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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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reference:
Composition 0
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
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: HsdCpb:Wu
- Source: Harlan/Winkelmann GmbH, 33178 Borchen, Germany
- Age at study initiation: 10-12 weeks approximately
- Weight at study initiation: 157-186 g
- Fasting period before study: Food was withheld from the animals for approximately 16-24 h before administration of the test item, and animals were fed again approximately 2-4 h after administration.
- Housing: The animals were group caged conventionally in polycarbonate cages on low dust wood granulate bedding.
- Diet and water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 5
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
other: acetone/corn oil 1/10
Details on oral exposure:
- Administration volume: 10 mL/kg bw
- Concentration in vehicle: The test substance was formulated in acetone / corn oil 1/10 (test compound + 1 ml acetone ad 10 ml with corn oil)
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
6 (3 for 1st and 3 for 2nd testing)
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Examinations: Deaths and overt signs of toxicity were recorded repeated times on day of dosing and daily for 14 days. Bodyweights were recorded on the day of dosing and on days 7 and 14. At the end of the study all animals were subject to gross necropsy, macroscopic abnormalities were recorded, no tissues were retained.
Statistics:
The LD50 value was estimated according to OECD TG 423 (2001).
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Mortality:
A dose of 2000 mg/kg bw was tolerated by female rats without mortalities.
Clinical signs:
No clinical signs were observed.
Body weight:
There were no toxicological effects on body weight or body weight gain.
Gross pathology:
No gross pathology findings.
Executive summary:

In an acute oral toxicity study according to OECD TG 423 female rats were given a limit dose of 2000 mg/kg bw. No mortalities, no clinical signs, no effects on weight gain and no gross pathological findings were observed. The LD50 for the test substance was therefore > 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:
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 pick-list as this should be the maximum score for read-across.
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
(1981)
GLP compliance:
yes
Test type:
standard acute method
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar rats Bor: WISW (SPF-Cpb)
- Source: Winkelmann, Borchen, Germany.
- Age at study initiation: 10 to 12 weeks
- Weight at study initiation: approx. 160 - 220 g
- Housing: in groups of 5 in conventional Makrolon® Type III cages
- Diet and water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Mode of exposure: Animals were head-nose exposed to the aerosolised test article.
- Generation of atmospheres: Atmospheres were generated under dynamic conditions using a piston pump and a binary nozzle.
- Generation of aerosol: The test substance was nebulized neat using conditioned (dry, oil-free) compressed air. The liquid containing parts were maintained at 80 °C, in order to decrease the viscosity of the test item. To increase the efficiency of the generation of respirable particles and to prevent larger particles from entering the chamber a preseparator/ baffle system was used. The inhalation chamber had a volume of 40 L.
- Conditioning of compressed air: The air supply was approx. 15 L/min. Control devices were employed to control supply pressure. The ratio between the air supplied and exhausted was chosen so that approx. 80% of the supplied air was removed via the exhaust system. The achieved air exchange was 15- up to max. 30-times/hour.
- Exhaust air treatment: The exhaust air was purified via filter systems.
- Temperature measurements revealed a temperature range of 23-25 °C.

