Methylidynetri-p-phenylene triisocyanate

Endpoint summary

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

version of 1996

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

SPF-bred Wistar rats of the strain HdsCpd:WU
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 225-244 g (males) and 158-164 g (females)
- Fasting period before administration of test substance: about 17 h

Route of administration:

oral: gavage

Vehicle:

corn oil

Doses:

2000 mg/kg

No. of animals per sex per dose:

3

Control animals:

no

Sex:

male/female

Dose descriptor:

discriminating dose

Effect level:

ca. 540 mg/kg bw

Based on:

act. ingr.

Remarks on result:

other: calculated from the LD50: > 2500 mg/kg (cut off) determined for the trade product (= a solution of 27% Triphenylmethane-4,4',4''-triisocyanate in ethyl acetate (w/w)

Sex:

male/female

Dose descriptor:

other: LD50 (cut off value)

Effect level:

> 2 500 mg/kg bw

Based on:

test mat.

Remarks on result:

other: a dose of 2000 mg/kg bw was tolerated without any findings

Mortality:

no deaths

Clinical signs:

other: none

Gross pathology:

no findings

Other findings:

none

Dose (mg/kg bw)	Toxicological results	Onset and duration of signs	Onset of mortality
male
2000	0 / 0 / 3	---	---
female
2000	0 / 0 / 3	---	---

Toxicological results:

number of dead animals / number of animals with signs after treatment / number of animals treated

Executive summary:

The acute oral toxicity of the Triphenylmethane-4,4',4''-triisocyanate (administered as a 27% solution in ethyl acetate) was evaluated in a GLP-compliant study on male and female Wistar rats according to OECD TG 423 of 1996. The oral dose of 2000 mg solution/kg bw was tolerated without mortalities, clinical signs, effects on weight gain or gross pathological findings. According to OECD TG 423 the LD50 cut-off of the solution can therefore be determined with > 2500 mg solution/kg bw. With regard to the content of Triphenylmethane-4,4',4''-triisocyanatet this is equivalent to approx. 540 mg/kg .

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

540 mg/kg bw

Quality of whole database:

Triphenylmethane-4,4',4''-triisocyanate obtained in a solvent-based manufacturing process and is not isolated throughout the process. A solvent-free Triphenylmethane-4,4',4''-triisocyanate is a solid which would significantly complicate the manufacturing process. Beyond that Triphenylmethane-4,4',4''-triisocyanate is not marketed as such but as a solution to facilitate handling of the substance by downstream users. A solution of 27% Triphenylmethane-4,4',4''-triisocyanate in ethyl acetate (w/w) represents the trade product for which a acute oral study was already available. As displayed in IUCLID section 1.4 removal of the solvent from the trade product invariably leads to the formation of higher molecular weight species, due to the inherent reactivity of the isocyanate moieties. Further, analysis of the toxicity profile of the solvent in the trade product (= ethyl acetate is non toxic after acute oral exposure) demonstrates that the available study with the trade product is sufficient for an adequate hazarad characterisation of Triphenylmethane-4,4',4''-triisocyanate (= active ingredient in the trade product). In view of this and the need to consider animal welfare, no further testing was done. The discriminating dose for Triphenylmethane-4,4',4''-triisocyanate was thus calculated from 2000 mg trade product/kg bw, which was tolerated without any findings, to be approx. 540 mg/kg bw.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 39 (2009)

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Nederland, AD Horst, Netherlands
- Strain: HsdCpb:Wu (SPF)
- Age at study initiation: approx. 2 months
- Weight at study initiation: at the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex
- Housing: singly in conventional Makrolon® Type IIIH cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-60
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: conditioned dry air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Mode of exposure: Animals were exposed to the aerosolized test substance in Plexiglas exposure restrainers applying a directed-flow nose-only exposure principle.

- Aerosol generation: Under dynamic conditions the test substance is atomized into the baffle (pre-separator) of the inhalation chamber. For atomization a binary nozzle and conditioned compressed air (15 L/min) was used. The representative dispersion pressure was approximately 600 kPa. The neat test article was fed into the nozzle system using a digitally controlled pump (Harvard PHD 2000 infusion pump).

