2,4-dioxo-1,3-diazetidine-1,3-bis(methyl-m-phenylene) diisocyanate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May to June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dioxo-1,3-diazetidine-1,3-bis(methyl-m-phenylene) diisocyanate

EC Number:

247-953-0

EC Name:

2,4-dioxo-1,3-diazetidine-1,3-bis(methyl-m-phenylene) diisocyanate

Cas Number:

26747-90-0

Molecular formula:

C18H12N4O4

IUPAC Name:

bis(3-isocyanato-4-methylphenyl)-1,3-diazetidine-2,4-dione

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 221305
- Purity: > 98.0 %
- Expiration date of the lot/batch: 2014-07-27

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

Mass median aerodynamic diameter (MMAD):

>= 3.04 - <= 3.85 µm

Geometric standard deviation (GSD):

>= 1.72 - <= 1.76

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: Dry conditioned air was used to generate the test substance atmospheres. The test atmosphere was conveyed through openings in the inner concentric cylinder of the chamber, directly towards the rats' breathing zone. This directed-flow arrangement minimizes re-breathing of exhaled test atmosphere. Each inhalation chamber segment was suitable to accommodate 20 rats at the perimeter location. All air flows were monitored and adjusted continuously by means of calibrated and computer controlled mass-flow-controllers. A digitally controlled calibration flow meter was used to monitor the accuracy of mass-flow-controller. The ratio between supply and exhaust air was selected so that ca. 90% of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. Gas scrubbing devices were used for exhaust air clean-up. The slight positive balance between the air volume supplied and extracted ensured that no passive influx of air into the exposure chamber occurred (via exposure restrainers or other apertures). The slight positive balance provides also adequate dead-space ventilation of the exposure restrainers. The pressure difference between the inner inhalation chamber and the exposure zone was 0.02 cm H20 (Pauluhn, 1994). The exposure system was accommodated in an adequately ventilated enclosure. Temperature and humidity are measured by the Data Acquisition and Control System using calibrated sensors. The sensors were located in the inhalation chamber.
- Optimization of respirability: In order to increase the efficiency of the generation of fine particles and to prevent larger particles from entering the chamber a preseparator/baffle system was used (Tillery et al., 1976) in combination with the Wright Dust Feeder.
- 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 (28 L/min x 60 min/3.8 L = 442, continous generation of test atmosphere). Under such test conditions chamber equilibrium is attained in less than one minute of exposure. At each exposure port a minimal air flow rate of 1.4 L/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 measurementswere 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 not calculated from the ratio of the total quantity of test item consumed during the exposure period and the total throughput of air through the inhalation chamber as this would have required a dismantling of the dust generator.
- Total mass concentration: The test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Gottingen, Germany; digital balance). This method was used to define the actual concentration.
- 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 used to minimize particle bounce due to the adhesive properties of the test article. Each impactor stage was covered with an aluminum foil which was subjected to gravimetric analysis using a digital balance.
- Respirability: The particle-size distribution achieved was adequate to reach all potential target structures of the respiratory tract.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

target concentrations: 600, 1000, 5000 mg/m3
gravimetric concentrations: 591, 1120, 5983 mg/m3

No. of animals per sex per dose:

5

Control animals:

yes

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 AP. Rosiello, I.M. Essigmann, and G.N. Wogan (1977) as modified by Pauluhn (1983). This method is based on the maximurn-likelihood method of C.I. Bliss (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. (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

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 1 120 mg/m³ air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: NOAEL: < 591 mg/m3

Mortality:

Mortality occurred only at 5983 mg/m3 (solid aerosol, MMAD = 8.47 µm) up to the second postexposure day. Due to the fact that the aerosol of this concentration did not reach the MMAD (< 4 µm) required by OECD Guideline 403, the technical maximal attainable concentration fulfilling the requirements of the OECD Guideline was 1120 mg/m3. Mortality did not occur in the range of aerosol concentrations that were in compliance with OECD Guideline 403.

