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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: Guideline study without detailed documentation.
Principles of method if other than guideline:
Gavage administration of limit dose 5000 mL/kg bw to each 5 male and female rats; Investigation of clinical signs, body weights and mortality; Duration of observation period following administration: 14 days; Necropsy of survivors performed at termination of study
GLP compliance:
not specified
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Bor: WISW (SPF Cpb)
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: approx. 9 (males) and 14 (females) weeks
- Weight at study initiation (mean): males 199 g, females 169 g
- Fasting period before study: Food was withheld from the animals for approx. 16 h before test substance administration until approx. 4 h after test substance administration.
- Housing: in groups of five in Makrolon type III cages on low dust wood granulate bedding.
- Diet and water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1.5
- Humidity (%): 60 +/- 5
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Doses:
5000 mL/kg bw eq. to 6000 mg/kg bw (density 1.2 g/mL)
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 and weighing: Clinical signs and mortality were checked several times on the day of administration and twice daily thereafter (exemption: once on week-ends and holidays). Body weights were determined on the day of administration, after one week and at termination of the study.
- Necropsy of survivors performed: yes, all animals were necropsied at the end of study.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 5 000 mL/kg bw
Remarks on result:
other: eq. to > 6000 mg/kg (density 1.2 g/mL)
Mortality:
Mortalities were not observed during the study.
Clinical signs:
None of the male or female animals showed clinical signs during the study.
Body weight:
No effects on body weights reported.
Gross pathology:
Gross necropsy revealed no test-substance related findings.
Executive summary:

For assessment of acute oral toxicity a limit test is available. In this study 5 male and 5 female rats were gavaged once at a dose of 5000 mL/kg bw (eq. to 6000 mg/kg bw; density 1.2 mg/mL). Animals were observed for a 14 -days period for clinical signs and mortality, furthermore necropsy was performed at study termination.

Mortalities and clinical signs did not occur in the course of the study. Gross necropsy revealed no test-substance related findings. The LD50 was therefore concluded to be > 5000 mL/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
Value:
6 000 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
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF)
- Source: Harlan-Nederland, AD Horst, The Netherlands
- 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 (based on A. Spiegel and R. Goennert, Zschr. Versuchstierkunde, 1, 38 (1961) and G. Meister, Zschr. Versuchstierkunde, 7, 144-153 (1965).
- Diet and water: ad libitum
- Acclimation period: at least 5 days; during this period, rats were also acclimatized to the restraining tubes.


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
- Mode of exposure: Animals were nose-only exposed to the aerosolized test article in restrainers made of Plexiglas. Restrainers were chosen that accommodated the animals' size. The type of exposure principle is comparable with a directed-flow exposure design (Moss and Asgharian, Respiratory Drug Delivery IV, 1994, 197) and minimizes re-breathing of exhaled test atmosphere. The ratio between supply and exhaust air was selected so that 90% of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports.
- Exposure apparatus: Plexiglas exposure restrainers (TSE, Bad Homburg, Germany). Each inhalation chamber segment was suitable to accommodate 20 rats at the perimeter location. 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 (freed from water, dust and oil) automatically by a VIA compressed air dryer.
- System of generating particulates/aerosols: Under dynamic conditions the various concentrations of the test article were atomized into the baffle (pre-separator) of the inhalation chamber. For atomization a binary nozzle and conditioned compressed air (15 L/min) was used. The representative dispersion pressure was approximately 600 kPa (constant liquid feed through the nozzle maintained at room temperature). The test article was fed into the nozzle system using a digitally controlled pump (Harvard PHD 2000 infusion pump).
- Optimization of respirability: In order to increase the efficiency of the generation of fine particles through evaporation of the vehicle and to prevent larger particles from entering the chamber a 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 chamber equilibrium is attained in less than one minute of exposure. At each exposure port a minimal air flow rate of 0.75 L/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 Messgeräte GmbH, Ettlingen, Germany). The position of the 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).
- 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 analysed using a BERNER critical orifice cascade impactor. Aerosol mass < 3 µm: 89.3 % (109 mg/m³), 86.6 % (166 mg/m³), and 86.1 % (428 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) throughout the groups was 1.59 – 1.68 µm, the geometric standard deviation (GSD) was 1.67 – 1.73.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
gravimetric analysis
Duration of exposure:
4 h
Concentrations:
109, 166, and 428 mg/m³
No. of animals per sex per dose:
5
Control animals:
other: yes, air-control
Details on study design:
- Duration of observation period following administration: 2 weeks
- Frequency of observations and weighing: Bodyweights were recorded prior to exposure and on days 1, 3, and 7, and weekly thereafter. The appearance and behaviour of each rat were examined carefully several times on the day of exposure and at least once daily thereafter.
- Necropsy of survivors performed: yes
- Other examinations performed: Reflexes were tested, based on recommendations made by Irwin (Psychopharmacologica 13, 1968, 222-257). Rectal temperatures were measured shortly after cessation of exposure (approximately within ½hour after the end of exposure) using a digital thermometer with a rectal probe for rats.
Statistics:
For necropsy findings: pair-wise Fisher test after the R x C chi-squared test.
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, 797) as modified by Pauluhn (1983). It is based on the maximum likelihood method of Bliss (1983; Q.J.Pharm.Pharmacol., 11, 192).
Sex:
female
Dose descriptor:
LC50
Effect level:
ca. 135 mg/m³ air
Exp. duration:
4 h
Sex:
male/female
Dose descriptor:
LC50
Effect level:
ca. 161 mg/m³ air
Exp. duration:
4 h
Mortality:
Mortality occurred at 109, 166 and 428 mg/m³ (males: 0/5 at 0 mg/m³, 1/5 at 109 mg/m³, 1/5 at 166 mg/m³, 5/5 at 428 mg/m³; females: 0/5 at 0 mg/m³, 1/5 at 109 mg/m³, 4/5 at 166 mg/m³, 5/5 at 428 mg/m³).
Female rats seem to be more susceptible than male rats due to the fact that at 166 mg/m³ only one male rat out of five died whereas four of five female rats died at this concentration. All rats exposed to 428 mg/m³ died on day 0.
Clinical signs:
other: All rats exposed to 109 and 166 mg/m³ showed clinical signs (e.g. irregular breathing, bradypnea, laboured breathing, dyspnea, breathing sounds, stridor, nose red encrusted, nostrils with red encrustations, motility reduced, tremor, atony, high-legged gai
Body weight:
Significant decreased body weights were found at 109 mg/m³ and above.
Gross pathology:
Necropsy findings were predominantly unremarkable in surviving rats whereas in rats that succumbed in the course of study the following findings of toxicological importance at 109 mg/m³ and above were observed (e.g. nostrils: brownish encrustations; nose: red encrusted, whitish viscous content; trachea: white foamy content; lung: less collapsed, light coloured, dark-red, dark-red marbled; pleural cavity: clear liquid content).
Other findings:
Significant decreased body temperatures were found at 109 mg/m³ and above.

