HDI oligomers, allophanate

Endpoint summary

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

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

(2001)

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system 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- Fasting period before study: Feed was withdrawn from the animals at least 16 hours before administration, but water was available ad libitum.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:

other: No clinical observations were observed during clinical examination.

Body weight:

other body weight observations

Remarks:

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 from a substance with a very similar chemical composition (comparable allophanate-type HDI oligomerisation product, EC 900 -066 -9, CAS 197393 -84 -3) is applied. The read across is based on physicochemical and toxicological similarity of the two substances. Especially a recently conducted comparative pulmonary irritant potency study based on the recommendations of TRGS 430 (Technical Rule for Hazardous Substances 430, published by the German Federal Ministry of Labour and Social Affairs, last update 2009), confirmed for both allophanate-type HDI oligomerisation products the same toxicological mode of action and a nearly identical potency (both NOAEL at 3.4 mg/m³). 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 administration. 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

Value:

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

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

(2009)

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

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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:

other: see "Other findings"

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.

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.

Executive summary:

A study on the acute inhalation toxicity of the substance on rats 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). Three 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 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 discharge and 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

Value:

264 mg/m³ air

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For acute oral toxicity a read across from a substance with a very similar chemical composition (comparable allophanate-type HDI oligomerisation product, EC 900 -066 -9, CAS 197393 -84 -3) is applied. The read across is based on physicochemical and toxicological similarity of the two substances. Especially a recently conducted comparative pulmonary irritant potency study based on the recommendations of TRGS 430 (Technical Rule for Hazardous substances 430, published by the German Federal Ministry of Labour and Social Affairs, last update 2009), confirmed for both allophanate-type HDI oligomerisation products the same toxicological mode of action and a nearly identical potency (see below). 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 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 concluded to be > 2000 mg/kg bw.

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

For acute inhalation toxicity a study in accordance with OECD TG 403 is available for the substance. In this study 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 observation 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 evaluating the applicability of the read across a direct comparison of the pulmonary irritant potency with both of the two allophanate-type HDI oligomerisation products (EC 933 -048 -4, CAS 28182 -81 -2 and EC 900 -066 -9, CAS 197393 -84 -3) was conducted. The study design was based on the recommendations of TRGS 430. In this study male rats were exposed 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. For this purpose 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 (e.g. bradypnea, laboured and irregular breathing pattern, piloerection, high-legged gait, reduced motility) occurred for both substances immediately after exposure and up to 5 postexposure days at 15 mg/m³ and higher. Rectal temperatures and body weights were slightly and transiently decreased for both substances, whereas 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. Necropsy revealed disoloured lungs with gray areas from dose group 15 mg/m³ onwards for the substance and only in dose group 50 mg/m³ for the comparable product (EC 900 -066 -9, CAS 197393 -84 -3).

The findings suggested that both test articles have a quite similar lung irritation profile with an interpolated NOAEL of 3.4 mg/m³. This PoD was estimated based on the most direct and sensitive target organ-specific endpoint (pulmonary irritation and plasma protein extravasation).

Generally, for diisocyanates the NOAELs from pulmonary irritant potancy studies have been shown to be equivalent with the NOAELs of a 90 -day subchronic inhalation study (Pauluhn, J. Appl. Toxicol., 24, 2004, 231 -247). In fact, the pulmonary irritant potency study for the two allophanate-type HDI oligomerisation products revealed that the NOAELs derived in this study for the two substances corresponded almost exactly to the NOAEL of a 90 -day study for a further HDI-derived oligomerisation product, that is HDI oligomerisation product, isocyanurate type (EC 931-274-8, CAS 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 LC50-study available. Additionally, studies investigating the pulmonary irritant threshold ("TRGS 430-studies") are available and are also of high value for the evaluation of acute inhalation toxicity, but were not selected here, since a study providing a LC50-value is requested.

Justification for classification or non-classification

No classification is required for acute oral and dermal 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 but Cat 4 due to the following reasons:

The Guidance on the Application of Regulation (EC) No 1272/2008 (chapter 3.1.2.3.2) acknowledges 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 Pauluhn¹ there is an option for a modified Classification and Labelling (C&L), called "split-entry"-concept. The current guidelines for acute inhalation toxicity testing prescribe an artificial predetermined particle size of 1 -4 µm MMAD in the breathing zone of exposed animals, in order to allow a robust relative ranking of the acute lethal toxic potency of different substances by ensuring best possible thoracic penetration. The "split-entry"-concept is extrapolating the potential human hazard based on an alignment to the realistic particle size distribution and by this a determination of the critical percentage of particles (=> thoracic fraction) present in a product as commercialized and used. This "split-entry"-concept is acknowledged in the ECHA-guidance on the application of the CLP criteria².

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, 2014 and 2011). The irritant 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 (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; including HDI oligomerisation product, isocyanurate type, iminooxadiazindione type, biuret type, and uretdione type) consistently demonstrates the above discussed toxicological mode of action.

For split entry the general concept established for the classification of mixtures applies (cp. Regulation (EC) No 1272/2008 (CLP))³.

The aerosolized substance is seen as virtual mixture with one relevant “ingredient” that has to be put into context with the respective concentration limits as defined by CLP. This relevant “ingredient” is the thoracic percentage of HDI oligomerisation product, allophanate-type. This thoracic percentage of the aerosol under spray use condition(s) need to be determined in order to make use of the relevant calculation rule in CLP. Recently recorded data on particle size during worst-case end-use on multiple types of HDI homopolymer-based formulations and technical applications have indicated a thoracic percentage of 12% to be an equally conservative and generic value. With a thoracic percentage of 12% and a LC50 (rat, 4h) of 0.264 mg/L an Acute Toxicity Estimate for the aerosolized mixture (ATEmix) > 1.0 mg/L is obtained. Thus, according to the respective concentration limits as defined by CLP classification as Acute Inhalation Toxicity (dusts and mists) Cat. 4 is proposed for HDI oligomerisation product, allophanat-type⁴.

 

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

²Guidance on the Application of the CLP Criteria (ECHA-13-G-10-EN), Version 4.0, Nov. 2013, p. 265. 

³Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, Annex I 3.1.3.6. Classification of mixtures based on ingredients of the mixture (Additivity formula)

⁴Pauluhn J, HDI-Homopolymers (HDI-Trimers) in Coating Formulations, Expert Opinion for GHS Classification and labelling using the EU-Split-Entry Principle, Bayer Pharma AG, 2014.