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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study meets generally accepted scientific principles, acceptable for assessment: non-GLP, limited documentation, no data on purity of test substance, no necropsy.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
yes
Remarks:
, no necropsy performed
GLP compliance:
no
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Wistar TNO W 74
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 9-14 weeks
- Mean weight at study initiation: males 182 g, females 167 g
- Housing: in groups (5 animals per cage) in Makrolon Type III cages
- Diet and water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1.5 °C
- Humidity (%): 60 +/- 5 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Doses:
5 mL/kg bw
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: several times on day of administration, twice daily within 14-days postobservation period, except for weekends (once daily)
- Frequency of weighing: on day of administration and at the end of the 14-days postobservation period
- Necropsy of survivors performed: no
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 5 mL/kg bw
Remarks on result:
other: corresponding to > 5665 mg/kg bw (relative density at 20°C 1.133 g/mL)
Mortality:
No mortality observed
Clinical signs:
No clinical signs observed
Body weight:
No effect on body weight gain observed
Gross pathology:
not performed
Executive summary:

In an acute oral toxicity study similar to OECD TG 401 5 male and 5 female rats received each a limit dose of 5 ml/kg bw. No mortality, no clinical signs and no effect on body weight gain was observed during treatment and the 14-days postobservation period. Necropsy was not performed in the study. The LD50 was determined to be > 5 ml/kg bw (corresponding to > 5665 mg/kg bw; relative density at 20°C 1.133 g/mL)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
Value:
5 665 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)
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF)
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approximately 2 months
- Weight at study initiation: At the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex
- Housing: singly in conventional Makrolon® Type IIIH cages
- Diet and water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- 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
- Mode of exposure: Animals were exposed to the aerosolized test article in restrainers made of Plexiglas. The design of the directed-flow inhalation chamber prevents rebreathing of the test atmosphere.
- Generation of atmosphere: Atmospheres were generated under dynamic conditions using a digitally controlled Harvard S/N infusion pump and a modified Schlick-nozzle Type 970, form-S 3 (Schlick GmbH, COburg, Germany).
- Generation of aerosol: The test substance was nebulized neat using conditioned (dry, oil-free) compressed air (dispersion pressure approximately 600 kPa, 10-30 µl/min solution of the test substance, 15 L air/min and inhalation chamber segment (1 segment used). The respective concentration was achieved by applying extraction/dilution cascades.
- Inhalation chamber: One segment of the aluminum inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 L). Details of this modular chamber and its validation have been published previously (Pauluhn, Journal of Applied Toxicology, 14, 55-62, 1994).
- Optimization of respirability: In order 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.
- Conditioning the compressed air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- Inhalation chamber steady-state concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (ca. 474 x, continuous generation of test atmosphere). Under such test conditions steady state is attained within the first minute of exposure (t99% = 4.6 x chamber volume/flow rate). The ratio between the air supplied and exhausted was chosen so that approximately 90% of the supplied air is removed via the exhaust system. The remainder provides adequate dead-space ventilation for the exposure tubes. 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.
- Exhaust air treatment: The exhaust air was purified by passage through aseries of aerosol filters.
- Temperature and humidity measurements are also performed by the computerized HP 3852A Data Acquisition and Control System using FTF sensors (ELKA ELEKTRONIK, Lüdenscheid, Germany). The position of the measuring probe was at the exposure location of rats.

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).
- Samples taken from breathing zone: yes
- Brief description of analytical method used: gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
- Particle size distribution: The particle-size distribution was analyzed using a BERNER-TYPE AERAS lowpressure criticai orifice cascade impactor (Hauke, Gmunden, Austria). Aerosol mass < 3 µm: 85.9 % for 65 mg/m³, 87.3 % for 117 mg/m³, 85.2 % for 168 mg/m³, 88 % for 357 mg/m³.
- MMAD (Mass median aerodynamic diameter): < 4 µm (mean MMAD = 1.5-1.8 µm) / GSD (Geometric st. dev.): 1.7
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
65, 117, 168, and 357 mg/m³
No. of animals per sex per dose:
5
Control animals:
other: yes, controls were exposed to conditioned dry air
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Body weights were measured before exposure, on days 3 and 7, and weekly thereafter. 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).
- Necropsy of survivors performed: yes
- Other examinations performed: The rectaI temperatures were measured shortly after cessation of exposure using a digital thermometer with a rectal probe for rats. A battery of reflex measurements was made on the first postexposure day.
Statistics:
For necropsy findings: pair-wise Fisher test after the R x C chi-squared test in accordance with Gad and Weil (Statistics for Toxicologists. Principles and Methods of Toxicology, ed. AW. Hayes, Raven Press, New York, 280, 1982). For statistical evaluation of the body weight gain for each group a one-way ANOVA (vide infra) is used. Data of rectaI temperature measurements are statistically evaluated using the ANOVA procedure (vide infra). For LC50 calculation: method of Rosiello et al. (J. Tox. and Environ. Health, 3, 797-809, 1977) as modified by Pauluhn (Computer-Aided Estimation of the LD50/LC50 BAYER AG Report No. 11835, dated May 18, 1983).
Sex:
male/female
Dose descriptor:
LC50
Effect level:
ca. 158 mg/m³ air
Exp. duration:
4 h
Sex:
male/female
Dose descriptor:
other: NO(A)EL
Effect level:
< 65 mg/m³ air
Exp. duration:
4 h
Mortality:
Mortality did not occur at 65 mg/m³ whereas at higher concentration rats succumbed on the exposure day or first postexposure days in a concentration-dependent manner.
Animals that died (onset of mortality): males 0/5 at 65 mg/m³, 0/5 at 117 mg/m³, 3/5 at 168 mg/m³ (0d, 1d), 5/5 at 357 mg/m³ (0d, 1d); females 0/5 at 65 mg/m³, 1/5 at 117 mg/m³ (1d), 3/5 at 168 mg/m³ (1d), 5/5 at 357 mg/m³ (0d, 1d).
Clinical signs:
other: Dose group 65 mg/m³: all rats tolerated the exposure without specific signs. Rats exhibited clinical signs in dose group 117 mg/m³ and above. The following signs occured: irregular breathing patterns, labored breathing patterns, tachypnea, piloerection, h
Body weight:
Comparisons between the control and the exposure groups revealed significant, although transient changes (decreases) in body weights.
Gross pathology:
Necropsy findings were unremarkable in surviving rats whereas in rats that succumbed intercurrently the following findings predominated: nose: serous discharge, lung less collapsed and edema, hydrothorax, consolidation, discoloration of parenchymatous organs.
Other findings:
Reflexes: In comparison to the rats of the control group, some rats of group 3 and 4 displayed a reduced righting response.
Rectal temperature: Statistical comparisons between the control and the exposure groups revealed significant changes in body temperatures indicative of hypothermia.

