Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
1) Ames-test, 1990, strains TA 1535, TA 100, T 1537 and TA 98, according to OECD 471, tested up to precipitation, negative / non mutagenic 2) Prediction via OECD Toolbox - Chromosome aberration, negative 3) Prediction via OECD Toolbox - gene mutation, negative 4) Read across from TDI, Ames-test, strains TA98, TA100, TA 1535, TA 1537, similar or equivalent to OECD 471, tested up to precipitation, positive / mutagenic 5) Read across from TDI, in vitro mammalian chromosome aberration test, primary lymphocytes, equivalent or similar to OECD 473, positive / mutagenic 6) Read across from TDI, forward mutation assay at the thymidine kinase locus in mammalian cells, L5178Y tk+/tk mouse lymphoma cells, similar or equivalent to OECD 476, tested up to precipitation, positive / mutagenic 7) Prediction via Toxtree - in vivo micronucleus test, possibly positive as one strucutral alert was identified
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-08-03 - 1989-11-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, only 4 Salmonella strains tested (TA98, TA100, TA1535 and TA1537)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
yes
Remarks:
only 4 Salmonella strains tested (TA98, TA100, TA1535, TA1537)
Qualifier:
according to
Guideline:
other: EEC Directive 84/449/EEC B.14. Other Effects - Mutagenicity Salmonella typhimurium Reverse Mutation Test
Qualifier:
according to
Guideline:
other: New and Revised Health Effects Test Guidelines October 1984. (U.S.) Environmental Protection Agency Washington, DC (PB 84-233295). HG - Gene Muta - S. typhimurium, October 1984
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
his-
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
other: histidine-auxotrophic strains
Metabolic activation:
with and without
Metabolic activation system:
The 9000 g fraction of homogenized mammalian livers: The rat S9 mix comprised 30% S9 fraction, 70% cofactor solution.
Test concentrations with justification for top dose:
Doses up to and including 5000 µg per plate

The following doses of Triisopropyldiisocyanatobenzene were evaluated:

Negative control: 0
Triisopropyldiisocyanatobenzene: 5000, 1000, 200, 40, 8 pg per plate
Positive control:
Na-azide 10 (only TA 1535) pg per plate
NF 0.2 (only TA 100) pg per plate
4-NPDA 10 (only TA 1537) pg per plate
4-NPDA 0.5 (only TA 98) pg per plate
2-AA: 3 pg per plate

Due to the substance's toxicity and precipitation, doses ranging from 62.5 µg to 2000 µg per plate were chosen for the repeat tests.
Vehicle:
- Vehicle(s)/solvent(s) used: Ethylene glycol dimethylether (EGDE) and for positive controls dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The used solvent was chosen out of the following solvents, in the order given: water, ethanol, acetone, DMSO, DMF, and ethylene glycol dimethylether according to information given by the internal sponsor.
Negative controls:
yes
Remarks:
Solvent minus test substance
Solvent controls:
yes
Remarks:
EGDE(Triisopropyldiisocyanatobenzene) & DMSO (positive control)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene
Remarks:
The positive controls sodium azide, nitrofurantoin and 4-nitro-1,2-phenylene diamine were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.
Details on test system and conditions:
The original strains were obtained from Prof. Bruce Ames and arrived at the testing laboratory on December 12, 1986.

Mammalian metabolism, which is of great significance in chemical mutagenesis, is simulated in this test by the 9000 g fraction of homogenised mammalian livers. It was made from the livers of at least six adult male Sprague Dawley rats. The S9 mix was freshly prepared (Ames et al., 1973a) and used only on the same day. It was placed in a vessel with a double glass wall until used. The hollow wall was filled with ice to keep the S9 mix permanently cold.
Together with co-factors, this forms the "S9 mix" which represents the metabolic model in this test. S9 mix consists of a cofactor solution* and the corresponding volume of S9 fraction. In all tests, the S9 mix comprised 30% (v/v) S9 fraction.

* Seventy mL of cofactor solution are composed as follows:
MgCl2 x 6 H2O: 162.6 mg
KCl: 246.0 mg
Glucose-6-phosphate, disodium salt: 179.1 mg
NADP, disodium salt: 315.0 mg
Phosphate buffer: 100.0 mM

The count was made after the plates had been incubated for 48 hours at 37 ° C. If no immediate count was possible, plates were temporarily stored in a refrigerator.
Evaluation criteria:
The toxicity of the substance was assessed in three ways. The first was a gross appraisal of background growth on the plates for mutant determination. If a reduction in background growth was observed, it was indicated in the tables by the letter "b" after the mutant count. Where only a single "b", without any other values, is noted for a concentration, this "b" represents four plates with background growth. (The same applies to the signs "c", "v", "p", "n" or "-" which may also be used in the tables.) Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titre was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.
The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratories' own historical data.
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience.
c) Titre determinations had to demonstrate sufficient bacterial density in the suspension.

