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Toxicological information

Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012-04-24 to 2012-05-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
adopted September 7, 2009
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Series on Testing and Assessment No. 125, Document No. ENV/JM/MONO (2010) 16, June 01, 2010.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-11-12
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: males: 7 weeks; females: 9 weeks
- Weight at study initiation: males: 242 - 256 g; females: 217 - 240 g
- Fasting period before study: feeding was discontinued approx. 16 hours before exposure; only tap water was then available ad libitum.
- Housing: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. During the 14-day observation period, the animals are kept by sex in groups of 2 - 3 animals in MAKROLON cages (type III plus).
- Diet (e.g. ad libitum): commercial diet, ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: at least 5 adaptation days

The animals were randomised before use. They were acclimatised to the test apparatus for approx. 1 hour on 2 days prior to testing. The restraining tubes did not impose undue physical, thermal or immobilization stress on the animals.

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the study was carried out using a dynamic inhalation apparatus (RHEMA-LABORTECHNIK, 65719 Hofheim/Taunus, Germany) (air changes/h (≥ 12 times)) with a nose-only exposure of the animals according to KIMMERLE & TEPPER. The apparatus consists of a cylindrical exposure chamber (volume 40 L) which holds 10 animals in pyrex tubes at the edge of the chamber in a radial position.

- System of generating particulates/aerosols: the dust of the test material was generated with a rotating brush dust generator (RBG 1000, PALAS GmbH Partikel und Lasermesstechnik,76229 Karlsruhe, Germany).
The generator was fed with compressed air (5.0 bar) from a compressor (ALUP Kompressorenfabrik, 73257 Köngen, Germany) (air was taken from the surrounding atmosphere of the laboratory room and filtered using at in-line disposable gas-filter).
At the bottom of the exposure chamber, the air was sucked off at a lower flow rate than it was created by the dust generator in order to produce a homogenous distribution and a positive pressure in the exposure chamber (inflow 900 L/h, outflow 800 L/h).
A manometer and an air-flow meter (ROTA Yokogawa GmbH & Co. KG, 79664 Wehr/Baden, Germany) were used to control the constant supply of compressed air and the exhaust, respectively. Flow rates were checked hourly and the corrected if necessary.
The exhaust air was drawn through gas wash-bottles.

- Method of particle size determination: an analysis of the particle size distribution was carried out twice during the exposure period using a cascade impactor according to MAY (MAY, K. R. Aerosol impaction jets, J. Aerosol Sci. 6, 403 (1975), RESEARCH ENGINEERS Ltd., London N1 5RD, UK.).
The dust from the exposure chamber was drawn through the cascade impactor for 5 minutes at a constant flow rate of 5 L/min. The slides were removed from the impactor and weighed on an analytical balance (SARTORIUS, type 1601 004, precision 0.1 mg). Deltas of slides’ weight were determined.
The mass median aerodynamic diameter (MMAD) was estimated by means of non-linear regression analysis. The 32 μm particle size range and the filter (particle size range < 0.5 μm) were not included in the determination of the MMAD in order not to give undue weight to these values.
The Geometric Standard Deviation (GSD) of the MMAD was calculated from the quotient of the 84.1%- and the 50%-mass fractions, both obtained from the above mentioned non-linear regression analysis.
In addition, a sample of approx. 10 g test material was taken from the exposure chamber to determine the median physical particle size with a CILAS 715 by My-Tec, 91325 Adelsdorf, Germany. This determination was non-GLP.

- Temperature, humidity, pressure in air chamber, oxygen content and carbon dioxide concentration: the oxygen content in the inhalation chamber was 21%. It was determined at the beginning and at the end of the exposure with a DRÄGER Oxygen-analysis test set (DRÄGER Tube Oxygen 67 28 081). Carbon dioxide concentration did not exceed 1%.
Temperature (22.5°C ± 0.4°C (main study) or 20.5°C ± 0.1°C (satellite group)) and humidity (61.9% ± 0.9% (main study) or 66.8% ± 0.1% (satellite group)) were measured once every hour with a climate control monitor (testo 175-HZ data logger).

