XDI.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
October - December 1977
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test performed according to published methods, performed before GLP introduction. Concentrations used not up to limit concentrations and not all required strains used. No independent repeat experiment included. Unprocessed data of H6XDI in reversion test are missing in the translated report.
Principles of method if other than guideline:
Reversion test according to Ames et al. (Proc. Nat. Acad. Sci. USA, 70: 782 (1973). Rec test according to Kada et al. (Mutation Research 16: 165 (1972). Some deviations were made (see in materials and methods description).
GLP compliance:
no
Remarks:
performed before GLP was in place
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
other: TA1537, TA98, TA100
Species / strain / cell type:
other: WP2hcr-
Species / strain / cell type:
other: Bacillus subtilis strains H17 (rec+) and M45 (rec-)
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenate, S9-mix, prepared from male Sprague Dawley rats pre-treated with phenobarbital and b-naphthoflavone
Test concentrations with justification for top dose:
reversion assay:
60, 120 and 240 ug XDI/plate without S9 mix; 120, 240 and 480 ug XDI/plate with S9 mix;
6.3 to 50 nl H6XDI/ plate
rec assay:
0.25, 2.5 and 25 uL H6XDI/disk;
30 mg XDI/ disk
Vehicle / solvent:
Vehicle used: DMSO
Postive and negative controls were dissolved in sterile distilled water or DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: ENNG for WP2hcr-, MNNG for TA100, 9AA for TA1537, 2NF for TA98, DAPA for all
Remarks:
For E.coli and S.Typhimurium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: DEN for WP2hcr-, DMN for TA100, 9AA for TA1537, AAF for TA98, 2AA for all E.Coli
Remarks:
For E.coli and S.Typhimurium
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: DAPA for M45, Kanamycin and AF-2 for all
Details on test system and experimental conditions:
Reversion tests:
Performed according to Ames et al. with the following modification: The reaction mixture was preincubated for 20 min at 37 degrees Celsius before mixed with soft agar and poured on the plate.
Rec-assay:
Performed according to Kada et al. except for the following: 0.1 ml of each bacterial culture was mixed with sof (top)agar and poured on the surface of nutrient agar plate. Paper disk impregnated with 0.025 ml of a solution of each agent was plased in the center of the top agar. The inhibition zone for each agent was measured after incubating the plate for 24 hr at 37 degrees Celsius.
Evaluation criteria:
Rec assay:
When the difference between the inhibition zones for rec+ and rec- strains was more than 2 mm, the rec-effect was indicated as positive.
Reversion assay:
Not specifically indicated
Species / strain:
other: TA100, TA1537, TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: WP2hcr-
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: Bacillus subtilis H17 and M45
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
other: valid for AF-2 and DAPA, not valid for Kanamycin
Additional information on results:
No cytotoxicity has been observed in the reversion assay.
Conclusions:
H6XDI gave negative results at doses 0.25 to 25 ul/disk in the rec-assay (Bacillus subtilis straisn H17 (rec+) and M45 (rec-). The substance gave negative results with and without metabolic activation in E coli strain WP2hcr-, and in S. typhimurium strains TA100, TA1537 and TA98. H6XDI was tested at doses ranging from 6.3 to 50 nl/ plate.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

With a comparable isocyanate, reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane (NBDI, CAS number: 74091-64-8), which tested negative in an AMES study, but gave a positive result in a mouse lymphoma, a combined in vivo Micronucleus/ COMET assay was performed. The outcome of this reliable GLP-study was negative. Based on this in vivo study it was in retrospective concluded that the positive outcome in the mouse lymphoma study was indeed false positive. It is of note that this in vivo study was evaluated by the Member States and the results in stomach tissue were rejected. A follow-up study confirmed that the outcome in stomach tissue is negative.