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
No further data on statistics available
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: substance's toxicity and precipitation above 2000 µg per plate
Negative controls valid:
other: yes, No "untreated" negative control was set for EGDE, since sufficient evidence was available in the literature and from experience indicating that this solvent had no influence on the spontaneous mutant counts of the bacterial strains used.
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 500 µg per plate, the substance started to precipitate. Therefore doses of 1000 µg per plate and above could only be used to a limited extent for assessment purposes.
- Other confounding effects: There was no indication of a bacteriotoxic effect of Triisopropyldiisocyanatobenzene at 8 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted. Higher doses had a weak, strain-specific bacteriotoxic effect.
None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the repeat tests.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1, 2-phenylene diamine and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Table 1: Summary of the Results With Triisopropyldiisocyanatobenzene in the Salmonella/Microsome Test

 S9 mix  TA 1535 TA 100 TA 1537 TA 98 
 without  negative (-ve)  (-ve)  (-ve)    (-ve)
 with   (-ve)  (-ve)   (-ve)   (-ve)

Table 2: Summary of tabulated data without S-9 Mix

Summary of Mean Values Without S9 Mix From Tables 1-8
Table and group µg/plate Strain
TA 1535 TA 100  TA 1537 TA98
1-4 0 16 114 8 20
8 15 105 8 21
40 17 91 8 20
200 15 98 7 23
1000 17 85 - 23
5000 -- 107 - --
Na-a2id 799      
NF   370    
4-NPDA     41 62
5-8 0 19 98 9 23
62.5 13 80 7 22
125 12 76 7 22
250 12 76 10 22
500 13 57 6 21
1000 14 61 5 23
2000 14 74 20
Na-acid 782      
NF   376    
4-NPDA     38 126

Table 3: Summary of tabulated data with S-9 Mix

Summary of Mean Values With S9 Mix From Tables 1-8
Table and group µg/plate Strain
TA 1535 TA 100  TA 1537 TA98
1-4    30 % S-9 0 19 146 13 36
8 22 126 11 35
40 21 133 8 33
200 20 101 10 28
1000 23 123 - 36
5000 --- - --
2-AA 121 639 60 395
5-8 30 % S-9 0 15 86 7 33
62.5 16 89 5 26
125 16 92 8 24
250 14 88 8 26
500 14 101 9 28
1000 15 106 7 20
2000 -- 83 - --
2-AA 100 539 60 435
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The study was performed according to the OECD Guideline 471 with deviations (only 4 Salmonella strains tested) and considered to be of good quality (reliability Klimisch 2). The vehicle and the positive control substances fulfilled validity criteria of the test system. The Salmonella/microsome test, employing doses up to 5000 µg per plate, showed Triisopropyldiisocyanatobenzene to produce only weak bacteriotoxic effects at 40 µg per plate and above. Substance precipitation occurred at 500 µg per plate and above. The test material did not induce significant increases in the frequency of revertant colonies in the bacterial strains TA98, TA100, TA1535 and TA1537. No indications of mutagenic effects of Triisopropyldiisocyanatobenzene could be found at assessable doses up to 5000 µg per plate in any of the Salmonella typhimurium strains used.
Executive summary:

Triisopropyldiisocyanatobenzene was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, T 1537 and TA 98.

The study was performed according to the OECD Guideline 471 with deviations (only 4 Salmonella strains tested) and considered to be of good quality (reliability Klimisch 2). 8 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a weak, strain-specific bacteriotoxic effect. Substance precipitation occurred at the dose 500 µg per plate and above. Therefore this range could only be used to a limited extent up to 5000 µg per plate for assessment purposes.

Evidence of mutagenic activity of triisopropyldiisocyanatobenzene was not seen. No biologically relevant increase in the mutant count, in comparison with the negative control was observed.

