Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Klimisch reliability of study is 1 (GLP guideline study); according to ECHA Practical Guide 6 rel. 2 is selected from the IUCLID pick-list as this should be the maximum score for read-across.
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
(2001)
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Rat / Wistar / CrlGlxBrlHan:WI
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: Young adult animals (female animals approx. 14 - 18 weeks)
- Weight at study initiation: 194 - 213 g
- Fasting period before study: Feed was withdrawn from the animals at least 16 hours before administration, but water was available ad libitum.
- Housing: Single housing in stainless steel wire mesh cages, type DK-lll (Becker & Co., Castrop-Rauxel, FRG)
- Diet: Kliba-Labordiät, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Tap water ad libitum
- Acclimation period: Acclimatization for at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
VEHICLE
Olive oil Ph.Eur./DAB
- Substance concentration in vehicle: 40g/100mL
- Administration volume: 5 mL/kg
- Justification for choice of vehicle: lnhomogeneous in watery preparation. Olive oil Ph.Eur./DAB had to be used to ensure homogeneity of the preparation.

Rationale for the selection of the starting dose: Based on the physical and chemical characteristics of the test substance and its composition, no pronounced acute oral toxicity was expected. Therefore, a starting dose of 2000 mg/kg body weight (limit test) has been chosen in the first step with 3 female animals.
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
6 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: at least 14 days
- Frequency of observations and weighing:
Body weight determination: Individual body weights shortly before administration (day 0), weekly thereafter and at the end of the study.
Mortality: A check for any dead or moribund animal was made twice each workday and once on Saturdays, Sundays and on public holidays.
Signs and symptoms: Recording of signs and symptoms several times on the day of administration, at least once each workday for the individual animals.
- Necropsy with gross-pathology examination on the last day of the observation period after killing with CO2.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred.
Clinical signs:
No clinical observations were observed during clinical examination.
Body weight:
The mean body weights of the administration groups increased throughout the study period.
Gross pathology:
No macroscopic pathologic abnormalities were noted in the animals examined at termination of the study.

For acute oral toxicity a read across to a substance with a very similar chemical composition (EC No. 900 -066 -9; comparable allophanate-type HDI oligomerisation product) is applied. The read across is based on physicochemical and toxicological similarity of the two substances. Especially a comparative pulmonary irritant potency study according to TRGS 430 (Technical Rule for Hazardous Substances 430; published by the German Federal Ministry of Labour and Social Affairs, 2009), as the key study for justification of read-across, confirmed for both substances the same toxicological mode of action and nearly identical potency. For further justification of the grouping and read-across according to regulation (EC) No 1907/2006, Annex XI, 1.5 see document attached to chapter "Assessment Reports".

 

Executive summary:

An acute oral toxicity study according to OECD TG 423 was conducted on female Wistar rats receiving a single dose of 2000 mg substance/kg bw via gavage. No mortality occurred. No clinical signs and findings were observed, and the mean body weights of the administration groups increased throughout the study period. No macroscopic pathologic abnormalities were noted in the animals examined at the end of the observation period. Thus, the LD50 was found to be > 2000 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
2 000 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:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
(2009)
Qualifier:
according to
Guideline:
other: OECD Guidance Document No. 39 (2009)
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF) wistar strain
- Source: Harlan-Nederland, AD Horst, Netherlands
- Age at study initiation: approximately 2 months
- Weight at study initiation: males: 178 - 187 g; females: 168 - 183 g
- Housing: singly in conventional Makrolon Type IIIH cages
- Diet: standard fixed-formula diet (KLIBA 3883), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 60
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Plexiglas exposure restrainers (TSE, Bad Homburg, Germany)
- Mode of exposure: Animals were exposed to the aerosolized test article in restrainers made of Plexiglas. Restrainers were chosen that accomodated the animals' size. Each inhalation chamber segment was suitable to accomodate 20 rats at the perimeter location. The design of the directed-flow inhalation chamber minimizes re-breathing of exhaled test atmosphere. For validation see Pauluhn, Journal of Applied Toxicology 14, 1994, 55-62 and Pauluhn & Thiel, Journal of Applied Toxicology 27, 2007, 160-167.
- Source and rate of air: Dry conditioned air, 15 L/min
- Method of conditioning air: Compressed air was supplied by Boge compressors and was conditioned (free from water dust and oil) automatically by a VIA compressed air dryer.
- System of generating particulates/aerosols: Under dynamic conditions the targeted concentrations were achieved by atomization using the nozzle-baffle system and inhalation chamber. For atomization a binary nozzle (Schlick water jacketed nozzle which was connected to a thermostat, 25°C, using a digitally controlled water bath) and conditioned compressed air was used (15 L/min). The representative dispersion pressure was approximately 600 kPa. The test article was fed into the nozzle system using a digitally controlled infusion pump (Harvard PHD 2000 infusion pump).
- 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-pre-separator/baffle system was used.
- 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 used chamber equilibrium is attained in less than one minute of exposure. At each exposure port a minimal air flow rate of 0.75 I/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.
- Method of particle size determination: Cascade impactor (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). The position of the probe was at the exposure location of rats. Mean temperatures from 20.7 to 22°C, 5% relative humidity.