The whole exposure system was mounted in an inhalation facility to protect the laboratory staff from possible hazards.

Exposition started by locating the animals into the exposure chamber after equilibration of the chamber concentration for at least 15 minutes (t95 approximately 8 minutes).

Before initiating the study with the animals, a pre-test was carried out with the exposure system in order to verify that under the experimental settings chosen, the limit concentration of 5 mg/L air could be achieved by gravimetric analysis.

The tests with the main study animals and the recovery animals were conducted in the same inhalation chamber but on different days. Between the exposure times the chamber was cleaned carefully.

TEST ATMOSPHERE
- Brief description of analytical method used: the actual dust concentration in the inhalation chamber was measured gravimetrically with an air sample filter (Minisart SM 17598 0.45 μm) and pump (Vacuubrand, MZ 2C (Membrane Pump,Vacuubrand GmbH + Co. KG, 97877 Wertheim/Main, Germany)) controlled by a rotameter. Dust samples were taken once every hour during the exposure. For that purpose, a probe was placed close to the animals' noses and air was drawn through the air sample filter at a constant flow of air of 5 L/min for 1 minute. The filters were weighed before and after sampling (accuracy 0.1 mg).
Individual chamber concentration samples did not deviate from the mean chamber concentration by more than 1%.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):
Main study: 1.953 µm (GSD: 2.94)
Satellite group: 2.037 µm (GSD: 3.06)
No smaller GSDs could be obtained with the test item supplied.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
see above ("Details on inhalation exposure")
Duration of exposure:
4 h
Concentrations:
Main study (limit test):
- actual concentration: 5.08 ± 0.03 mg/L air
- nominal concentration: 6.67 mg/L air
Satellite group:
- actual concentration: 5.05 ± 0.04 mg/L air
- nominal concentration: 6.67 mg/L air
No. of animals per sex per dose:
Main study (limit test):
3 males / 3 females
Satellite group:
3 males / 3 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 24 hours (satellite group) and 14 days (main study)

- Frequency of observations and weighing: during and following exposure, observations were made and recorded systematically; individual records were maintained for each animal. Careful clinical examinations were made at least once daily until all symptoms subsided, thereafter each working day. Observations on mortality were made at least once daily (in the morning starting on test day 2) to minimize loss of animals to the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals.
Cageside observations included, but were not limited to: changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, as well as somatomotor activity and behaviour pattern.
Particular attention was directed to observation of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The animals were also observed for possible indications of respiratory irritation such as dyspnoea, rhinitis etc.
Individual weights of animals were determined once during the acclimatisation period, before and after the exposure on test day 1, on test days 3, 8 and 15. Changes in weight were calculated and recorded when survival exceeded one day. At the end of the test, all animals were weighed and sacrificed.

- Necropsy of survivors performed: yes
Necropsy of all main study and satellite animals (3 + 3 males and 3+3 females) was carried out and all gross pathological changes were recorded:
- Satellite animals: necropsy at 24 hours after cessation of exposure, as this is likely to be the time at which any signs of respiratory irritation would have manifested;
- Main study animals: necropsy at the end of the 14-day observation period, also to assess whether any respiratory tract irritation persists or abates.