The positive control sodium azide, nitrofurantoin, 4 -nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Therefore, Triisopropyldiisocyanatobenzene was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

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

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity data for TRIDI

The substance triisopropylbenzenediisocyanate was investigated for its potential to induce mutation in Salmonella typhimurium (histidine-auxotrophic strains TA 1535, TA 100, T 1537 and TA 98) with and without metabolic activation (according to OECD 471 with the deviation that only 4 strains were tested, Herbold et al., 1990). The concentrations used were 8, 40, 200, 1000 and 5000 µg / plate. The solvent employed for TRIDI was ethylene glycol dimethylether (EGDE) and for the positive controls dimethylsulfoxid (DMSO). There was no indication of a bacteriotoxic effect of TRIDI at 8 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted. Higher doses (40 µg per plate and above) had a weak, strain-specific bacteriotoxic effect. Substance precipitation occurred at 500 µg per plate and above. Therefore doses of 1000 µg per plate and above could only be used to a limited extent for assessment purposes. None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the repeat tests. Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility), revealed no biologically relevant variations from the respective negative controls. No indications of mutagenic effects of the test substance could be found at assessable doses up to 5000 µg per plate in any of the Salmonella typhimurium strains used.

Prediction for TRIDI using the OECD QSAR Toolbox

The chemical 2,4,6-triisopropyl-m-phenylene diisocyanate (CAS 2162 -73 -4) was evaluated by the QSAR OECD Toolbox software for its potential to induce gene mutations in bacterial strains. The prediction was based on the measured values of chemicals assigned into the category.

The target chemical was classified as "Diisothiocyanates" by US EPA New Chemical Categories. Therefore, at first chemicals with the same classification were searched. 21 chemicals were assigned into the category. The prediction was performed based on the experimental data for Bacterial Reverse Mutation Assay for 10 chemicals. Thereafter, the chemicals in the category were scanned for the differences to the target relevant for the genotoxicity endpoint. All chemicals in the category possessed the same organic functional group isocyanate which can trigger genotoxicity events. All chemicals possessed the same DNA binding mechanism. The chemical was predicted to be negative.

The chemical 2,4,6-triisopropyl-m-phenylene diisocyanate (CAS 2162 -73 -4) was evaluated by the QSAR OECD Toolbox software for its potential to induce chromosome aberrations in mammalian cells. The prediction was based on the measured values of chemicals assigned into the category.

The target chemical was classified as "Diisothiocyanates" by US EPA New Chemical Categories. Therefore, at first chemicals with the same classification were searched. 21 chemicals were assigned into the category. The prediction was performed based on the experimental data for in vitro Chromosome Aberration for 4 chemicals. Thereafter, the chemicals in the category were scanned for the differences to the target relevant for the genotoxicity endpoint. No further subcategorization was performed. All chemicals in the category possessed the same DNA binding mechanism and the same organic functional group (isocyanate) which can trigger genotoxicity events. The chemical was predicted to be negative.

Prediction for TRIDI using TOXTREE

Genetic toxicity potential of 2,4,6-triisopropyl-m-phenylene diisocyanate was assessed using the Toxtree (version 2.1.0.) modelling tool. Toxtree was developed by IDEA Consult Ltd (Sofia, Bulgaria) and is approved and recommended by the EU Joint Research Center in Ispra (Italy) (LINK:http://ecb.jrc.ec.europa.eu/qsar/qsar-tools/index.php?c=TOXTREE). According to the modelling results of Toxtree, at least one structural alert was identified for 2,4,6-triisopropyl-m-phenylene diisocyanate, which might induce positive response in in-vivo micronucleus assay.

Genetic toxicity data for TDI

Mutagenicity data in bacterial cells (Ames Test)

The substances toluene diisocyanate (TDI) and diphenylmethanediisocyanate (MDI) were investigated for their mutagenic potential in an Ames test with and without metabolic activation using the Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 (Seel et al.,1999). Previously published data has shown that any Salmonella / microsome tests, where the substance was dissolved in dimethylsulfoxide (DMSO) showed uniformly mutagenic effects. This is due to the instability of aromatic diisocyanates in DMSO this solvent (metabolism to toluene diamine and other metabolites), and therefore it was replaced by ethyleneglycol dimethylether (EGDE). It was found that TDI endured the dissolving and was still available for the subsequent bacterial tests. No aromatic diamines (TDA or MDA) could be detected in EGDE prior to the start of the assays. To gain deeper insight into the chemical changes that occurred during the Salmonella / microsome test, the possible reactions were modelled by mixing predissolved diisocyanates with a defined surplus of water and monitoring the progress of the chemical reactions by analytical methods. Additionally, the quality of the model was checked by exposing solutions of 2,6-TDI to the real biological test environment. In both cases, the reaction patterns of TDI were different to those of MDI. Within 1 min, which is the maximum time needed to mix the predissolved compounds with water before they are poured onto the agar plate, the TDI content was reduced in favour of different ureas and TDA. In addition water was replaced by the complete set of test ingredients. The amount of residual TDI was reduced considerably. Reactions of MDI were markedly slower than those of TDI. Evidently, chemical reactions continue during incubation. The accelerated disappearance of the TDI and the MDI in the presence of the complete set of test ingredients can be explained by the consumption of the aromatic diisocyanates through the rapid reactions with the biological macromolecules. These findings indicate that the chemical interactions between reactive test compounds and solvents or test media need to be considered in the interpretation of the relevance of test results.

