Methylidynetri-p-phenylene triisocyanate

Acute Toxicity: inhalation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Methylidynetri-p-phenylene triisocyanate

EC Number:

219-351-8

EC Name:

Methylidynetri-p-phenylene triisocyanate

Cas Number:

2422-91-5

Molecular formula:

C22H13N3O3

IUPAC Name:

1-[bis(4-isocyanatophenyl)methyl]-4-isocyanatobenzene

Test animals

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

Administration / exposure

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.

Results and discussion

Effect levels

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).