2,4,6-triisopropyl-m-phenylene diisocyanate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-01-04 - 1990-11-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

only 10 air changes per hour

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-triisopropyl-m-phenylene diisocyanate

EC Number:

218-485-4

EC Name:

2,4,6-triisopropyl-m-phenylene diisocyanate

Cas Number:

2162-73-4

Molecular formula:

C17H22N2O2

IUPAC Name:

2,4-diisocyanato-1,3,5-tris(propan-2-yl)benzene

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Strain Bor: WISW (SPPVCpb)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Young adult, healthy specific-pathogen free Wistar rats (strain Bor: WISW (SPF Cpb)) from Winkelmann, Borchen, Kreis Paderborn, were used.
- Identification of animals: Individual colour codes and cages numbers.
- Randomisation: After the acclimatisation period (before beginning of the experiment) all animals were examined regarding their physical conditions. Afterwards the animals were randomly assigned to the different groups.
- Age/weight at study initiation: 170 - 210 g. Animals with these weights are usually 2 -3 months old (young adult), females were nulliparous and not pregnant
- Housing conditions: During the adaptation, as well as test time, the animals were housed conventionally in Makrolon cages type III (5 animals per cage) (according to Spiegel, A., GONNERT, R, Zschr. Laboratory Animal Science 1, 38 (196l) and MASTER , GQ, Zschr. Laboratory Animal Science 7, 144-153 (1965)). The cages (including water bottles and unused food) were changed at least once a week.
- Bedding: as bedding material low-dust wood granules type S 8 / 15 from Ssniff, D4 770 Soest, Westphalia were used. The wood pellets were randomly tested for harmful substances. The analysis results are stored. The analysis results revealed no evidence for an influence of the objective.
- Animal room: all animals of one concentration were housed in one animal room. From time to time for reasons of capacity rats from other toxicological studies were placed in the same animal room. By an adequate physical separation (different cage racks), a clear animal and cage identification and organisational measures within the work flow a mix of animals was avoided.
- Diet/water: Fixed-formula - diet ( "Altromin®1324 - Haltungsdiät für Ratten und Mäuse" (Producer: Altromin GmbH, Lage, Germany)) and tap water, ad-libitum.
- Acclimation period: 5 days

- Cleaning, disinfection, pest control:
The animal room was cleaned at least once a week and disinfected with Zephirol-10 % (1 % in water). A contamination of the food and contact with animals has been excluded. Pest controls have not been conducted in the animal room during the study.

ENVIRONMENTAL CONDITIONS

- Temperature (°C): 20 - 24 °C
- Humidity (%): ca. 50 % (relative)
- Air changes (per hr): ca. 10-times per hour
- Photoperiod (hrs dark / hrs light): 12-hour, from 6 to 18 o'clock MEZ (artificial illumination)

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

other: acetone was partially used as vehicle for production of the aerosol

Details on inhalation exposure:

During the experiment rats were exposed to the aerosol in an exposure-tube (made from plexiglas). The size of those tubes was adapted to the size of the rats. The tubes (Rhema Labortechnik) were built in a way, which guaranteed, that the tail of the rat was outside of the tube. That bears the advantage that the risk of hyperthermia can be minimised. The exposure procedure was equivalent to head-nose-exposure. Any contamination of the fur can be avoided with using this type of exposure condition.
The inhalation chambers used are commercially available (Rhema Labortechnik, Am Stegskreuz 2, Hofheim).

Exposure technique
The aerosol was sprayed under dynamic conditions in a cylindrical inhalation chamber with pre-cleaner ('Baffle'). Made from PVC, the inhalation chamber had the following dimensions: diameter = 30 cm, height = 28 cm (volume: 20 litres).
In the studies, the relationship between supply and exhaust air was such that approximately < 80 % was extracted of the dynamic air supplied via an aerosolfilter (cotton-containing cylinder). The exposure system was therefore able to form an air flow towards the rats. The inhalation chambers were operated in deductions. The intake air was purified through an absolute filter.

