Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 21 to December 14, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test conducted following internationally accepted testing guidelines and performed according to the GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Justification of Species: the rat is a representative rodent species preferred by various regulatory agencies for use in an acute inhalation studies.
- Source: Texas Animal Specialities, Humble, TX.
- Age at study initiation: 51 days.
- Weight at study initiation: males: 250-277 g; females: 182-195 g.
- Housing: one per cage.
- Cage type: suspended, wire bottom, stainless steel.
- Diet: PMI TM Lab Diet Formula #5008; available ad libitum except during the exposure period.
- Water: municipal water supply analyzed by TNRCC Water Utilities Division; from automatic water system, available ad libitum except during the exposure period.
- Quarantine period: 5 days.

ENVIRONMENTAL CONDITIONS
Set to maintain
- Temperature: 22 ± 3 °C.
- Humidity: 30 - 70 %.
- Air changes: 10-12 air change/hour.
- Photoperiod: 12 hours light/dark cycle.

IN-LIFE DATES
Animlas born in October 01, 2001. They were received by the laboratory at November 21 and were treated with the test substance on November 30. The in-life portion of the study was termined on December 14, 2001.

No contaminants were expected to have been present in the feed or water which would have interfered with or affected the results of the study.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
PRESTUDY TESTING
Trial assays were conducted to determine which method(s) of aerosolizing the test substance into the exposure chamber would produce an acceptable concentration and mass median aerodynamic diameter (MMAD).

EXPOSURE CHAMBER
A 500 l nose-only stainless steel, dynamic flow inhalation chamber was utilized in this experiment. The body of the chamber has 25 ports in 5 rows. Polycarbonate tubes are inserted into 10 designated individual ports. The test substance is introduced through the opening in the top of the chamber. The bottom section has a corresponding air outlet and a drain valve for cleaning the chamber. The individual polycarbonate tubes are tapered at one end to fit the shape of the animal's head and the back portion is sealed with a rubber cap. The tubes containing the animals fit tightly into the ports, and are sealed with "O" rings.

GENERATION OF TEST ETMOSPHERE
The aerosol was generated by a Gem T Trost Air Mill which aspirated the test substance from a motorized revolving disc delivery system coupled to the mill, then sprayed the resulting aerosol directly into the exposure chamber. Air flow into the chamber was maintained through the use of a calibrated orifice plate at a rate of 22.4 air changes per hour. Air flow was recorded at 30 minute intervals during the exposure period, and was sufficient to ensure an oxygen content of at least 19 % of the exposure atmosphere.
Temperature and humidity were recorded at 30 minute intervals during the exposure period from a Taylor wet bulb/dry bulb hygrometer located in the exposure chamber.

TEST SUBSTANCE ADMINISTRATION
Healthy albino rats were released from quarantine, and five males and five females were selected for testing. The test substance was ground for 24 hours and sifted prior to exposure. The animals were exposed to an aerosol generated from the undiluted test substance (fine powder) for a period of four hours. When 99 % concentration (t-99) was attained, the animals which were individually housed in polycarbonate exposure tubes were inserted into a 500 l stainless steel nose-only inhalation chamber for the specified exposure period. At the termination of the exposure period, the animals were returned to their stock laboratory cages.

DETERMINATION OF CONCENTRATION
The concentration of test substance in the exposure atmosphere (taken from the breathing zone of the animals) was determined gravimetrically twice per hour and nominally at the end of the exposure. The gravimetric concentration was determined by passing a known volume of exposure air through a pre-weighed filter and dividing the amount of test substance deposited on the filter by the volume of air which passed through the filter. The nominal concentration was determined by dividing the loss in weight of the test substance after the exposure by the total volume of air which passed through the chamber.

PARTICLE SIZE DISTRIBUTION
Particle size, taken from the breathing zone of the animals, was determined twice during the exposure, using a cascade impactor, at a rate of 7.1 l/minute for a duration of 20 seconds. The MMAD and particle size distributions are calculated from these data by a computer program utilizing probit analysis.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
Determined gravimetrically twice per hour and nominally at the end of the exposure.
Duration of exposure:
ca. 4 h
Concentrations:
2.51 mg/l, with an average MMAD of 2.0 µm.
No. of animals per sex per dose:
5 males and 5 females (nulliparous and non-pregnant) x dose
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observation for mortality and signs of pharmacologic and/or toxicological effects were made frequently an the day of exposure and at least once daily thereafter for 14 days (Day 0 is day of exposure). Individual body weights were recorded just prior to the inhalation exposure and on Days 7 and 14.
- Necropsy of survivors performed: yes. On Day 14 after exposure, each animal was euthanized by an intraperitoneal injection of Fatal Plus@ (Vortech Pharmaceuticals, Dearbom, MI 48126). All study animals were subjected to gross necropsy, and all abnormalities were recorded.
Statistics:
In order to calculate a mean exposure, the Mean Value Theorem of Calculus was used to properly weight the concentration, since the concentrations could not be measured continuously. This method weights concentrations based on the time span of each concentration. A concentration can be calculated for each minute, which better represents the exposure concentration received by each animal.

Results and discussion

Preliminary study:
Trial assays were conducted to determine which method(s) of aerosolizing the test substance into the exposure chamber would produce an acceptable concentration and mass median aerodynamic diameter (MMAD).
Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 2.51 mg/L air (nominal)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
There was no mortality during the study. As indicated by the data, the acute inhalation LC50 for Lysozyme Technical is greater than 2.51 mg/l.
Clinical signs:
other: Prominent in-life observations included activity decrease and pilo-erection in both sexes on the day of dosing, and decreased defecation in one female on Day 2.
Body weight:
Body weight gain was unaffected by the administration of the test substance.
Gross pathology:
The gross necropsy conducted on each animal at termination of the study revealed no observable abnormalities.

Any other information on results incl. tables

Reaction and severity

Time after exposure begins

Hours Days
0.5 1.0 2.5 4.5 6.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
MALES
Pilo-erection (v-s) 0 0 0 5 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Activity decrease (v-s) 0 0 0 5 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0
FEMALES
Pilo-erection (v-s) 0 0 0 5 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Activity decrease (v-s) 0 0 0 5 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Decrease defection 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0

Applicant's summary and conclusion

Interpretation of results:
other: not assessable, according to the CLP Regulation
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
LC50 is greater than 2.51 mg/l in males and females
Executive summary:

The test substance was evaluated for its acute inhalation toxicity potential in albino rats. Five males and five females were exposed for four hours to an aerosol generated from the undiluted test substance (fine powder) at a level of 2.51 mg/l.

There was no mortality during the study. Clinical signs included activity decrease and pilo-erection on the day of dosing, and decreased defecation on Day 2. Body weights were unaffected by exposure. The gross necropsy revealed no observable abnormalities.

Conclusion

The acute inhalation LC50 is greater than 2.51 mg/l

According to the CLP Regulation (EC 1272/2008), the higher classification threshold limit for acute inhalation toxicity is fixed at 5 mg/l for dust and mist, thus the test result can be used only in order to exclude a classification as toxic if inhaled (category 3: 0.5 < ATE ≤ 1.0).