TEST ATMOSPHERE
- The integrity end 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: gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
- Samples taken from breathing zone: yes
- Particle size distribution: The particle size distribution was analyzed using a BERNER critical orifice cascade impactor. Aerosol mass < 3 µm: 87.5% for 235 mg/m³, 86.9% for 284 mg/m³, 84.6% for 314 mg/m³.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The aerosol was generated so that it was respirable to rats, i.e. the average mass median aerodynamic diameter (MMAD) was 1.8 µm, the average geometric standard deviation (GSD) was approx. 1.6.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Target concentrations: 200 mg/m³, 275 mg/m³, 300 mg/m³
Analytical concentrations: 235 mg/m³, 284 mg/m³, 314 mg/m³
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The appearance and behavior 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 (e.g. spasms, abnormal movements, and severe respiratory signs). Body weights were measured before exposure, on days 1, 3 and 7, and weekly thereafter.
- Necropsy of survivors performed: yes
- Other examinations performed: Reflexes were tested (visual placing response, grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflex, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and touch (back).
Rectal temperatures were determined shortly after cessation of exposure.
Statistics:
Analysis of variance (ANOVA) was used for statistical evaluation.
Calculation of LC50 was performed according to Rosiello et al. (1977; Rosiello, Essigmann and Wogan, Tox and Environ. Health, 3, pp797) as modified by Pauluhn (1983). It is based on the maximum likelihood method of Bliss (1983; Q.J.Pharm.Pharmacol., 11, pp192).
Sex:
male
Dose descriptor:
LC50
Effect level:
264 mg/m³ air
95% CL:
240.53 - 290.7
Exp. duration:
4 h
Sex:
female
Dose descriptor:
LC50
Effect level:
> 314 mg/m³ air
Exp. duration:
4 h
Mortality:
No animal died in the lowest dose group of 235 mg/m³. Mortality occured at 284 mg/m³ and higher (4 males and 2 females at 284 mg/m³, 5 males and 1 female at 314 mg/m³). Rats succumbed on the exposure day or were found dead in the morning of the first postexposure day.
Clinical signs:
Clinical observation showed evidence of respiratory irritation typical of lower respiratory tract sensory irritation. The following signs were observed (exposure day up to postexposure day 10): bradypnoea, labored breathing patterns, crepitations, irregular breathing patterns, motility reduced, atony, high-legged gait, tremor, gait uncoordinated, hair-coat ungroomed, piloerection, cyanosis, nasal discharge (serous), nose red encrustations, muzzle red encrustrations, stridor, nostrils: red encrustrations, eye lids: red encrustrations, decreased body weights, decreased reflexes, and hypothermia.
Body weight:
Treated animals showed transient reduced body weights as compared to control animals.
Gross pathology:
Necropsy of animals that died during the study revealed nasal/muzzle with red encrustations, nostrils with foamy content/discharge, lung less collapsed with foamy whitish content in trachea, hydrothorax, abdomen bloated and discoloration/bloodless appearance of parenchymatous organs.
Necropsy of surviving animals at the end of the study period did not reveal any abnormal finding compared to control.
Other findings:
All treated animals showed significantly reduced rectal temperatures (hypothermia).
A battery of reflex measurements was made on the first post-exposure day. ln comparison to the rats of the control group, substance treated rats exhibited impaired reflexes.
Executive summary:

A study on the acute inhalation toxicity of the substance on rats has been conducted in accordance with OECD TG 403. Test procedures were adapted so as to comply also with the EU Directive 92/69/EEC, and especially OECD GD39 (2009). Three groups of rats were nose-only exposed to the liquid aerosol of the test article in actual concentrations of 235, 284 and 314 mg/m³. The aerosol was generated so that it was respirable to rats, i.e. the average mass median aerodynamic diameter (MMAD) was 1.8 µm, the average geometric standard deviation (GSD) was approx. 1.6.

Mortality occured at 284 mg/m³ and higher. Rats succumbed on the exposure day or were found dead in the morning of the first postexposure day. Clinical observation showed evidence of respiratory irritation typical of lower respiratory tract sensory irritation, e.g. bradypnoea, labored and irregular breathing patterns, reduced motility, atony, high-legged and uncoordinated gait, tremor, ungroomed hair-coat, piloerection, cyanosis, serous nasal dischargeand encrustations of nose and eye.

For males the LC50 was determined to be 264 mg/m³. Females were found less susceptible than males, resulting in a LC50 of > 314 mg/m³. The NOAEL for both male and female rats was concluded to be < 235 mg/m³.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
264 mg/m³

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study on the acute inhalation toxicity of HDI oligomerisation product, allophanate-type was conducted in accordance with OECD TG 403. Test procedures were adapted so as to comply also with the EU Directive 92/69/EEC, and especially OECD GD39 (2009). Groups of rats were nose-only exposed to the liquid aerosol of the test article in actual concentrations of 0, 235, 284 and 314 mg/m³. The aerosol was generated so that it was respirable to rats, i.e. the average mass median aerodynamic diameter (MMAD) was 1.8 µm, the average geometric standard deviation (GSD) was approx. 1.6.

Mortality occurred at 284 mg/m³ and higher. Rats succumbed on the exposure day or were found dead in the morning of the first postexposure day. Clinical signs were observed from exposure day up to day 10. These signs showed evidence of respiratory irritation typical of lower respiratory tract sensory irritation, e.g. bradypnoea, laboured and irregular breathing patterns, reduced motility, serous nasal discharge and encrustations of nose and eye.

The LC50 was determined to be 264 mg/m³ for males. Females were found less susceptible than males, resulting in a LC50 of > 314 mg/m³. The NOAEL for both male and female rats was concluded to be < 235 mg/m³.

For acute oral toxicity a read across to a substance with a very similar chemical composition (EC No. 900 -066 -9; comparable allophanate-type HDI oligomerisation product) is applied. The read across is based on physicochemical and toxicological similarity of the two substances. Especially a comparative pulmonary irritant potency study according to TRGS 430 (see below), as the key study for justification of read-across, confirmed for both substances the same toxicological mode of action and nearly identical potency. For further justification of the grouping and read-across according to regulation (EC) No 1907/2006, Annex XI, 1.5 see document attached to chapter "Assessment Reports".

The acute oral toxicity study with the read-across substance (EC No. 900 -066 -9) was conducted according to OECD TG 423. Female Wistar rats received a single dose of 2000 mg/kg bw via gavage administration. No mortality occurred, no clinical signs and findings were observed, and the mean body weights of the animals increased throughout the study period. No macroscopic pathologic abnormalities were noted in the animals examined at the end of the observation period. Thus, the LD50 was found to be > 2000 mg/kg bw.

No data on acute dermal toxicity are available for HDI oligomerisation product, allophanate-type.

For assessing the applicability of read across and comparing the pulmonary irritant potency of the two allophanate-type HDI oligomerisation products (EC nos 933 -048 -4 and 900 -066 -9) a comparative investigation was conducted. In this study the methodological aspects as called for by TRGS 430 (Technical Rule for Hazardous Substances 430, German Federal Ministry of Labour and Social Affairs, 2009) were observed. Test procedures were adapted so as to comply also with OECD TG 403 (2009) and especially with OECD GD39 (2009).