Conditioning the air:
The compressed air was generated with two parallel Boge Compressors (type SB 270/15/350D). The compressed air was fully automatically conditioned with a downstream VIA-air dryer (type A 110), meaning to free it of water, dust and oil. The operating control pressure of the compressors is 8 to 10 bar (800 to 1000 kPa). The working pressures were set by reducers.

Aerosol generation:
With the aid of a nozzle (two-fluid nozzle, Rhema Labortechnik) and a Braun infusion pump with a 50 mL glass-cut syringe and with the aid of conditioned air (10 litres of air per minute, dispersion pressure approximately 500 kPa) the test substance atomised under dynamic conditions into a 2 L three-necked flask. For means of deposition of larger particles additionally a glass pre-cleaner (Pitt No. 1, see ASTM method) was used. The glass pre-cleaner was located between the three-necked flask, and the inhalation chamber.
The three-necked flask (baffled) and the pre-separator increase (Pitt No. 1) the efficiency of the aerosol generation and the respirable mass fraction. For aerodynamic reasons the income of the conditioned air was either before or after the pre-separator. This had an influence on the recovery rate.
The acetone test atmosphere (group 2) was generated as follows: 3 mL of acetone were placed in a tube and impinger led by a TELAB BF411 Mini-dosing pump into the air incoming to the inhalation chamber.

Air exchange:
The generation of the aerosol conditions afforded a 30-hour air changes in the inhalation chamber. Under conditions of this test arrangement, the equilibrium is ('steady state') in about 6 minutes operation achieved (. tqc-* = 3 * Chamber volume / air flow, McFarland, 1976).

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Analysis of the particle distribution of the aerosol in the breathing zone of the rats showed a high inhalable mass fraction. The analytical verification revealed differences from nominal to analytical concentrations and were included in interpretation.

Duration of exposure:

4 h

Concentrations:

Nominal concentrations (mg/m3 air):

Group 1: 0 (air control)
Group 2: 0 (acetone control, acetone: 212 mg/m³)
Group 3: 300
Group 4: 306
Group 5: 500
Group 6: 700
Group 7: 1000
Group 8: 625
Group 9: 1250

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 4 weeks
- Frequency of observations and weighing: Records were made twice daily (mornings and evenings), bodyweights were determined manually before exposure, on day 3 and 7 post exposure, and then weekly.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, histopathology, reflex testing, rectal temperatures

Statistics:

Analytical concentrations: From all single measures of the analytical air concentrations the mean was calculated.

Calculation of LC50: Where it was possible to calculate a mean lethal concentration (LC50), it was conducted computer-assisted after the method of A.P. Rosiella, J.M. Essigmann and G.N. Wogna (1977); modified after Pauluhn (1983) based on the Maximum-Likelihood-Method from C.I. Bliss (1938).

Body weights: arithmetic mean and standard deviation. The averages were shown graphically as a function of time - separately for male and female rats
The evaluation of the body weight differences was done with the developments described in the analysis of variance (ANOVA).
In this method, a parametric normal distribution of data by comparison of median and mean will be reviewed. The groups were compared on the level of confidence = 95 % (p = 0.05). An examination of the homogeneity of variances between groups was performed using the Box test. This test is preferred for small sample size compared to the Bartlett's test. In case, that the results in the F-test show that the variance is greater in the group than between groups, this is indicated in the notes as "no statistical difference between the groups". If a difference is found, a pairwise post hoc comparison of groups (one-and two-sided) for the Games and Howell's modification of Tukey-Kramer significance test is conducted.
The software for the analysis of variance comes from the BCTIC Computer Code Collection, modified by Pauluhn. The validation the software was done by data sets from the literature (GAD and Weil, 1982; BCTIC). The calculations are performed AG on a HP 3000 computer system, Department of Toxicology, BAYER.
Rectal temperature values were statistically analysed with ANOVA program.

Results and discussion

Preliminary study:

No information on preliminary studies available.

Effect levels

Effect levels 1

Sex:

male/female

Dose descriptor:

LC50

Effect level:

110 mg/m³ air (analytical)

Based on:

test mat.

