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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
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:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Sprague Dawley CD (Crl: CD (SD) IGS BR)
- Source: Charles River Ltd, Margate, Kent, UK.
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: The bodyweights fell within an interval of +/- 20% of the mean initial bodyweight of the first treated group.
- Housing: in groups of 3 in suspended solid-floor polypropylene cages furnished with woodflakes.
- Fasting period before study: An overnight fast immediately before dosing and for approximately three to four hours after dosing.
- Diet and water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%):30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
ADMINISTRATION VOLUME APPLIED: 10 mL/kg

VEHICLE
- Arachis oil BP; For the purpose of the study the test material was freshly prepared, as required, as a solution at the appropriate concentration in arachis oil BP.
- Concentration in vehicle: 200 mg/mL

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: After surveying all available information on the toxicity of the test material, 2000 mg/kg was chosen as the starting dose.
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
6 (3 animals per step)
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: 30 min., 1, 2 and 4 hours after dosing and subsequently once daily
- Frequency of weighing: prior to dosing and seven and fourteen days after treatment or at death.
- Necropsy of survivors performed: yes
Statistics:
Using the mortality data obtained, an estimate of the acute oral median lethal dose of the test material was made according to the scheme of OECD TG 423.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 500 mg/kg bw
Mortality:
There were no deaths.
Clinical signs:
There were no signs of systemic toxicity.
Body weight:
All animals showed expected gains in bodyweight over the study period.
Gross pathology:
No abnormalities were noted at necropsy.
Executive summary:

An acute oral toxicity study according to the Acute Toxic Class Method (OECD TG 423) was performed on 3 + 3 female rats, receiving each a single dose of 2000 mg/kg of the substance. No mortalities, no signs of systemic toxicity and no effects on body weight gain were observed. At necropsy no abnormalities were noted. The LD50 of the test material was estimated according to the flow chart of the OECD TG 423, Annex 2d to be > 2500 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
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
(1981)
GLP compliance:
yes
Test type:
standard acute method
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.
Executive summary:

An acute (4h) inhalation toxicity study 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

An acute oral toxicity study on rats according to OECD TG 423 (Acute Toxic Class Method) revealed no mortalities, no signs of systemic toxicity and no effects on body weight gain up to the limit dose of 2000 mg/kg bw. At necropsy no abnormalities were noted. The LD50 of the test material was therefore > 2000 mg/kg bw.

 

An acute dermal toxicity study on rats according to OECD TG 402 revealed no mortalities, no signs of systemic toxicity and no effects on body weight gain up to the limit dose of 2000 mg/kg bw. No signs of dermal irritation were seen and no abnormalities were noted at necropsy. Another acute dermal toxicity study was conducted on rabbits according to EPA Health Effects Test Guideline (40 CFR 798) and revealed also no mortality, but effects on body weight gain for one male, and abnormalities at necropsy for one male (dark red lungs) and two females (one with light tan kidneys and one with pale tan lungs). Local skin reactions including scabs, desquamation and alopecia were reported in the rabbit-study, but could at least partly be attributed to the resinous properties of the substance, which constitutes a clear, hard sample residue at the end of contact period (In fact, the layer of the gauze sheeting adhered completely to the skin and could not be removed). The LD50 for both dermal studies was > 2000 mg/kg bw.

 

For an assessment of acute inhalation toxicity an aerosol inhalation study according to OECD TG 403 is available. This study was conducted on 5 up to 10 rats per sex and dose group with head/nose-only exposure. The aerosol was of adequate respirability for the animals (90 % particles < 5 µm; MMAD 2.5 -3 µm, GSD approx. 1.7).

In this study concentrations of 150 mg/m³ and higher were followed by concentration-dependent signs of respiratory irritation. Effects on 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 oedema) 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. The LC50 (4h) was calculated to be 543 mg/m³ for male and approx. 390 mg/m³ for female rats.

A second study on rats (OECD TG 403, head/nose-only exposure) with a test article containing 90 % of the substance (= active ingredient) and 10 % solvent confirmed this result qualitatively and quantitatively (LC50 (4h): 573 mg/m³ for male rats (i.e. 516 mg/m³ based on active ingr.), 472 mg/m³ for female rats (i.e. 425 mg/m³ based on active ingr.).

Furthermore, studies investigating the pulmonary irritant potency on rats are available. These studies are based on the recommendations of the TRGS 430 (Technical Rule for Hazardous Substances 430; published by the German Federal Ministry of Labour and Social Affairs). The objective of this kind of study is to analyse concentration- and time-dependent changes in bronchoalveolar lavage (BAL) endpoints as markers of pulmonary irritation following a single 6 hours inhalation exposure.

In the key-TRGS 430-study female rats were exposed to target concentrations of 0 (air control), 4, 15, 60, and 120 mg/m³ of the aerosolised test item. The liquid aerosol was generated so that it was respirable to rats (MMAD approx. 1.9 µm, GSD 1.5). BAL fluid was sampled twice on exposure day (directly and 3h after cessation of exposure) and on post-exposure days 1, 3, and 7 using each six rats per group.

Rats exposed to 60 mg/m³ and above elaborated concentration-dependent clinical signs related to respiratory tract irritation. Necropsy findings in rats exposed to 60 mg/m³ and above provided evidence of concentration-dependent macroscopic alterations, such as lung consolidation and lung oedema. One out of 30 rats exposed to 120 mg/m³ succumbed following exposure.

Analysis of BAL fluid from animals exposed to 15 mg/m³ and above revealed significantly increased BAL endpoints (total protein, ACE) immediately after cessation of exposure, which is indicative for an injury of the bronchoalveolar region. A marked time-dependent exacerbation could not be observed, expect a transiently delayed influx of cells at exposure concentration 120 mg/m³. In most instances, changes returned to the level of control on day 7.

Effects that did not return to the level of control appeared to be related to an up-regulation of protective mechanism such as increased levels of glutathione in lung tissue and acid phosphatase in BAL-cells. The relative comparison of effects revealed that the most sensitive marker indicative of a dysfunction of the air-blood barrier was the increase in ACE activity in BAL fluid.

The study is of high value for assessment purposes, as it indicates the toxicological mode of action of the respirable substance-aerosol: the aerosol appears to interacts directly with the air-blood barrier function causing an increased extravasation of plasma proteins as a result of increased, transient permeability of the capillary endothelial cells. 4 mg/m³ did not evoke any remarkable changes except a transient and borderline, however, statistically significant, increase of protein in BAL fluid. Statistical analysis of this endpoint derived the NO(A)EL at 3 mg/m³.

 

For discussion of RD50-study see Endpoint Summary in chapter Irritation/corrosion.


Justification for selection of acute toxicity – oral endpoint
One of three fully reliable studies selected as key. The result is in any case 2000 mg/kg bw as the discriminating dose.

Justification for selection of acute toxicity – inhalation endpoint
One of the fully reliable LC50-studies selected. This study was found of more value than the others, as it was conducted with the substance as such (not with the dissolved trade product) and obtained a definite value as LC50 (not a range; cp. study of 2011). Moreover, the studies investigating the pulmonary irritant threshold ("TRGS 430-studies") are also of high value for the assessment of acute inhalation toxicity of the substance, but are not selected here, since a study providing a LC50-value is requested.

Justification for selection of acute toxicity – dermal endpoint
None of the key-studies is selected, 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 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 the substance, i.e. the substance is a liquid with a very low volatility causing local toxicity (irritation) to the lower respiratory tract, but no systemic toxicity after inhalation exposure to rats (Bayer AG, 2001/2007, 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, biuret, uretdione, iminooxadiazindione, 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