TEST ATMOSPHERE
- Lower concentrations of the aerosol were characterised by using real-time laser-velocimeter (TSI APS 33).
- Samples taken from breathing zone: yes
- Brief description of analytical method used: gravimetric (analysis of glass-fibre-filter) and HPLC analysis (HPLC-method: Nitro-reagent-treated glass fibres were exposed to the test atmosphere. The content of isocyanate was analytically detected via HPLC.). Gravimetrically determined concentrations were essentially similar to concentrations determined by nitro-reagent derivatisation technique.
- Particle size distribution: The particle-size distribution was analysed using an Anderson or Berner cascade impactor. Approx. 90 % of the particles were < 5 µm and therefore respirable for the rat.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 2.5-3 µm / 1.7
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
150, 162, 283, 402, 438, 833, and 1033 mg/m³
No. of animals per sex per dose:
5 up to 10/sex and dose group
Control animals:
no
Details on study design:
- Duration of observation period following administration: 3 weeks
- Frequency of observations and weighing: Observations were done several times on testing day and twice per day thereafter. Bodyweights were recorded prior to exposure and once weekly thereafter.
- Necropsy of survivors performed: yes
Statistics:
LC50 calculation: According to A.P. Rosiello, J.M. Essigmann and G.N. Wogan (J. Tox. and Environ. Health 3, 797 -809, 1977). This method is based on the Maximum likelyhood method of C.I. Bliss (Q. J. Pharm. Pharmacol. 11, 192 -216, 1938).
Sex:
male
Dose descriptor:
LC50
Effect level:
543 mg/m³ air
95% CL:
431 - 687
Exp. duration:
4 h
Sex:
female
Dose descriptor:
LC50
Effect level:
390 mg/m³ air
Exp. duration:
4 h
Remarks on result:
other: approximate value
Sex:
male/female
Dose descriptor:
LC50
Effect level:
462 mg/m³ air
95% CL:
404 - 529
Remarks on result:
other: Value for combined sexes not reported in study, but published in Pauluhn, Toxicol. Sciences 58, 2000, 173-181.
Mortality:
Exposure to concentrations up to and including 162 mg/m³ were tolerated without mortality. Aerosol exposure starting from 283 mg/m³ and higher induced test substance related mortality. Deaths occurred in all groups on the day of exposure and first postexposure days, and in some cases up to the fourth postexposure day.
Mortality data (concentration - no. of deaths/total number of animals (time of death):
Males: 150 mg/m³ - 0/5, 162 mg/m³ - 0/10, 283 mg/m³ - 1/5 (1d), 402 mg/m³ - 3/10 (1d-4d), 438 mg/m³ - 3/10 (1d), 833 mg/m³ - 3/5 (7h-1d), 1033 mg/m³ - 10/10 (4h-1d)
Females: 150 mg/m³ - 0/5, 162 mg/m³ - 0/10, 283 mg/m³ - 0/5, 402 mg/m³ - 6/10 (4h-4d), 438 mg/m³ - 8/10 (1d), 833 mg/m³ - 5/5 (7h-1d), 1033 mg/m³ - 10/10 (4h-1d)).
Clinical signs:
Concentrations of 150 mg/m³ and higher were followed concentration-dependent by signs such as dyspnoea, bloody snouts, salivation, limp, constricted or closed eyelids, ataxia, reduced motility, ungroomed hair-coat and piloerection. In most instances, signs resolved completely within the first four postexposure days. At the latest, all clinical signs were resolved for the surviving animals after 13 days.
Body weight:
Loss of body weight was observed at exposure concentrations of 402 mg/m³ and higher.
Gross pathology:
Animals which died during post-observation period: lungs distended, red-gray to dark-red foci and colloidal areas in lungs, serous liquid in thorax and lung, lobulation of liver, gastro-intestinal tract with yellowish mucous content, duodenum reddened, kidneys pale and marmorated.
Animals killed at the end of post-observation period: lungs distended, red-gray to dark-red foci and colloidal areas in lungs.
No other findings than at the respiratory tract observed.

For acute inhalation toxicity a read across to HDI oligomers, isocyanurate type (EC 931 -274 -8) is applied. This substance is a close structural analogue to HDI oligomers, iminooxadiazindione type, also derived from catalytic oligomerisation of 1,6 -hexamethylene diisocyanate (HDI; CAS 822 -06 -0) and also belonging to the CAS number 28182-81-2 (Hexane, 1,6 - diisocyanato-, homopolymer). The read across is based on physicochemical and toxicological similarity. In fact, comparison of the toxicological endpoints, that are available for both of the two substances (Acute oral toxicity, Acute inhalation toxicity, Skin and Eye Irritation/Corrosion, Skin Sensitisation, Bacterial mutagenicity (Ames)) reveal good correlation. With respect to Inhalation Toxicity an expert statement is available justifying the read across (Pauluhn, Comparison of pulmonary irritation potency..., Bayer HealthCare AG, 2008).

Therefore, test results obtained for HDI oligomers, isocyanurate type can be transferred to HDI oligomers, iminooxadiazindione type and the results of the acute inhalation toxicity study of HDI oligomers, isocyanurate type are also valid for HDI oligomers, iminooxadiazindione type. This approach is in accordance with Annex XI, section 1.5 of the REACH Regulation (Regulation (EC) No 1907/2006).