95% CL:

>= 96 - <= 126.9

Exp. duration:

4 h

Effect levels 2

Sex:

male/female

Dose descriptor:

other: NOEL

Remarks:

used as NOEC

Effect level:

10 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Effect levels 3

Sex:

male/female

Dose descriptor:

other: NOAEC

Effect level:

20 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

The rats died in a two-phase manner, either during the first three post observation days or delayed during the 12th to 20th post observation day.

Clinical signs:

other: GROUP 1 - 3: all rats tolerated the exposure without clinical symptoms. GROUP 4: reduced motility and piloerection. After day one after application all animals were clinically without symptoms. GROUP 5: reduced and laboured breathing, hyperfrequent breath

Body weight:

In all animals dosed with concentrations higher than 61.2 mg/m³ the body weights were reduced.

Gross pathology:

Rats that were sacrificed at the end of the observation period (4-weeks):
Gross pathology revealed in animals dosed with concentrations higher than 61.2 mg/m³ signs of macroscopically recognisable lung injuries (pale and liver-like lungs, bronchial airways partly filled with highly viscous mucus).

Rats killed at the end of follow up: the pathological examinations revealed from group 5 on signs of macroscopically detectable lung damage (lung pale and liver-like appearance, bronchial tubes partially filled with highly viscous mucus).

Rats died intercurrent: hydrothorax; serous nasal discharge; airways filled with highly viscous mucus; lung inflated, reddish to liver-like (hepatisation) and oedematous; pale spleen, liver and kidneys; liver with lobular marking; gastrointestinal tract (mucosa), red, in the lumen of a yellow / bloody phlegm.

Other findings:

REFLEX TESTING
Conducted on day 0, 1 or day 2, no treatment-related observations were noted.

RECTAL TEMPERATURE
At the end of the observation period, from group 5 (61 mg/m³ air) and higher concentrations a toxicological relevant hypothermia was noted.

INTERCURRENT DECEASED RATS
hydrothorax, serous nasal discharge, bronchial airways filled with highly viscous mucus, distended lungs, hepatisation of the lungs, oedema of the lungs. Spleen, liver and kidneys were pale, lobular liver details, reddened GIT-mucosa, GIT filled with yellowish bloody mucus.

Any other information on results incl. tables

The results, which were obtained using the aerosol (4 hours of exposure), are depicted in the following table (group number is indicated with N)

Table 1:Toxicological results - exposure.: 1x4h

N	concentration (mg/m^3 air)		toxikol. result	duration of symptom	time of death	rel. mass < 3 - 5µm (%)
	nominal	analytical
rat (male)
1	air control		0/0/5	--	--	-
2	acetone-control		0/0/5	--	--	-
3	300	10.4	0/0/5	--	--	-**
4	306	20.0	0/5/5	4h - 6h	--	95
5	500	61.2	0/5/5	4h - 28d	--	95
6	700	79.1	1/5/5	4h - 28d	15d	79
7	1000	132.1	5/5/5	4h - 2d	1d - 2d	98
8	625	151.3	3/5/5	4h - 28d	2d	100
9	1250	249.9	5/5/5	4h - 1d	0d - 1d	96
rat (female)
1	air-control		0/0/5	--	--	-
2	acetone-control		0/0/5	--	--	-
3	300	10.4	0/0/5	--	--	-**
4	306	20.0	0/5/5	4h - 6h	--	95
5	500	61.2	0/5/5	4h - 28d	--	95
6	700	79.1	0/5/5	4h - 11d	--	79
7	1000	132.1	5/5/5	4h - 20d	1d - 20d	98
8	625	151.3	4/5/5	4h - 28d	12d - 13d	100
9	1250	249.9	5/5/5	4h - 1d	0d - 1d	96

**evaluation not possible; approximation: 1µL = 1mg

In the table in the column toxicol. result the first number indicates the number of dead animals, the second number indicates the number of animals with symptoms and the third number indicates the total number of animals.

LC50 (male./female) summarised:

LC50= 110 mg/m³ air*

confidence interval (95%)= 96.0 - 126 .9 mg/m³ air

slope = 3.01

NOEC = 10 mg/m³ air*

*These concentrations indicate the analytical concentrations in the airways of the rats.