NBDI is a diisocyanate like 1,3-H6XDI. Both substances have two isocyanatomethyl-groups. For 1,3-H6XDI these are substituted to a saturated cyclohexane core, whereas NBDI is a bicyclo[2.2.1]heptane, i.e. cyclohexane with additional methylene bridge. No other functional groups next to the isocyanate group are present in either of the analogues. The isocyanate groups will readily react with water to yield a carbamic acid, which decarboxylates to produce CO2 and an amine. The latter is highly reactive and considered to be responsible for the false positive results when tested in vitro in mammalian cells. Since the isocyanate groups are expected to be the main determinant for this effect, it is justified to read across the data on NBDI to 1,3-H6XDI.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: 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:
In vitro testing with isocyanates resulted in false positive outcome (e.g. toluene diisocyanate; NBDI as discussed below). Therefore as a false positive outcome is expected, performance of in vitro testing using mammalian cells with 1,3-H6XDI was considered not justified. For Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane (NBDI, CAS number: 74091-64-8), which tested negative in an AMES study, but gave a positive result in a mouse lymphoma, a combined in vivo Micronucleus/ COMET assay was performed. The outcome of this reliable GLP-study was negative. Based on this in vivo study it was in retrospective concluded that the positive outcome in the mouse lymphoma study was indeed false positive. It is of note that this in vivo study was evaluated by the Member States and the results in stomach tissue were rejected. A follow-up study was performed, which confirmed the negative outcome in stomach tissue (the additional study is summarized in this dosier as well).
NBDI is a diisocyanate like 1,3-H6XDI. Both substances have two isocyanatomethyl-groups. For 1,3-H6XDI these are substituted to a saturated cyclohexane core, whereas NBDI is a bicyclo[2.2.1]heptane, i.e. cyclohexane with additional methylene bridge. No other functional groups next to the isocyanate group are present in either of the analogues. The isocyanate groups will readily react with water to yield a carbamic acid, which decarboxylates to produce CO2 and an amine. The latter is highly reactive and considered to be responsible for the false positive results when tested in vitro in mammalian cells. Since the isocyanate groups are expected to be the main determinant for this effect, it is justified to read across the data on NBDI to 1,3-H6XDI.
Reason / purpose:
read-across source
Key result
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. The result is read across to 1,3-H6XDI.
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. The result is read across to 1,3-H6XDI.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
IIn vitro testing with isocyanates resulted in false positive outcome (e.g. toluene diisocyanate; NBDI as discussed below). Therefore as a false positive outcome is expected, performance of in vitro testing using mammalian cells with 1,3-H6XDI was considered not justified. For Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane (NBDI, CAS number: 74091-64-8), which tested negative in an AMES study, but gave a positive result in a mouse lymphoma, a combined in vivo Micronucleus/ COMET assay was performed. The outcome of this reliable GLP-study was negative. Based on this in vivo study it was in retrospective concluded that the positive outcome in the mouse lymphoma study was indeed false positive. It is of note that this in vivo study was evaluated by the Member States and the results in stomach tissue were rejected. A follow-up study was initiated, and based on preliminary results the outcome in stomach tissue is negative. As soon as the final report is issued, the current dossier will be updated to reflect the full data-set.
NBDI is a diisocyanate like 1,3-H6XDI. Both substances have two isocyanatomethyl-groups. For 1,3-H6XDI these are substituted to a saturated cyclohexane core, whereas NBDI is a bicyclo[2.2.1]heptane, i.e. cyclohexane with additional methylene bridge. No other functional groups next to the isocyanate group are present in either of the analogues. The isocyanate groups will readily react with water to yield a carbamic acid, which decarboxylates to produce CO2 and an amine. The latter is highly reactive and considered to be responsible for the false positive results when tested in vitro in mammalian cells. Since the isocyanate groups are expected to be the main determinant for this effect, it is justified to read across the data on NBDI to 1,3-H6XDI.
Reason / purpose:
read-across source
Key result
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. This result is read across to 1,3-H6XDI.
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. This result is read across to 1,3-H6XDI.

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:
In vitro testing with isocyanates resulted in false positive outcome (e.g. toluene diisocyanate; NBDI as discussed below). As a false positive outcome is expected, performance of in vitro testing using mammalian cells with 1,3-H6XDI was considered scientifically not justified. For Reaction mass of 2,5-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane and 2,6-bis(isocyanatomethyl)-bicyclo[2.2.1]heptane (NBDI, CAS number: 74091-64-8), which tested negative in an AMES study, but gave a positive result in a mouse lymphoma, a COMET assay in stomach was performed to verify the results of the combined COMET/ in vivo Micronucleus study. The outcome of this reliable GLP-study was also negative. Based on this in vivo study it was in retrospective concluded that the positive outcome in the mouse lymphoma study was indeed false positive. NBDI is a diisocyanate like 1,4-H6XDI. Both substances have two isocyanatomethyl-groups. For 1,4-H6XDI these are substituted to a saturated cyclohexane core, whereas NBDI is a bicyclo[2.2.1]heptane, i.e. cyclohexane with additional methylene bridge. No other functional groups next to the isocyanate group are present in either of the analogues. The isocyanate groups will readily react with water to yield a carbamic acid, which decarboxylates to produce CO2 and an amine. The latter is highly reactive and considered to be responsible for the false positive results when tested in vitro in mammalian cells. Since the isocyanate groups are expected to be the main determinant for this effect, it is justified to read across the data on NBDI to 1,3-H6XDI.
Reason / purpose:
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 1,3-H6XDI.
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 1,4-H6XDI.

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

Additional information

The mutagenic potential of 1,4-bis(isocyanatomethyl)cyclohexane was assessed in an Ames test, performed under GLP principles. The test item was tested in several strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and in one strain of Escherichia coli (WP2uvrA) in the absence and in the presence of a metabolic activation system (S9 -mix). A dose-range finding study and two independent main studies were conducted. Concentrations up to and including 5000 µg/plate were used in the dose-range finding study. Based on the results of the dose-range finding study, the concentrations were determined for the main studies: the top dose was the minimum dose at which growth inhibition was observed in the dose-range finding study.

The test item precipitated on the plates at the top dose of 5000 μg/plate in the absence of S9 -mix and at a dose of 1250 µg/plate in the presence of S9 -mix. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all tester strains at different dose levels, with and without S9 -mix. In all strains, in the absence and in the presence of S9-mix, no increase in the number of revertants was observed. The results of the solvent control and the positive controls were within the historical range of the test facility.

In conclusion, 1,4 -bis(isocyanamethyl)cyclohexane is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, with and without metabolic activation. This result is read across to 1,3-H6XDI.

H6XDI gave negative results at doses 0.25 to 25 ul/disk in the rec-assay (Bacillus subtilis straisn H17 (rec+) and M45 (rec-). The substance gave negative results with and without metabolic activation in E coli strain WP2hcr-, and in S. typhimurium strains TA100, TA1537 and TA98. H6XDI was tested at doses ranging from 6.3 to 50 nl/ plate.

The results of an in vivo Micronucleus/COMET assay performed with NBDI, an isocyanate with a comparable structure, were read across to 1.3 -H6XDI. The results of this study demonstrated that there are no indications the isocyanate groups have genotoxic properties. This results in stomach tissue was confirmed in a second COMET assay. Both in vivo tests were done up to and including toxic levels.

Justification for classification or non-classification

Based on the available studies, 1,3-H6XDI is not classified for genotoxic properties according to the CLP Regulation (EC) No. 1272/2008.