- Inhalation chamber: The chambers used are commercially available (TSE, 61348 Bad Homburg) and the performance as well as their validation has been published (Pauluhn and Thiel, Journal of Applied Toxicology 27: 160-167, 2007; Pauluhn, Journal of Applied Toxicology 14: 55-62, 1994).

- Optimization of respirability: In order to increase the efficiency of the generation of fine particles likely to evaporate and to prevent larger particles from entering the chamber a glass-preseparator/ baffle system was used (Tillery et al., Environmental Health Perspectives 16, pp. 25-40, 1976).
- Conditioning the compressed air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.

- Inhalation chamber equilibrium concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour [15 L/min x 60 min/(3.8 L) = 237, continuous generation of test atmosphere]. Under such test conditions chamber equilibrium is attained in less than one minute of exposure. At each exposure port a flow rate of 0.75 I/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.

- Exhaust air treatment: The exhaust air was purified via cotton-wool/HEPA filters. These filters were disposed of by Bayer Pharma AG.

- Temperature and humidity measurements: Temperature and humidity measurements are also performed by the computerized Data Acquisition and Control System using HC-S3 sensors (Rotronic, http://www.rotronic-usa.com/prod_oem/hc2%20probes/hc2_main.htm). The position of the probe was at the exposure location of rats. Temperature and humidity data are integrated for 30 seconds and displayed accordingly. The humidity sensors are calibrated using saturated salt solutions according to Greenspan (1977) and Pauluhn (1994) in a two-point calibration at 33% (MgCI2) and at 75% (NaCI) relative humidity. The calibration of the temperature sensors is also checked at two temperatures using reference thermometers.

ANALYSIS OF TEST ATMOSPHERE
- Nominal concentration: The nominal concentration was calculated from the ratio of the total quantity of test item consumed and not captured by the pre-separating system during the exposure period and the total throughput of air through the inhalation chamber. For calculation of the consumed content of neat test article a specific density of 1 g x cm-3 was used. The lower analytical concentrations compared with the nominal concentrations are attributed to the efficient removal of larger particles in the baffle/pre-separator system.

- Gravimetric concentration: The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Göttingen, Germany; digital balance).The mass collected by the filter was converted to the test substance taking into account the concentration of constituents contained in it that are prone to evaporate subsequent to nebulization. The relative proportion of constituents prone to evaporate is determined as follows: aliquots of the test substance were added onto glass fiber filters and the filters were allowed to dry under specified conditions (at 70°C drying temperature) over a time period of maximal 3 hours. During this time course that time of drying will be defined at which stable conditions are attained. This evaluation revealed that the test article contained volatile constituents. Accordingly, the filter samples were dried and corrected for 23.5% of volatile constituents as follows: filter mass x 100/(100-76.5). Filter analysis used drying after sampling (30 min, 70°C).
- Samples taken from breathing zone: yes
- Particle size distribution: The particle-size distribution was analyzed using a BERNER critical orifice cascade impactor. An adhesive stage coating (silicone spray) was not used to minimize particle bounce due to the adhesive properties of teh test article. Gravimetric analyses of filters used a digital
balance.

- Respirability: The particle-size distribution achieved was adequate to reach all potential target structures of the respiratory tract.

RESULTS OF PARTICLE-SIZE ANAL
- Particle size distribution: In the 1839 mg/m3 exposure group 66.3 % of particles were < 3 µm.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): In the 1839 mg/m3 exposure group MMAD was 2.21 µm (GSD: 2.08).

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

The test article (Desmodur RE) contained volatile constituents . Therefore, concentrations were given as total mass (non-volatile active ingredient= Triphenylmethane-4,4',4''-triisocyanate) and back-calculated to the actual test article.


target concentration: 300/450/600 mg/m³
gravimetric concentration: 317/447/603 mg/m³
analytical concentration: 961/1570/2082 mg/m³

No. of animals per sex per dose:

exposure groups: 5
control groups: 5

Control animals:

other: Comparison with an appropriate historical control were performed. This control was exposed to an atmosphere using essentially similar exposure conditions as were used for the test substance (15 L air/min; conditioned air)

Details on study design:

- Duration of observation period following administration: 2 weeks
- Frequency of observations and weighing: clinical signs were examined several times on the day of exposure and at least once daily therafter; body weights were measured before exposure, on days 1, 3 and 7, and weekly thereafter.
- Necropsy of survivors performed: yes
- Other examinations performed: rectaI temperatures were measured shortly after cessation of exposure (approx. within half an hour after the end of exposure); a battery of reflex measurements was made on the first post-exposure day.