Executive summary:

An acute (4h) inhalation toxicity study with according to OECD TG 403 was conducted with groups of 5 to 10 male and 5 to 10 female Wistar rats. In this study animals were head-nose exposed to the aerosolised test substance in concentrations of 150 up to 1033 mg/m³. The aerosol was of adequate respirability for the rats (90 % particles < 5 µm; MMAD 2.5-3 µm / GSD 1.7).

Exposure to concentrations up to and including 162 mg/m³ were tolerated without mortality. Aerosol concentrations starting from 283 mg/m³ and higher induced test substance related mortality. Deaths occurred in all groups on the day of exposure and first postexposure days; in some cases up to the fourth postexposure day. Exposure to concentrations of 150 mg/m³ and higher were followed by concentration-dependent signs suggestive of irritation to the respiratory tract (e.g. dyspnoea, bloody snouts, salivation) and non-specific signs such as limp, reduced motility, and loss of body weight. In most instances, signs resolved completely within the first four postexposure days. At necropsy changes in lung parenchyma (toxic lung oedema) were reported and were seen in the context of local irritating properties of the substance. No other necropsy findings than at the respiratory tract were observed. The LC50 (4 h) in rats was calculated to be 543 mg/m³ for males and approx. 390 mg/m³ for females.

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

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
2 000 mg/kg bw

Additional information

The acute oral LD50 of the substance was > 2000 mg/kg bw in female rats (OECD TG 423). No mortality, no clinical signs, no effects on body weight and no gross pathology findings were observed.

For assessment of acute dermal and acute inhalation toxicity a read across to HDI oligomers, isocyanurate type (EC 931 -274 -8) is applied. This substance is a close structural analogue to HDI oligomers, iminooxadiazindione type, also derived from catalytic oligomerisation of 1,6 -hexamethylene diisocyanate (HDI; CAS 822 -06 -0) and also belonging to the CAS number 28182-81-2 (Hexane, 1,6 - diisocyanato-, homopolymer). The read across is based on physicochemical and toxicological similarity. In fact, comparison of the toxicological endpoints, that are available for both of the two substances reveal good correlation.

 Toxicological endpoint  HDI oligomers, isocyanurate type  HDI oligomers, iminooxadiazindione type
 Acute oral toxicity  > 2000 mg/kg  > 2000 mg/kg
 Acute inhalation toxicity (pulmonary irritant study) NOAEL 3 mg/m³   NOAEL 2.1 mg/m³
 Skin Irritation/Corrosion  slight irritation/no classification required  slight irritation/no classification required
 Eye Irritation/Corrosion  very slight irritation/no classification required  very slight irritation/no classification required
 Skin Sensitisation  classification required  classification required
 Bacterial Mutagenicity (Ames)  negative  negative

With respect to Inhalation Toxicity an expert statement is available justifying the read across (Pauluhn, Comparison of pulmonary irritation potency..., Bayer HealthCare AG, 2008; attached to this endpoint summary).

Therefore, test results obtained for HDI oligomers, isocyanurate type can be transferred to HDI oligomers, iminooxadiazindione type and data an acute dermal and acute inhalation toxicity of HDI oligomers, isocyanurate type are also valid for HDI oligomers, iminooxadiazindione type. This approach is in accordance with Annex XI, section 1.5 of the REACH Regulation (Regulation (EC) No 1907/2006).

Read across to the structural analogue HDI oligomers, isocyanurate type give an acute dermal LD50 of > 2000 mg/kg bw from either an OECD TG 402-study on rats and a study according to EPA Health Effects Test guideline on rabbits. No mortalities occurred for both species during the studies, but for the more sensitive rabbits, in contrast to the rats, particularly skin reactions were observed.

Read across to the structural analogue give also the LC50 for acute aerosol inhalation toxicity (4h, head/nose only exposure) with approx. 390 mg/m³ air for female and 543 mg/m³ air for male rats (OECD TG 403; LC 50 for both sexes combined = 462 mg/m³). In this study exposure to concentrations of 150 mg/m³ and higher were followed by concentration-dependent signs of respiratory irritation. Effects of surviving animals were in most cases completely resolved within the first four post-exposure days. At necropsy changes in lung parenchyma were reported (toxic lung edema) and were seen in the context of the local irritant properties of the substance. Findings on organs other than the respiratory tract were not observed.