The rats displayed signs suggestive of respiratory irritation. Necropsy findings support the conclusion that mortality is causally related to acute lower respiratory tract irritation, i.e., lung edema.

Executive summary:

An Acute Inhalation Toxicity study according to OECD TG 403 was conducted on 5 rats per sex, which were nose-only exposed to concentrations of 65, 117, 168 and 357 mg/m³ test substance aerosol. The aerosol was of adequate respirability for the rats (mean MMAD 1.5 -1.8 µm / GSD 1.7).

Mortality did not occur at 65 mg/m³ whereas at higher concentration rats succumbed on the exposure day or first postexposure days in a concentration-dependent manner. Based on the result of the probit analysis, the approximate LC50 was 158.3 mg/m³. The rats displayed signs suggestive of respiratory irritation. Necropsy findings (lung edema) support the conclusion that mortality is causally related to acute lower respiratory tract irritation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
Value:
158 mg/m³

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The acute oral LD50 of the substance was > 5 mL/kg bw in male and female rats in a study similar to OECD TG 401 (corresponding to > 5665 mg/kg bw, rel. density 1.133 g/mL at 20 °C). No mortality, no clinical signs and no effects on body weight were observed. Gross pathology was not performed.

Acute dermal toxicity studies are not available for the substance.

In an acute aerosol inhalation study on rats according to OECD TG 403 (nose-only exposure) the LC50 (4 h) was determined to be approximately 158 mg/m³. The aerosol was of adequate respirability for the rats (mean MMAD 1.5 -1.8 µm / GSD 1.7).

In this study mortality did not occur at a concentration of 65 mg/m³ whereas at higher concentrations rats succumbed on the exposure day or first postexposure days in a concentration-dependent manner. The rats displayed signs suggestive of respiratory irritation. Necropsy findings (lung edema) support the conclusion that mortality is causally related to acute lower respiratory tract irritation.

Additionally available is a pulmonary irritant potency study based on TRGS 430 (Technical Rule for Hazardous Substances 430, published by German Federal ministry of Labour and Social Affairs, last update in 2009). In this study male rats were exposed once for 6 hours to concentrations of 0 (air control), 0.56, 2.8, 18.5, and 50.8 mg/m³ of the aerosolized test item. The liquid aerosol was generated so that it was respirable for rats (MMAD ranging from 1.2 - 1.5 µm; GSD approximately 1.7). Following exposure bronchoalveolar lavage (BAL) fluid was analysed for endpoints indicative for pulmonary irritation. For this purpose BAL fluid was sampled on exposure day (approx. 3 hours after cessation of exposure) and on postexposure days 1, 3, and 7 using six rats per group.

As result, the study suggested that the respirable substance aerosol appears to interact directly with the air-blood barrier causing a temporarily increased extravasation of plasma proteins as a result of an increased, transient permeability of the capillary endothelial cells. The data generated show unequivocally that the concentration of respirable particulates is most important for the elicitation of irritant related pulmonary response. At high exposure levels (18.5 mg/m³ and above) clinical evidence existed that the aerosol elicited both a lower as well as an upper respiratory tract irritation potential whereas at 2.8 mg/m³ and below clinical evidence of respiratory tract irritation was not observed. With respect to pulmonary irritation 2.8 mg/m³ was considered to be the threshold concentration (NO(A)EL).


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

Justification for selection of acute toxicity – inhalation endpoint
Only one fully reliable LC50-study available. Additionally, an acute inhalation toxicity study investigating the pulmonary irritant threshold ("TRGS 430-study") is available and is also of high value for the evaluation of the substance, but is not selected here, since a study providing a LC50-value is requested.

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 158 mg/m³ the substance is not classified as Cat. 2 for acute inhalation toxicity, but as Cat. 3 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 i 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) in the lower respiratory tract, but no systemic toxicity after inhalation exposure to rats (Bayer AG, 2010, 2006, and 2002). 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, iminooxadiazindione, biuret, 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 0.158 mg/L an Acute Toxicity Estimate for the aerosolized mixture (ATEmix) of > 0.5 mg/L is obtained. According to the respective concentration limits as defined by CLP classification as acute inhalation toxicity (dusts and mists) Cat. 3 is thus proposed for the substance.

 

[1] Guidance on the Application of the CLP Criteria (ECHA-13-G-10-EN), Version 4.1, June 2015, p. 247. 

[2] Pauluhn J, Experimental and Toxicologic Pathology 60, 2008, 111 -124