Statistics:

-Necropsy flndings: If specific findings occur from the respiratory tract of surviving rats they are evaluated statistically using the pairwise Fisher test after the R x C chi-squared test.

-Body weights: Means and single standard deviations of body weights are calculated. Since in acute studies individual group means may differ prior to commencement of the first exposure, the body weight gain was statistically evaluated for each group. For these evaluations a one-way ANOVA (vide infra) is used.

-Calculation of the LC50: If calculation of a median lethal concentration (LC50) is possible, it is performed by computer (PC) according to the method of Rosiello et al.,J. Tox. and Environ. Health~, pp. 797-809 (1977) as modified by Pauluhn (1983). This method is based on the maximurn-likelihood method of C.I. Bliss, Q.J. Pharm. Pharmacol. 11, 192-216 (1938). If only 2 pairs of values with greater than 0% lethality and less than 100% are available then the first linear approximation is based on these values and a homogeneity test is not performed. In this case the interpolated concentration at 50% lethality is designated at approximate LC50. Additionally, the moving average interpolation according to Schaper et al., Arch. Toxico!. 68:332-337 (1994) is used for calculation, if applicable.

-Analysis of variance (ANOVA): This parametric method checks for normal distribution of data by comparing the median and mean The groups are compared at a confidence level of (1-alpha)= 95% (p=0.05) The test for the between-group homogeneity of the variance employed Box's test if more than 2 study groups were compared with each other. If the above F-test shows that the intra-group variability is greater than the inter-group variability, this is shown as "no statistical difference between the groups". If a difference is found then a pairwise post-hoc comparison is conducted (1- and 2-sided) using the Games and Howell modification of the Tukey-Kramer significance test.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

437 mg/m³ air

Based on:

act. ingr.

95% CL:

393 - 487

Exp. duration:

4 h

Remarks on result:

other: NOAEL: < 317 mg/m³ air

Mortality:

For details see Table 1 below

Clinical signs:

other: Controls: All rats tolerated the exposure without specific signs. Exposure group (males): 300 mg/m³: irregular breathing pattern, labored breathing pattern, bradypnea, tachypnea, breathing sounds, motility reduced, atony, tremor, staggering gait, highle

Body weight:

Comparisons between the control and the exposure groups revealed transient changes in body weights of toxicological significance in all exposure groups.

Gross pathology:

A qualitative description, only of findings of toxicological importance and for toxicological evaluation, is given below:

- Animals sacrificed at the end of the observation period: The macroscopic findings of extrapulmonary organs were essentially indistinguishable amongst exposure and control groups. In the previous lungs showed a higher incidence of discolorations.

-Animals that succumbed during the observation period: Nostrils with foamy and/or consolidated yellowish content/discharge, lung less collapsed with foamy whitish content in trachea, hydrothorax, heart consolidated with thickened ventricle walls, intestines with yellowish mucus, and discolorationl blood-less appearance of parenchymatous organs.

Other findings:

- Rectal temperature:
Statistical comparisons between the control and the exposure groups revealed significant changes in body temperature (for details see Table 1 below).

- Reflex measurements:
In comparison to the rats of the control group, rats of all exposure groups exhibited impaired reflexes.