- Histopathology:
All main study and satellite animals were subjected to the same level of histopathological examination upon necropsy at the end of the respective observation period. During histopathology, attention was paid to alterations that might be indicative of respiratory irritation, such as hyperaemia, oedema, minimal inflammation, thickened mucous layer.
The following organs of all animals were fixed in 10% (nose, i.e. head without brain, eyes and lower jaw) or 7% (other organs) buffered formalin for histopathological examination:
- Nasal cavity, nasopharynx and paranasal sinus:
The tip and level 1 of the nose were taken from a cut just anterior to the incisor teeth. With tip removed, level 2 was taken approximately 2 mm posterior to free tip of the incisor teeth. Level 3 was cut through the incisive papilla. Level 4 was cut through the middle of the second palatal ridge, which is located just anterior to the molar teeth. Level 5 was cut through the middle of the molar teeth. All sections were embedded face down to yield a section from the anterior section, except the nose tip was embedded posterior surface down.
- Larynx
- Trachea
- Lungs (five levels)
Paraffin sections were prepared of all above mentioned organs and stained with haematoxylin-eosin.
Statistics:
Since no animal died prematurely, the calculation of an LC50 was not required.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.08 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: mortality: 0/10; temporary slight clinical signs attributed to inert dust exposure, no clinical signs of substance specific irritation, macroscopic findings (marbled lungs associated with small black foci) without histopathological correlation
Mortality:
No animal died prematurely.
Clinical signs:
other: Under the present test conditions, a 4-hour inhalation exposure to zinc dilaurate at a concentration of 5.08 mg/L air revealed slightly reduced motility, slight ataxia and slightly reduced muscle tone on test day 1 immediately after the end of exposure un
Body weight:
No influence on body weight gain was observed.
Gross pathology:
Macroscopic changes in the nasal cavity and lungs. Marbled lungs were observed in all animals of the main study (14-day sacrifice) and in all satellite animals (24-hour sacrifice). Small black foci were observed in 2 of 3 male and 1 of 3 female satellite animals.
Other findings:
- HISTOPATHOLOGY
Microscopic changes in the nasal cavity and lungs:
1. Test item-related histopathological changes: the histomorphological examination of the trachea, larynx, lungs and the nose of male and female rats after inhalation of zinc dilaurate did not reveal any morphological changes, considered to be related to the inhalation of the test item, in the main study animals and in the satellite animals.
2. Non-test item-related histopathological changes:
Male and female animals of the main study and the satellite group:
- Observations made for the nose (five levels): the nasal cavity of level 1 showed a normal squamous epithelium and a normal respiratory epithelium with goblet cells. A mild congestion was noted in all animals.
The level 2 and 3 showed a similar normal morphological finding with congestion compared to level 1.
The normal respiratory epithelium partially with cilia contained three major cell types, the basal cells above the basement membrane, the ciliated epithelial cells and the secretory goblet cells.
A normal olfactory epithelium with 5 to 7 nuclear layers, normal basal cells, olfactory sensory cells and sustentacular cells was noted in the male and female animals.
The level 4 and 5 of the nose showed focally an olfactory epithelium with mild vacuolization and a loss of epithelial cells (artefacts) on the nasal surface in the dorsal region of the nasal cavity. A normal olfactory epithelium was noted in the other region of the nasal cavity.
Minimal to mild lympho-histiocytic infiltrations or follicular lymphoid hyperplasia in the subepithelial region of the respiratory epithelium (naso-pharynx region) was observed as a normal finding.
- Observations made for the lungs (five levels): the most localisations of the lungs in the main study (14-day sacrifice) and in the satellite group (24-hour sacrifice) were morphologically normal without pathological findings.
Some localisations showed minimal to mild alveolar pulmonary emphysema and mild congestion. In some animals a focal haemorrhage was observed.
The larynx showed in some rats a minimal subepithelial lympho-histiocytic infiltration.
In the trachea some animals showed a focal mild lympho-histiocytic infiltration or a tracheal gland dilatation.
Only in 7 from 30 lung localisations a focal minimal inflammatory oedema with few lymphocytes and neutrophilic granulocytes and a mild congestion was observed.

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LC50 (rats; 4 hours) > 5.08 mg/L air (actual concentration)
Based on the results of the histopathological and macroscopic investigations, zinc dilaurate does not require classification for respiratory irritation.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not classified as acute toxic via the inhalation route or as respiratory irritant.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as acute toxic via the inhalation route or as specific target organ toxicity - single exposure.