Rats were exposed once for 6 hours to target concentrations of 0 (air control), 1.5, 5, 15, and 50 mg/m³ of the aerosolised test substances. The liquid aerosols were generated so that they were respirable to rats. Following exposure bronchoalveolar lavage (BAL) fluid was analysed for endpoints indicative for pulmonary irritation. Therefore BAL fluid was sampled on postexposure days 1, 3, and 7 using six rats per group.

No deaths were recorded throughout the study. All rats tolerated the exposure without effects up to 5 mg/m³, whereas clinical signs occurred for both substances immediately after exposure and up to 5 postexposure days at 15 mg/m³ and higher. Rectal temperatures, body weights and gross necropsy examination appeared to be indistinguishable between test groups and controls. Lung weights and BAL endpoints where concentration-dependently increased at 5 mg/m³ and above. Despite the marked effect observed, rapid reversibility occurred up to 15 mg/m³.

As far as differences between the two test substances occurred they appear to be within the variability of this bioassay.

Based on statistical analysis of the most sensitive endpoint, BAL-protein, a NOAEL of 3.4 mg/m³ was derived for both of the two substances.

Moreover, the study revealed that the NOAELs derived for the two substances corresponds almost exactly to the NOAEL for another substance with a similar composition, that is HDI oligomerisation product, isocyanurate type (EC No 931-274-8; CAS No 28182 -81 -2).


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

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

 

Despite the 4 hour LC50 of 264 mg/m³ for male rats the substance is not classified according to Regulation (EC) No 1272/2008, Annex I for acute inhalation toxicity Cat. 2 due to the following reasons:

The Guidance on the Application of Regulation (EC) No 1272/2008 acknowledges (chapter 3.1.2.3.2) that special consideration is required if a substance is tested in a form (i.e. specific particle size distribution) that is different from the forms in which the substance is placed on the market and in which it can reasonably be expected to be used. According to Pauluhn1there is an option for a modified Classification and Labelling (C&L), called “split-entry concept”. While the current test principles for acute inhalation uses a predetermined particle size (MMAD 1 - 4 µm) in the breathing zone of exposed animals in order to allow a robust relative ranking of the acute lethal toxic potency of different substances, split entry takes into account for an assessment of potential human hazards the actual percentage of the critical percentage of particles (=> thoracic fraction) present in the product as commercialized and used. This “split-entry concept” was already discussed on EU level, at the latest on 21stJanuary 2008 (Ispra, Italy) and is acknowledged in the recently published version of the ECHA-guidance on the application of the CLP criteria.2

Based on the currently available data the prerequisites for employing split-entry1are given for HDI oligomerisation product, allophanate-type, e.g. the substance is a liquid with a very low volatility causing local toxicity (irritation) in the lower respiratory tract, but no systemic toxicity after acute inhalation exposure to rats (Bayer AG, 2013 and 2011). The irritating potential on the portal-of-entry is confirmed by other studies on animals with the substance, where only minor indications for systemic toxicity could be observed at all (e.g. positive skin sensitisation potential) that are not regarded to be of relevance for the assessment of acute toxicity. Moreover, the database with acute and repeated animal studies for substances with a similar composition (HDI homopolymers, CAS No. 28182 -81 -2; e.g. HDI oligomerisation product, isocyanurate type, EC No. 931 -274 -8) consistently demonstrates this toxicological mode of action.

Currently available data on particle size during worst-case end-use of HDI homopolymers indicate a thoracic percentage below the requested threshold justifying a modified C&L for acute inhalation toxicity for the substance. On this basis the classification is for HDI oligomerisation product, allophanate-type, for acute inhalation toxicity Cat. 4, H332: Harmful if inhaled. Further studies regarding the particle size distribution are presently ongoing.

 

1Pauluhn J, Experimental and Toxicologic Pathology 60, 2008, 111-124

2Guidance on the Application of the CLP Criteria (ECHA-12-G-14-EN), Version 3.0, Nov. 2012, p. 199.