Clinical signs:

other: All rats exposed up to 5983 mg/m3 showed clinical signs (e.g. irregular breathing pattern, labored breathing pattern, bradypnea, motility reduced, atony, high-legged gait, piloerection, cyanosis, nose/muzzle: red encrusted, eyelids: red encrusted, reduced

Body weight:

Significant decreased body weights were found at 591 mg/m3 and above.

Gross pathology:

Necropsy revealed no findings of toxicological importance at 591 and 1120 mg/m3. In rats exposed to 5983 mg/m3 evidence of oral uptake of test substance was observed (e.g. white deposits in the stomach). In addition, in these high dose animals the following necropsy findings were seen: nose: red encrusted, red discharge, nasal cavity with white deposits; larynx/pharynx: white deposits; trachea: white dusty content; lung: light colored areas, hepatoid areas, less collapsed, dark-red discoloration, lung: dark-red marbled; kidneys: renal pelvis bilaterally red; thymus: white discoloration; pleural cavity: colorless-clear liquid, -0.5 ml; liver: light colored areas; spleen: reduced in size, light discoloration; heart: reduced in size, hardened, light discoloration, ventricles exsanguinous, ventricle walls thickened; stomach: bloated; intestines: bloated.

Other findings:

Significant decreased body temperatures were found at 591 mg/m3 and above.

Any other information on results incl. tables

Table 1: Summary of acute inhalation toxicity (4 h, solid aerosol) of Metalink U (Desmodur TT)

Sex	Target concentration (mg/m3)	Toxicological results	Onset and duration of signs	Rectal temperature (°C)	Onset of mortality
male	0 (Control)	0 / 0 / 5	---	37.7	---
	600	0 / 5 / 5	0d - 4d	35.6	---
	1000	0 / 5 / 5	0d - 4d	35.2	---
	5000	1 / 5 / 5	0d - 8d	31.7	2d
female	0 (Control)	0 / 0 / 5	---	38.2	---
	600	0 / 5 / 5	0d - 8d	34.6	---
	1000	0 / 5 / 5	0d - 6d	33.6	---
	5000	1 / 5 / 5	0d - 9d	31.1	1d (< 24h)

Toxicological results:

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

Applicant's summary and conclusion

Executive summary:

A study on the acute inhalation toxicity of Metalink U on male and female rats has been conducted in accordance with OECD TG 403. Three groups of rats were nose-only exposed to solid aerosols of the test substance once for 4h in actual concentrations (gravimetric analysis) of 591, 1120 and 5983 mg/m3. Rats of the control groups were exposed to conditioned air under identical circumstances. The post-exposure period was 2 weeks. Only the test atmospheres generated with the Wright Dust Feeder (WDF) at concentrations of 591 and 1120 mg/m3 fulfill the MMAD < 4 µm required by OECD guideline 403. The respirability of these aerosols was adequate and in compliance with the test guideline (MMAD: 3.04 - 3.85 µm; GSD: 1.72- 1.76). 1120 mg/m3 was the maximal technical concentration producible by using the WDF. Higher concentrations than 1120 mg/m3 could be generated by using an Exactomat 4200, but these aerosols were not in compliance with the criteria required by OECD guideline 403 (MMAD 1-4 µm). Therefore the overall maximal technical attainable concentration in compliance with OECD TG 403 was 1120 mg/m3.

All rats exposed to 591 and 1120 mg/m3 showed clinical signs (e.g. irregular breathing pattern, labored breathing pattern, bradypnea, motility reduced, atony, high-legged gait, piloerection, cyanosis, nose/muzzle: red encrusted, eyelids: red encrusted, reduced reflexes). Significant decreased body temperatures and body weights were found at 591 mg/m3 and above. Mortality did not occur up to 1120 mg/m3. Necropsy revealed no findings of toxicological importance at 591 and 1120 mg/m3.

In summary, Metalink U (solid aerosol) proved to have essentially low acute inhalation toxicity in male and female rats (LC50, 4h: > 1120 mg/m3).