The mortality patterns were typical of an irritation-related acute lung edema (e.g.: white foamy discharge in the nose, white foamy content in the trachea, less collapsed and dark-red marbled lungs, bradypnea, labored breathing, breathing sounds, stridor, cyanosis, pallor).

Executive summary:

An acute inhalation toxicity study in rats was recently conducted for reasons of product safety (the substance is used in spray applications). In this study, conducted according to OECD TG 403 and OECD GD 39, male and female rats were nose-only exposed to the liquid aerosol of the substance at 0 (air control), 109, 166, and 428 mg/m³. The respirability of the aerosol was in compliance with the respective test guidelines (the MMAD throughout the groups was 1.59 – 1.68 µm, the GSD was 1.67 – 1.73).

All rats exposed to 109 and 166 mg/m³ showed clinical signs (e.g. irregular breathing, bradypnea, stridor, nose and nostrils with red encrustations, motility reduced, tremor, atony, piloerection, haircoat ungroomed, emaciation, nasal discharge (serous), reduced reflexes). Significant decreased body temperatures and body weights were found at 109 mg/m³ and above. Mortality occurred at 109, 166 and 428 mg/m³. Female rats seem to be more susceptible than male rats due to the fact that at 166 mg/m³ only one male rat out of five died whereas four of five females died at this concentration. All rats exposed to 428 mg/m³ died on day 0. The mortality patterns were typical of an irritation-related acute lung oedema (e.g.: white foamy discharge in the nose, white foamy content in the trachea, less collapsed and dark-red marbled lungs, bradypnea, labored breathing, breathing sounds, stridor, cyanosis, pallor). Necropsy revealed findings of toxicological importance at 109 mg/m³ and above (e.g. nose: red encrusted, whitish viscous content; trachea: white foamy content; lung: less collapsed, light coloured, dark-red, dark-red marbled).

Due to the fact that a gender-specific mortality pattern was observed at 166 mg/m³, the LC50 for the more susceptible females is taken forward: LC50 (4 h, females) 135 mg/m³.

 

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

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For assessment of acute oral toxicity a limit test is available. In this study 5 male and 5 female rats were gavaged once at a dose of 5000 mL/kg bw (eq. to 6000 mg/kg bw; density 1.2 mg/mL). Animals were observed for a 14 -days period for clinical signs and mortality, furthermore at study termination necropsy was performed.

Mortalities and clinical signs did not occur in the course of the study. Gross necropsy revealed no test-substance related findings. The LD50 was therefore concluded to be > 5000 mL/kg bw.

An acute inhalation toxicity study in rats was recently conducted for requirements of product safety (the substance is used in spray applications). In this study, conducted according to OECD TG 403 and OECD GD 39, male and female rats were nose-only exposed to the liquid aerosol of the substance at 0 (air control), 109, 166, and 428 mg/m³. The respirability of the aerosol was in compliance with the respective test guidelines (the MMAD throughout the groups was 1.59 – 1.68 µm, the GSD was 1.67 – 1.73).

