Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
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
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Reaction mass of 2,2'-iminodiethanol and 2-{[bis(2-hydroxyethyl)amino]methyl}-4-[2-(4-hydroxyphenyl)propan-2-yl]phenol and 2,2-bis(4-hydroxyphenyl)propane and 2-{[bis(2-hydroxyethyl)amino]methyl}-4-[2-(3-{[bis(2-hydroxyethyl)amino]methyl}-4-hydroxyphenyl)propan-2-yl]phenol
- Appearance: viscous, clear, yellowish liquid

Test animals

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 (mean): males 187.4-193.2 g, females 176.8-179.4; 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 with gnawing sticks.
- 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

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: ethanol
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 85-90 % of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports.
- Exposure apparatus: The chambers used are commercially available (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, 55-62, 1994, and Pauluhn & Thiel, Journal of Applied Toxicology 27, 160-167, 2007.
- 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 BEKO RA 55 compressed air dryer.
- System of generating particulates/aerosols: Atmosphere for inhalation exposure was generated under dynamic conditions using a digitally controlled Harvard PHD 2000 infusion pump or similar equipment and a modified Schlick nozzle Type 970, form-S 3 (Schlick GmbH, Coburg, Germany). The test article (solution) was nebulized under dynamic conditions using conditioned (dry, oil free) compressed air (dispersion pressure approximately 600 kPa), 15 L air/min and inhalation chamber segment.
- 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 (Tillery, Environmental Health Perspectives, 16, 25-40, 1976).
- 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: 84.4 % at the limit dose.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The respirability of the aerosol was adequate and in compliance with test guidelines, i.e. the average mass median aerodynamic diameter (MMAD) throughout the groups was 1.14 µm, the geometric standard deviation (GSD) was 2.63.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
gravimetric analysis
Duration of exposure:
4 h
Concentrations:
Target concentration: 5000 mg/m³
Analytical concentration: 4263 mg/m³ (technically maximal attainable concentration)
No. of animals per sex per dose:
5
Control animals:
other: yes, vehicle 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 behavior of each rat were examined carefully at least two 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:
Pair-wise Fisher test after the R x C chi-squared test used for necropsy findings (Procedure in accordance with Gad and Weil, Statistics for Toxicologists. Principles and Methods of Toxicology, ed. A.W. Hayes, Raven Press, New York, 280, 1982).
Analysis of variance (ANOVA) used for statistical evaluation (e.g. body weights, rectal temperatures).

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 4 263 other: mg/m³ (maximal technical attainable concentration)
Exp. duration:
4 h
Mortality:
One animal died at 4263 mg/m³. The mortality patterns were typical of a lung edema (e.g.: nose: colorless serous discharge; trachea: colorless foamy content).
Clinical signs:
other: All rats exposed to 4263 mg/m³ showed clinical signs (bradypnea, dyspnea, labored breathing, breathing sounds, motility reduced, atony, high-legged gait, piloerection, haircoat ungroomed, nasal discharge (serous), nose reddened, nose/muzzle red encrusted,
Body weight:
Compared to control significantly decreased body weights were found at 4263 mg/m³.
Gross pathology:
Necropsy findings were unremarkable in surviving rats whereas in the rat that succumbed in the course of study the following findings of toxicological importance at 4263 mg/m³ were observed (nose: colorless serous discharge; lung: red and dark-red areas; pharynx: ablation of mucosa; trachea: colorless foamy content; stomach: bloated; esophagus: clear colorless liquid content; heart: hardened, exsanguinous, ventricle walls thickened).
Other findings:
Compared to control significantly decreased body temperatures were found at 4263 mg/m³.

Applicant's summary and conclusion

Executive summary:

A study on the acute inhalation toxicity of the substance was conducted according to OECD TG 403 and OECD GD 39. In this study male and female rats were nose-only exposed to the liquid aerosol of the substance (vehicle ethanol) at the maximal technical attainable concentration of 4263 mg/m³. The respirability of the aerosol was adequate and in compliance with test guidelines (MMAD 1.14 µm, GSD 2.63). Rats of the control group were exposed to the vehicle ethanol under otherwise identical circumstances.

All rats exposed to 4263 mg/m³ showed clinical signs (bradypnea, dyspnea, labored breathing, breathing sounds, motility reduced, atony, high-legged gait, piloerection, haircoat ungroomed, nasal discharge (serous), nose reddened, nose/muzzle red encrusted, stridor, nostrils with red encrustation, tremor, salivation increased). Significantly decreased body temperatures and body weights were found at 4263 mg/m³. One animal died at 4263 mg/m³; the mortality patterns were typical of a lung edema. Necropsy findings were unremarkable in surviving rats.

The LC50 (4 h) was thus concluded to be > 4263 mg/m³.