Furthermore, a study with HDI oligomers, iminooxadiazindione type itself, investigating the pulmonary irritant potency on rats, is available. The study design is 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 in 2009). The objective of such a kind of study is to analyse concentration- and time-dependently changes in bronchoalveolar lavage (BAL) endpoints as markers of pulmonary irritation following a single 6 hours inhalation exposure. In this study male rats were exposed to concentrations of 0 (air control), 0.45, 3.3, 15.2, and 46.4 mg/m³ of the aerosolised test item. The liquid aerosol was generated so that it was of good respirability for the rats (MMAD 1.2 -1.5 µm, GSD approx. 1.7). BAL fluid was sampled on exposure day (approx. 3h after cessation of exposure) and on post-exposure days 1, 3, and 7 using each six rats per group.

Rats exposed to 15.2 mg/m³ and above elaborated concentration-dependent signs, most of them directly related to respiratory tract irritation. At these exposure levels clinical signs were observed up to postexposure day 7. At 3.3 mg/m³ and above lung weights and parameters in BAL fluid were significantly increased. The most sensitive endpoint, BAL-protein, was maximal on the exposure day and day 1. Essentially all changes directly related to pulmonary irritation declined rapidly towards postexposure day 3 and were indistinguishable from the control on postexposure day 7. Overall it can be concluded that at 0.45 mg/m³ there were no significant changes in any of the BAL endpoints or lung weights whilst they were significantly increased at 3.3 mg/m³. With respect to lower respiratory tract irritation, the extrapolated NO(A)EL is 2.1 mg/m³ (MLE benchmark NOAEL based on BAL-protein).

For study PH 28522 (Bayer Corp., 1999) see "Summary and discussion of irritation" in chapter "Irritation".


Justification for selection of acute toxicity – oral endpoint
Only one study available

Justification for selection of acute toxicity – inhalation endpoint
Read-across to the key LC50-study of the structural analogue HDI oligomers, isocyanurate type is applied. For HDI oligomers, iminooxadiazindione type itself a study investigating the pulmonary irritant threshold ("TRGS 430-study") is available and is also of high value for the assessment of acute inhalation toxicity, but is not selected here, since a study providing a LC50-value is requested.

Justification for selection of acute toxicity – dermal endpoint
Read-across to the two key LD50-studies of the structural analogue HDI oligomers, isocyanurate type is applied. None of the studies is selected here, because both key-studies on acute dermal toxicity, one conducted on rats and one on rabbits, are of equivalent value for assessment purposes. The result is in both cases 2000 mg/kg bw as the discriminating dose.

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex I, no classification is warranted for acute oral and dermal toxicity.

According to Regulation (EC) No 1272/2008, Annex I, classification for acute inhalation toxicity is warranted. Despite the 4 hour LC50 of 462 mg/m³ (females 390 mg/m³, males 543 mg/m³) the substance is not classified according as Cat. 2 for acute inhalation toxicity, but as Cat. 4 due to the following reasons:

Regulation (EC) No 1272/2008 and ECHA Guidance on the Application of the CLP Criteria [1] 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 Pauluhn there is an option for a modified Classification and Labelling (C&L) [2]. 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 concept is now to extrapolate 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 procedure is already acknowledged in the ECHA guidance on the Application of the CLP criteria and shall be referred to in the following as the "Acute Aerosol Assessment" (AAA).

Based on the currently available data the prerequisites [2] for employing AAA are given for HDI oligomers, iminooxadiazindione type, i.e. 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 and repeated inhalation exposure to rats (Bayer AG, 2004, 1993, 1987, and 1985). 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 oligomers, isocyanurate, biuret, uretdione, and allophanate type) consistently demonstrates the above discussed toxicological mode of action.

For AAA the general concept established for the classification of mixtures applies (cp. Regulation (EC) No 1272/2008). 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 the substance. 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 0.46 mg/L an Acute Toxicity Estimate for the aerosolized mixture (ATEmix) of 3.9 mg/L is obtained. According to the respective concentration limits as defined by CLP classification as acute inhalation toxicity (dusts and mists) Cat. 4 is thus proposed for the substance. [3]

 

[1] Guidance on the Application of the CLP Criteria (ECHA-15-G-05-EN), Version 4.1, June 2015, p. 247.
[2] Pauluhn J, Experimental and Toxicologic Pathology 60, 2008, 111-124
[3] Pauluhn 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