In the present investigations, 2,4-TDI, 2,6-TDI and TDI 80, all of which were dissolved in EGDE, showed a consistently negative response in the absence of S9 mix. Furthermore, no mutagenicity was observed in any of the TA 1535 strain experiments. However, clearly positive results were obtained in the other strains after metabolic activation of 2,4-TDI, 2,6-TDI and TDI 80. Consistently positive results were obtained in strain TA 98 with all types of TDI tested. In TA 1537, clearly positive results were obtained for 2,4-TDI and weak effects were observed for TDI 80 whereas no effects were found for 2,6-TDI. Weak positive results were also obtained for TDI 80 with TA 100. The positive findings for three types of TDI can be explained with the appearance of TDA—a known mutagen—that is one of the reaction products generated when solutions of TDI in EGDE are added to the aqueous test system. From the comparison of DMSO and EGDE as solvents for aromatic diisocyanates in bacterial tests, the conclusion can be drawn that the chosen solvent considerably influences the biochemical fate of reactive chemicals.

Mammalian Chromosome Aberration Assay

The substance toluene diisocyanate was investigated for its mutagenic potential in an in vitro mammalian chromosome aberration test with and without metabolic activation using primary human lymphocytes (Maki-Paakhanen et al.,1987). The study was conducted equivalent or similar to OECD Guideline 473 with deviations (exposure time to TDI only 1.5 hours, positive control in cells treated without S9 -mix also cyclophosphamide) and no data is available whether the good laboratory principles have been followed. However, the quality is considered to be good (Klimisch 2). Primary human lymphocytes have been used and as vehicle acetone. Both TDI and MDI increased the frequency of structural chromosome aberrations and the number of aberrant cells. Without metabolic activation, TDI produced a slight increase in aberrations (gaps included) at the highest two doses (0.075 and 0.15µL/mL) and in aberrant cells (gaps included) at 0.075 µL/mL. MDI gave a positive result at all doses tested. No clear dose-response was seen for either TDI or MDI. In the presence of S9 mix, both compounds increased the number of aberrations at one of the tested doses: TDI at 0.038 µL/mL and MDI at 4.3 µL/mL. Only MDI, at the highest dose available (4.3 µL/mL), gave a statistically significant increase in aberrant cells. Again, no dependence on dose was observed. The weak effects of the isocyanates in the pulse treatment may be assigned to the short treatment time, and, to the presence of the polymer particles on the slides (which precluded the analysis of high doses) rather than to inactivation by S9 mix.

Both with and without S9 mix, most aberrations induced by TDI and MDI were chromatid-type breaks and gaps. MDI, at the highest analysable dose (2.17 µL/mL), produced a marginal increase in SCEs with and without metabolic activation. TDI did not significantly affect the number of SCEs. In acetone-treated control cultures, the frequency of SCEs was somewhat higher (P<0.05) in the presence of S9 mix than in its absence.

Both MDI and TDI were less efficient inducers of chromosome aberrations and SCEs than cyclophosphamide especially in the presence of S9 mix.

Thus, to explain the positive results in the aberration test with MDI and TDI in the absence of S9 mix, it can be assumed that the amines are activated in the whole-blood test system without exogenous metabolising systems. Other possibilities are that the amine derivatives are direct mutagens in human cells or that other compounds instead of the amines are involved in the clastogenic effects of the isocyanates.

One possibility is that the observed cytogenetic effects resulted from indirect effects mediated by reactive radicals possibly created in the reaction of TDI and MDI with water or through uptake of the polymer particles to phagocytising leukocytes. The lack of a clear dose-response in the induction of chromosome damage by TDI and MDI may be related to the solubility of TDI, MDI or their reactive metabolites.

Gene Mutation Assay in mammalian cells in vitro

A study was conducted to evaluate the ability of 27 chemicals to induce forward mutations at the thymidine kinase locus (tk) in a mammalian cell, as assayed by colony growth of L5178Y clone 3.7.2C mouse lymphoma cells in the presence of 5-trifluorothymidine (TFT) (McGregor et al., 1991).