Table 1: Summary of acute inhalation toxicity (4 hrs, liquid aerosol) of the trade product (27 % active ingredient in ethyl acetate)

Sex	Target  concentration (mg/m³)	Toxicological results	Onset and duration of signs	Rectal temperature (°C)	Onset and duration of mortality
male	0	0 / 0 / 5	---	38.0	---
	300	0 / 5 / 5	0d - 5d	28.7 **	---
	450	2 / 4 / 5	0d - 6d	27.6 **	0d, 2d
	600	5 / 5 / 5	0d - 1d	27.3 **	0d, 1d, 2d
female	0	0 / 0 / 5	---	38.5	---
	300	0 / 5 / 5	0d - 10d	28.8 **	---
	450	3 / 5 / 5	0d - 5d	27.7**	0d, 1d
	600	5 / 5 / 5	0d	27.1**	0d, 1d

Toxicological results:

number of dead animals / number of animals with signs after cessation of exposure / number of animals exposed

** = p < 0.01

Executive summary:

An acute inhalation study with a solution (27 % active ingredient in ethyl acetate) according to OECD TG 403 was conducted on male and female rats, which were nose-only exposed to the liquid aerosol of the solution in actual concentrations of 317, 447, 603 mg/m³ ( gravimetric concentration of the active ingredient). The liquid aerosol was generated so that it was respirable to rats. Mortality occurred at 447 and 603 mg/m³ and rats succumbed on the exposure day or were found dead up to the morning of the second postexposure day. Clinical observation showed evidence of respiratory irritation typical of lower respiratory tract sensory irritation at >= 317 mg/m³. Animals of all exposure groups showed decreased body weights, decreased reflexes, and hypothermia. Gender-specific differences indicative of sex-specific susceptibilities were not observed. Therefore, the LC50 was calculated for the sexes combined.

In summary, the aerosolized solution (solid aerosol of the active ingredient) proved to have a high inhalation toxicity in rats (LC50: 437 mg/m³).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

437 mg/m³ air

Quality of whole database:

Triphenylmethane-4,4',4''-triisocyanate obtained in a solvent-based manufacturing process and is not isolated throughout the process. A solvent-free Triphenylmethane-4,4',4''-triisocyanate is a solid which would significantly complicate the manufacturing process. Beyond that Triphenylmethane-4,4',4''-triisocyanate is not marketed as such but as a solution to facilitate handling of the substance by downstream users. A solution of 27% Triphenylmethane-4,4',4''-triisocyanate in ethyl acetate (w/w) represents the trade product. As displayed in IUCLID section 1.4 removal of the solvent from the trade product invariably leads to the formation of higher molecular weight species, due to the inherent reactivity of the isocyanate moieties. On the other hand, in spite of generation a liquid aerosol of the trade product, the actual exposure would be to the solvent-free solid (=Triphenylmethane-4,4',4''-triisocyanate ), due to evaporation of the solvent. Therefore the trade product was choosen to assess the acute inahaltion toxicity.

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Triphenylmethane-4,4',4''-triisocyanate obtained in a solvent-based manufacturing process and is not isolated throughout the process. A solvent-free Triphenylmethane-4,4',4''-triisocyanate is a solid which would significantly complicate the manufacturing process. Beyond that Triphenylmethane-4,4',4''-triisocyanate is not marketed as such but as a solution to facilitate handling of the substance by downstream users. A solution of 27% Triphenylmethane-4,4',4''-triisocyanate in ethyl acetate (w/w) represents the trade product for which a base set of toxicological information was already available. As displayed in IUCLID section 1.4 removal of the solvent from the trade product invariably leads to the formation of higher molecular weight species, due to the inherent reactivity of the isocyanate moieties. Further, analysis of the toxicity profile of the solvent in the trade product (= ethyl acetate is non toxic after acute oral exposure) demonstrates that the available study with the trade product is sufficient for an adequate hazarad characterisation of Triphenylmethane-4,4',4''-triisocyanate (= active ingredient in the trade product) for acute oral toxicity. In view of this and the need to consider animal welfare, no further testing was done for the oral route. For the inhalation route the trade product was choosen to be the test item because in spite of generation a liquid aerosol of the trade product, the actual exposure will be to the solvent-free solid (= Triphenylmethane-4,4',4''-triisocyanate ), due to evaporation of the solvent.