All rats exposed to 109 and 166 mg/m³ showed clinical signs (e.g. irregular breathing, bradypnea, stridor, nose and nostrils with red encrustations, motility reduced, tremor, atony, piloerection, haircoat ungroomed, emaciation, nasal discharge (serous), reduced reflexes). Significant decreased body temperatures and body weights were found at 109 mg/m³ and above. Mortality occurred at 109, 166 and 428 mg/m³. Female rats seem to be more susceptible than male rats due to the fact that at 166 mg/m³ only one male rat out of five died whereas four of five females died at this concentration. All rats exposed to 428 mg/m³ died on day 0. The mortality patterns were typical of an irritation-related acute lung oedema. Necropsy revealed findings of toxicological importance at 109 mg/m³ and above (e.g. nose: red encrusted, whitish viscous content; trachea: white foamy content; lung: less collapsed, light coloured, dark-red, dark-red marbled).

Due to the fact that a gender-specific mortality pattern was observed at 166 mg/m³, the LC50 for the more susceptible females is taken forward: LC50 (4h, females) 135 mg/m³.

No acute dermal toxicity is available for the substance. Nevertheless an assessment of acute dermal toxicity is possible based on the available information.

The substance has a molecular weight of 380 g/mol (related to the idealised structure of the UVCB), which is not contradictive for systemic availability after dermal exposure. Due to the reactive isocyanate groups the substance is unstable towards nucleophiles, e.g. water or alcohols.

For acute inhalation the available study on rats reveals indications for “port-of-entry” irritant toxicity (clinical signs as irregular breathing, nose and nostrils with red encrustrations and serous nasal discharge; "irritation-related acute lung edema"), which is typical for aliphatic isocyanates and thus confirms the typical isocyanate-mode of action .

After acute oral exposure the substance was practically non-toxic (LD50 > 5000 mL/kg). In the respective study the substance caused no mortalities, clinical signs or findings at gross necropsy.

The available data on skin/eye irritation/corrosion and sensitisation (Buehler) confirm a slight irritant property for the substance. With the exception of skin sensitisation (Buehler and GPMT both have a positive result) the available data reveal no indications for systemic availability, however, due to the positive skin sensitisation it cannot be completely ruled out. Nevertheless, despite the irritant effect which may diminish the protective function of the epidermis (worst case assumption) it is not expected for a substance with such a profile (i.e. irritancy at the port-of-entry due to isocyanate-reactivity) that systemic availability after dermal exposure would be higher than after oral exposure.

Overall, with the mode of action outlined above, the oral LD50 > 5000 mL/kg and no overt signs of toxicity after oral exposure, it is not expected that testing on acute dermal toxicity would reveal adverse systemic effects. Local effects were already assessed by the respective skin irritation/corrosion studies. Thus, based on the weight of evidence a conclusion can be drawn that for acute dermal toxicity no hazard is expected. This is in line with recent publications [1][2], in which the benefit of performing acute dermal toxicity studies in general is questioned, in particular if there are no indications for a substance to be hazardous via the oral route. The European Competent Authorities for REACH and CLP (Caracal) have already acknowledged that. From publicly available information it can be derived that the committee agreed on proposals to amend REACH Annex VIII (8.5.3) as such that substances with no acute oral toxicity up to 2000 mg/kg would not require acute dermal toxicity data. [3][4]

[1] Moore, Regulatory Toxicology and Pharmacology, 2013, 66, 30-37

[2] Creton, St. et al, Critical reviews in Toxicology, 2010, Vol. 40 No.1, pages 50-83

[3] http://www.piscltd.org.uk/wp-content/uploads/2014/09/20140714-110033_CA_61_2014-Acute-toxicity-testing-proposal.pdf

[4] https://chemicalwatch.com/20557/caracal-agrees-need-to-drop-acute-dermal-toxicity-tests



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

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

Justification for classification or non-classification

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

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

With the mode of action outlined above, the expectation that systemic availability after dermal exposure will not exceed systemic availability after oral exposure, the oral LD50 > 5000 mL/kg and no signs of toxicity at all after oral exposure, it is not expected that testing on acute dermal toxicity would lead to a hazard classification for the substance. Therefore, it can be concluded that the available data are conclusive for non-classification of acute dermal toxicity.

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

Despite the 4 hour LC50 of 135 mg/m³ the substance is not classified as Cat. 2 for acute inhalation toxicity, but 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 all 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 the 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 acute inhalation exposure to rats (Bayer AG 2014). 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 studies for substances with similar compositions (HDI homopolymers, CAS No. 28182 -81 -2, including HDI oligomers, isocyanurate, iminooxadiazindione, 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 0.135 mg/L an Acute Toxicity Estimate for the aerosolized mixture (ATEmix) > 0.5 mg/L is obtained. According to the respective concentration limits as defined by CLP classificationas acute inhalation toxicity (dusts and mists) Cat. 3 is thus proposed for the substance.

 

[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