Reaction mass of 2-ethylhexyl (6-isocyanatohexyl)-carbamate and bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April-May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

(2009)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD GD 39 (Guidance Document on Acute Inhalation Toxicity Testing)

Version / remarks:

(2009)

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl (6-isocyanatohexyl)-carbamate

EC Number:

247-735-5

EC Name:

2-ethylhexyl (6-isocyanatohexyl)-carbamate

Cas Number:

26488-60-8

Molecular formula:

C16H30N2O3

IUPAC Name:

6-Isocyanatohexylamino 3-ethylheptanoate

Constituent 2

Reference substance name:

Bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

EC Number:

278-583-8

EC Name:

Bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

Cas Number:

76977-79-2

Molecular formula:

C24H48N2O4

IUPAC Name:

6-(2-Ethylhexyloxycarbonylamino)hexylamino 3-ethylheptanoate

impurity 1

Reference substance name:

6-[3-(6-Isocyanatohexyl)-2,4-dioxo-1,3-diazetidin-1-yl]hexylamino 3-ethylheptanoate

Molecular formula:

C24H42N4O5

IUPAC Name:

6-[3-(6-Isocyanatohexyl)-2,4-dioxo-1,3-diazetidin-1-yl]hexylamino 3-ethylheptanoate

impurity 2

Reference substance name:

Hexamethylene diisocyanate

EC Number:

212-485-8

EC Name:

Hexamethylene diisocyanate

Cas Number:

822-06-0

Molecular formula:

C8H12N2O2

IUPAC Name:

1,6-diisocyanatohexane

impurity 3

Reference substance name:

Unknown impurities

Molecular formula:

not applicable

IUPAC Name:

Unknown impurities

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF)
- Source: Harlan-Nederland, AD Horst, Netherlands
- Age at study initiation: approximately 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. The legal requirements for housing experimental animals (Directive 86/609 EEC) are followed.
- Diet and water: ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.4 - <= 1.46 µm

Geometric standard deviation (GSD):

>= 1.76 - <= 1.85

Remark on MMAD/GSD:

The respirability of the aerosol was adequate and in compliance with test guidelines.

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. Restrainers were chosen that accommodated the animals' size. The type of exposure principle is comparable with a directed-flow exposure design (Moss and Asgharian, Respiratory Drug Delivery IV, 1994, 197).
- Exposure apparatus: The chambers used are commercially available (TSE, Bad Homburg, Germany) and the performance as well as their validation has been published (Pauluhn, Journal of Applied Toxicology 14, 55-62, 1994, and Pauluhn & Thiel, Journal of Applied Toxicology 27, 160-167, 2007). Each inhalation chamber segment was suitable to accommodate 20 rats at the perimeter location. The ratio between supply and exhaust air was selected so that 90 % of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. The slight positive balance between the air volume supplied and extracted ensured that no passive influx of air into the exposure chamber occurred.
- Source and rate of air: Conditioned (dry, oil free) compressed air, 15 L/min
- Method of conditioning air: Compressed air was supplied by Boge compressors and was conditioned (freed from water, dust and oil) automatically by a VIA compressed air dryer.
- System of generating particulates/aerosols: Under dynamic conditions the various concentrations of the test substance were 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 test article was fed into the nozzle system using a digitally controlled pump (Harvard PHD 2000 infusion pump).
- Optimization of respirability: In order to increase the efficiency of the generation of fine particles and prevent larger particles from entering the chamber a pre-separator (baffle) system was used (Tillery, Environmental Health Perspectives, 16, 25-40, 1976).
- 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 minimal air flow rate of 0.75 L/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.
- Method of particle size determination: The particle-size distribution was analysed using a BERNER critical orifice cascade impactor.
- Treatment of exhaust air: The exhaust air was purified via filter systems.
- Temperature, humidity: Temperature and humidity measurements were performed by the computerized Data Acquisition and Control System using HC-S3 sensors (Rotronic Messgeräte GmbH, Ettlingen, Germany). The position of the probe was at the exposure location of rats.

TEST ATMOSPHERE
- The integrity and stability of the aerosol generation and exposure system was measured by using a RAS-2 real-time aerosol photometer (MIE, Bedford, Massachusetts, USA).
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: glass-fibre 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 analysed using a BERNER critical orifice cascade impactor. Aerosol mass < 3 µm: 91.1 % at 307 mg/m³, 90.1 % at 454 mg/m³, and 88 % at 597 mg/m³..
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The respirability of the aerosol was adequate and in compliance with test guidelines, i.e. the average mass median aerodynamic diameter (MMAD) was 1.4 µm at 307 mg/m³, 1.45 at 454 mg/m³, and 1.46 at 597 mg/m³; GSD 1.75-1.85.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Target conc.: 300, 450, and 600 mg/m³
Analytical conc. (gravimetric): 307, 454, and 597 mg/m³