Acute toxicity: oral

The discriminating dose for Triphenylmethane-4,4',4''-triisocyanate was thus calculated to be approx. 540 mg/kg bw based on the

the LD50 > 2000 mg/kg of the trade product which was determined according to OECD TG 423 (Krötlinger, 2002). The test dose of 2000 mg/kg was tolerated without any findings. Acute toxicity: inhalation An acute inhalation study with the trade product (27 % Triphenylmethane-4,4',4''-triisocyanate in ethyl acetate) according to OECD TG 403 was conducted on male and female rats, which were nose-only exposed to the liquid aerosol of the solution in actual concentrations of 317, 447, 603 mg/m³ ( gravimetric concentration of the active ingredient). The liquid aerosol was generated so that it was respirable to rats. Mortality occurred at 447 and 603 mg/m³ and rats succumbed on the exposure day or were found dead up to the morning of the second postexposure day. Clinical observation showed evidence of respiratory irritation typical of lower respiratory tract sensory irritation at >= 317 mg/m³. Animals of all exposure groups showed decreased body weights, decreased reflexes, and hypothermia. Gender-specific differences indicative of sex-specific susceptibilities were not observed.Therefore, the LC50 was calculated for the sexes combined.

In summary, the aerosolized solution (solid aerosol of the active ingredient) proved to have a high inhalation toxicity in rats (LC50: 437 mg/m³).

A pulmonary irritant potency study on male rats according to OECD TG 403/TRGS 430 has been conducted in order to analyze the concentration- and time-dependence onset of alveolar damage following a single 6-hour nose-only exposure to the aerosolized trade product. The aerosol was gererated so it was respirable to rats (MMAD 1.7 - 2.4 µm; GSD 2.0 - 2.5) in gravimetric concentrations of 1.9, 13.0, and 94.2 mg Triphenylmethane-4,4',4''-triisocyanate/m³ (active ingredient)

Interim sacrifices were performed on postexposure days 1, 3, and 7. On these days the bronchoalveolar lavage fluid (BALF) of the rat was examined. At 1.9 mg/m³ the exposure was tolerated without specific effects while the rats exposed to 13.0 and 94.2 mg/m³ experienced signs up to the fifth postexposure days. This signs included a labored and irregular breathing pattern, bradypnea, tachypnea, breathing sounds, motility reduced, atony, high-legged and staggering gait, piloerection, cyanosis, yellowish deposits in nostrils, nose/nuzzle: red encrustations, and serous discharge from the nose. Hypothermia and transiently decreased body weights occurred at 94.2 mg/m³ only. In bronchoalveolar lavage the cellular endpoints and especially PMNs in BAL were amongst the most sensitive endpoints and changes were maximal on the first postexposure day. A similar concentration-time profile existed for LDH, protein, y-GT, and lung weights. The most sensitive endpoint was polymorphonuclear cells (PMNs, absolute and relative) which was already statistically increased at 1.9 mg/m³ on the first postexposure day. Essentially all endpoints gained statistical significance at 13.0 and 94.2 mg/m³ (day 1 and partially also day 3) when compared to the time-matched concurrent control. Although most endpoints were reversible at the end of the 7 -day postexposure period, evidence of an increased neutrophilic inflammation was still apparent at this time point.

In summary, these findings suggest that the test article is a pulmonary irritant and first evidence of a transient pulmonary irritation occurred at a concentration of 1.9 mg/m³.

Justification for selection of acute toxicity – oral endpoint
Only one study available

Justification for selection of acute toxicity – inhalation endpoint
Only one study available

Justification for classification or non-classification

Acute toxicity: oral

The discrminating dose was calculated to be 540 mg/kg bw.

According to EU-Directive 67/548/EEC, Annex VI Triphenylmethane-4,4',4''-triisocyanate shall be classified as harmful.

According to Regulation (EC) No 1272/2008, Annex I, Triphenylmethane-4,4',4''-triisocyanate shall be allocated to Category 4.

Acute toxicity: inhalation

The LC50 (4 hours, aerosol), rat (male/female) was determined to be 437 mg/m³.

According to EU-Directive 67/548/EEC, Annex VI Triphenylmethane-4,4',4''-triisocyanate shall be classified as toxic.

According to Regulation (EC) No 1272/2008, Annex I, Triphenylmethane-4,4',4''-triisocyanate shall be allocated to Category 2.

Acute toxicity: dermal

no data available

No classification required according to EU-Directive 67/548/EEC, Annex VI.

No classification required according to Regulation (EC) No 1272/2008, Annex I.