Symptoms and mortality:

From 20 mg/m³ air on, unspecific symptoms (piloerection, reduced motility) were noted. From 61 mg/m³ air additionally typical isocyanat symptoms were noted (hypothermia, laboured breathing and in cases breathing noises, as well as cyanosis, reddened rhinarium, lethargy and reduced body weights). 10 mg/m³ air were tolerated without clinical symptoms.

The animals that died during the experiment, died biphasically (within the first 3 observation days, or after 10 to 12 days post applicationem). Death is considered to be mainly induced by the irritation potential of the test substance (free fluid in the pleural cavity, acute oedema of the lungs). The surviving rats (group 61 mg/m³ air) had highly viscous mucus in the bronchial branches.

Applicant's summary and conclusion

Interpretation of results:

Category 1 based on GHS criteria

Remarks:

Criteria used for interpretation of results: other: EU-GHS

Conclusions:

The study was performed according to the OECD Guideline 403 with only negligible deviations and therefore considered to be of the highest quality (reliability Klimisch 1). The validity criteria of the test system are fulfilled. The test material did induce signs of acute inhalation toxicity. The test material was considered to have a high acute inhalation toxicity for rats under the conditions of the test, which is based on the LC50 values derived from this study (LC50 = 110 mg/m³ air (confidence interval (95 %) = 96.0 - 126 .9 mg/m³ air); NOEC = 10 mg/m³ air). Having regard to the concentration of 10 mg/m³ air, which has been tolerated without any symptoms, and the low vapour pressure at room temperature (1.1 mg/m³ air saturation concentration) exposure to the product shows no increased potential danger for people.
The concentration of 20 mg/m³ associated with unspecific symptoms (piloerection, reduced motility) without pathological changes is considered to be the NOAEC.

Executive summary:

A study on the acute inhalation toxicity of 2,4-Triisopropylbenzoldiisocyanat (aerosol) for rats was conducted according to OECD Guideline No. 403.

The exposure was conducted via head-nose-only exposure of groups of five male and five female rats to a test atmosphere containing a concentration between 10 and 250 mg (analytical) of the test item per m³ for a single 4 -hour period. The Mass Median Aerodynamic Diameter was between 1.6 and 1.88 µm. After exposure the animals were observed for 4 weeks.

From 20 mg/m³ air on, unspecific symptoms (piloerection, reduced motility) were noted. From 61 mg/m³ air additionally typical isocyanat symptoms were noted (hypothermia, laboured breathing and in cases breathing noises, as well as cyanosis, reddened rhinarium, lethargy and reduced body weights). 10 mg/m³ air were tolerated without clinical symptoms.

The animals that died during the experiment, died biphasically (within the first 3 observation days, or after 10 to 12 days post applicationem). Death is considered to be mainly induced by the irritation potential of the test substance (free fluid in the pleural cavity, acute oedema of the lungs). In the necropsies performed in the animals of group 5 (61 mg/m³ air), a highly viscous mucus was found in the bronchial branches. It was concluded that the 4-hour LC50 value was higher than 110 mg/m³ for both sexes under the present test conditions. The present data suggests the conclusion that a 2,4 Triisopropylbenzoldiisocyanat aerosol has a high impact on most of the lung periphery or the terminal / central bronchioles.
The studies thus show that the test substance has a high acute inhalation toxicity. Considering the acute symptoms tolerated concentrations of 10 mg / m³ air and the low vapour pressure at room temperature (1.1 mg / m³ air saturation concentration) can be recognized to have no increased risk for acute toxicity for people involved in using the product. The concentration of 20 mg/m³ associated with unspecific symptoms (piloerection, reduced motility) without pathological changes is considered to be the NOAEL

Results:

LC50 - Inhalation-Aerosol (Exposition: 4h)

Rat (male/female): 110 (96 - 127) mg/m³ air (analytical conc.)

NOEC = 10 mg/m³ air (analytical conc.)

NOAEC = 20 mg/m³air (analytical conc.)