Twenty-seven chemicals were tested for their mutagenic potential in the L5178Y tk+/tk mouse lymphoma cell forward mutation assay. These experiments were conducted similar or equivalent to OECD Guideline 476. Cultures were exposed to the chemicals (i.e. 2,4 toluene diisocyanate and 2,6 toluene diisocyanate) for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 µg/ml. The chemicals were tested at least twice. Statistically significant responses were obtained with acid orange 10, aniline, benzaldehyde, o-chloroaniline, chlorodibromomethane, cytembena, 1,2-dibromo-4-(1,2-dibromomethyl) cyclohexane dieldrin, lithocholic acid, oxytetracycline, phenazopyridine HCI, 1 -phenyl-3-methyl- 5-pyrazolone, sodium diethyldithiocarbamate, solvent yellow 14, tetraethylthiuram disulfide (disulfiram), 2,4-toluene diisocyanate, and 2,6- toluene diisocyanate. Apart from phenazopyridine HCI, acid orange 10, and solvent yellow 14, rat liver S9 mix was not a requirement for the mutagenic activity of these compounds. Chemicals not identified as mutagens were N-4-acetylaminofluorene, chlorpheniramine maleate, chlorpropamide, 1,4-dioxane, endrin, ethylene glycol, iron dextran, methapyrilene, sodium(2- ethylhexyl)alcohol sulfate, and 2,3,7,8-tetrachlorodibenzo- p-dioxin. In summary the results of this experiments show that the test substances 2,4 toluene disocyanate and 2,6 toluene diisocyanate were able to induce forward mutations and are considered to be mutagenic, based on the results obtained with the mammalian cells gene mutation assay.

Genetic toxicity in vivo

According to good laboratory practice (GLP), a reliable study without restrictions (Klimisch 1) was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice (Mackay, 1992). The method used has been designed to comply with the OECD Guideline 474 and the corresponding validity criteria are considered as fulfilled.

A range-finding (Phase I, female mice: 7.5 ppm and 11.9 ppm, male mice: 11.8 ppm and 18.9 ppm) was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes.

In Phase II, male and female animals were given TDI at target concentrations of 7.5 and 11.9 ppm for females and 11.8 and 18.9 ppm for males.

Following the results of the cytogenetic analysis of Phase II, Phase III was conducted by giving male and female animals TDI at target concentrations of 3.7, 7.5 and 11.9 ppm for females and 5.9, 11.8 and 18.9 for males. Additionally, Phase II as well as Phase III was conducted by giving male and female animals vinyl chloride as positive control at a target concentration of 50000 ppm. Besides, further groups of mice were given a single 6 hour inhalation to air, to serve as vehicle,respectively.

The atmospheres for Phase I-III of the study were generated by passing clean dry air from the laboratory air supply via a flow controller and a flow meter to a jacketed bubbler containing the test material or the control. The frequency of treatment is described as a single 6 -hour inhalation period, respectively.

Animals were killed 24, 48 or 72 hours later, the bone marrow was extracted and smear preparations were made and stained. Polychromatic and normochromic erythrocytes were scored for the presence of micronuclei.

Although increases in the incidence of micronucleated PCEs were observed in both males and females exposed to TDI, these increases were small, not concentration-related and were not reproducible at concentrations limited by lethality in a repeated study. It is therefore considered that the increases observed are of no biological significance and do not indicate clastogenic activity of TDI in the mouse bone marrow micronucleus assay. TDI is not clastogenic in the mouse micronucleus test.

Conclusion on genetic toxicity data for TRIDI

For TDI, which contains as a structural alert the diisocyanate group the results obtained by experimental data were ambiguous in the bacterial reverse mutation assay, negative in the chromosome aberration test and positive in the mouse lymphoma cell forward mutation assay. Further, it was tested negative in the mammalian erythrocyte micronucleus test. However, these results are not taken at face value for the classification of TRIDI, because the predictions derived with the aid of the OECD Toolbox comprise more category members and are therefore more reliable than the information for TDI.

For TRIDI the results of the experimental bacterial reverse mutation assay and the Toolbox-derived prediction allow the non-classification for genetic toxicity.


Justification for selection of genetic toxicity endpoint
The test was conducted with TRIDI, the target substance.

Justification for classification or non-classification

According to the European regulation (EC) No. 1272/2008 the test material does not meet the criteria for classification and will not require labelling as a mutagen.