No. of animals per sex per dose:

5

Control animals:

yes

Remarks:

air control

Details on study design:

- Duration of observation period following administration: 2 weeks
- Frequency of observations and weighing: Body weights were measured before exposure (day 0), on days 1, 3, 7, and 14. The appearance and behaviour of each rat were examined carefully several times on the day of exposure and at least once daily thereafter. Weekend assessments were made once a day (morning). Assessments from restraining tubes were made only if unequivocal signs occurred.
- Necropsy of survivors performed: yes
- Other examinations performed: Reflexes were tested, based on recommendations made by Irwin (Psychopharmacologica 13, 1968, 222-257). Rectal temperatures were measured shortly after cessation of exposure (approximately within ½ hour after the end of exposure) using a digital thermometer with a rectal probe for rats.

Statistics:

Body weights: Means and single standard deviations of body weights are calculated. Mean body weights are also depicted graphically as a function of time. 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.
Particle size analysis: described in detail in report.
Physiological data: Data of rectal temperature measurements are statistically evaluated using the ANOVA procedure (vide infra).
Calculation of the LCso: 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 3, 797-809, 1977) as modified by Pauluhn (1983). This method is based on the maximumlikelihood method of Bliss (Pharm. Pharmacol. 11, 192-216, 1938)
Randomization: A computerized list of random numbers served the purpose to assign animals at random to the treatment groups.
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-a) = 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 in the Appendix 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. This program was originally obtained from BCTIC.

Results and discussion

Effect levels

Effect levels 1

Key result

Sex:

female

Dose descriptor:

LC50

Effect level:

ca. 521 mg/m³ air

Based on:

test mat.

Exp. duration:

4 h

Effect levels 2

Sex:

male

Dose descriptor:

LC50

Effect level:

ca. 857 mg/m³ air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Mortality occurred at 454 and 597 mg/m³ up to the first post exposure day.
Number of mortalities at conc. (onset of mortality): males - 0/5 at 0 mg/m³, 0/5 at 307 mg/m³, 0/5 at 454 mg/m³, 1/5 at 597 mg/m³ (0d); females - 0/5 at 0 mg/m³, 0/5 at 307 mg/m³, 1/5 at 454 mg/m³ (1 d), 4/5 at 597 mg/m³ (1 d).

Clinical signs:

other: All rats exposed to 307 mg/m³ and concentrations above showed clinical signs (bradypnea, labored breathing patterns, breathing sounds, motility reduced, atony, high-legged gait, tremor, hair-coat ungroomed, piloerection, cyanosis, pallor, nose and muzzle

Body weight:

Comparisons between the control and the exposure groups revealed significant changes in body weights at 307 mg/m³ and concentrations above.

Gross pathology:

Necropsy revealed treatment-related findings at 454 and 597 mg/m³ (e.g. nose: white foamy discharge; trachea: white foamy content; lung: less white coloured areas, less collapsed, dark-red coloured).

Other findings:

Differences between groups exposed to 454 and 597 mg/m³ (reduced tonus, impaired righting response and reduced grip strength) compared to the control group were recorded.
Statistical comparisons between the control and the exposure groups revealed significant changes in body temperature at 307 mg/m³ and above.

Any other information on results incl. tables

The mortality patterns were typical of an irritation-related acute lung edema (white foamy content in nose and trachea, lung findings, bradypnea, laboured breathing patterns, breathing sounds, cyanosis, and stridor). Due to the mortality pattern, female rats appear to be more susceptible than male rats.

Applicant's summary and conclusion

Executive summary:

An acute inhalation toxicity study with the test substance has been conducted in accordance with OECD TG 403 and OECD GD 39. In that study groups of rats were nose-only exposed to the liquid aerosol of the test item at 0 (air control), 307, 454, and 597 mg/m³. The respirability of the aerosol was adequate and in compliance with test guidelines (MMAD 1.40 -1.46 µm, GSD 1.76 -1.85).

All rats exposed to 307 mg/m³ and above showed clinical signs (e.g. bradypnea, laboured breathing patterns, motility reduced, high-legged gait, nasal discharge (serous), nostrils with red encrustation). Significant decreased body temperatures and body weights were found at 307 mg/m³ and above. Mortality occurred at 454 and 597 mg/m³. At this concentrations necropsy revealed findings of toxicological importance (e.g. nose: white foamy discharge; trachea: white foamy content; lung: less white coloured areas, less collapsed, dark-red coloured). The mortality patterns were typical of an irritation-related acute lung edema with white foamy content in nose and trachea, lung findings, bradypnea, labored breathing patterns, breathing sounds, cyanosis, and stridor. Due to mortality pattern females appear to be more susceptible than male rats (4/5 females exposed to 597 mg/m³ died whereas only 1/5 males died at this concentration). The LC50 was approx. 857 mg/m³ for male rats and approx. 521 mg/m³ for female rats.