Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An AMES test was performed with 1,4-bis(isocyanatomethyl)cyclohexane, which showed that the substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation. In vitro testing with isocyanates has been reported to result in false positive outcome. Therefore as a false positive outcome is expected, performance of in vitro testing using mammalian cells with 1,4-H6XDI was considered not justified.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 February 2009 - 13 April 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: Standards Established by the Minister of Labour in Accordance with the Industrial Safety and Health Law, Article 57-2-1
Version / remarks:
Notification No. 77, September 1, 1988, and Notification No. 67, June 2 1997, Ministry of Labour, Japan
Deviations:
no
Qualifier:
according to
Guideline:
other: Study Methods on New Chemical Substances, etc.
Version / remarks:
November 21, 2003; YakuShokuHatsu No. 1121002, Heisei 15-11-13 SeiKyoku No. 2, and KanHoKiHatsu No. 031121002, Revised finally on November 20, 2006
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Solubility in water: insoluble
Solubility in DMSO: ≥ 50 mg/mL
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from male rats induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Dose-range finding study (all tester strains, with and without S9): 1.22, 2.44, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate

Concentrations used in the main studies were selected based on the results of the dose-range finding study. As the maximum dose, the minimum dose at which growth inhibition was observed was selected.

First main study (without S9)
TA100: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
TA1535: 0.15, 0.31, 0.61, 1.22, 2.44 and 4.88 µg/plate
WP2uvrA: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA98: 0.15, 0.31, 0.61, 1.22, 2.44 and 4.88 µg/plate
TA1537: 0.04, 0.08, 0.15, 0.31, 0.61 and 1.22 µg/plate

First main study (with S9)
TA100: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
TA1535: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
WP2uvrA: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA98: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA1537: 0.15, 0.31, 0.61, 1.22, 2.44 and 4.88 µg/plate

Second main study (without S9)
TA100: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
TA1535: 0.15, 0.31, 0.61, 1.22, 2.44 and 4.88 µg/plate
WP2uvrA: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA98: 0.15, 0.31, 0.61, 1.22, 2.44 and 4.88 µg/plate
TA1537: 0.04, 0.08, 0.15, 0.31, 0.61 and 1.22 µg/plate

Second main study (with S9)
TA100: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
TA1535: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
WP2uvrA: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA98: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA1537: 0.15, 0.31, 0.61, 1.22, 2.44 and 4.88 µg/plate
Vehicle / solvent:
- Solvent: DMSO
- Justification for choice of solvent: the test item was soluble in DMSO at and above 50mg/mL (according to solubility test at Bozo Research Center Inc.)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: ICR-191; 2-aminoanthracene; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
Remarks:
For more details on positive controls see 'any other information on materials and methods'
Details on test system and experimental conditions:
A dose-range finding study and two independent main studies were performed.

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 20 minutes (while shaking)
- Exposure duration: 48 hours (dose-range finding), 50 hours (first main study) and 48.5 hours (second main study)

NUMBER OF REPLICATIONS: 2

METHOD OF SLIDE PREPARATION: D-biotin, L-histidine or L-tryptophan solutions were added to a soft agar solution to prepare top agar. Top agar were kept in a therostatic chamber set at 45°C to prevent fixation.

DETERMINATION OF CYTOTOXICITY
- Method: number of revertant colonies, counted with an automatic colony counter

- OTHER: the presence or absence of growth inhibition was observed using a stereoscopic microscope.
Evaluation criteria:
If a two-fold or more increase in the number of revertant colonies compared to that of spontaneous revertant colonies (solvent control) and dose-response and reproducibility were noted, or even if no clear dose-response was observed but there was at least a two-fold increase in comparison with the number of spontaneous revertant colonies and reproducibility was observed, the test item was considered to be positive.
Statistics:
No statistical method applied.
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.
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.

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

Genetic toxicity in vivo

Description of key information

The data obtained in an in vivo micronucleus/ COMET assay performed with a substance analogue were read across.

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 1,4-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. The results of the follow-up study confirm that the results in stomach tissue are negative (study summaru included in this dossier as well).
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 1,4-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 1,4-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 1,4-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:
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 1,4-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 1,4-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 1,4-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 1,4-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 1,4-H6XDI.

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

Additional information

Justification for classification or non-classification

Based on the available data, the substance is not classified for mutagenicity in accordance with CLP Regulation (EC) No. 1272/2008.