Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 14, 1999 to February 15, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Substance type: UVCB
- Physical state: liquid
- Lot/batch No.: PPQ 6460
- Expiration date of the lot/batch: 12 July 2009
- Stability under test conditions: The test material is stable for at least 24 hours at room temperature
- Storage condition of test material: Frozen (-20°C)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd, Manston Road, Margate, Kent UK.
- Age at arrival: 7-8 weeks old
- Housing: Five animals per cage. In holding cages (size 35 cm x 53 cm x 25 cm height).
- Diet (e.g. ad libitum): SDS rat and mouse diet (RM1), ad libitum, except during the 4 hr exposure
- Water (e.g. ad libitum): Tap water ad libitum, except during the 4 hr exposure
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: October 27, 1999 To: November 16, 1999

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 3.1 µm
Geometric standard deviation (GSD):
>= 2.19
Remark on MMAD/GSD:
Approximately 85% of the particles were considered of a respirable size (< 7 µm aerodynamic diameter).
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: ADG Developments Ltd., Hitchin, Hertfordshire, England
- Exposure chamber volume: 30 L
- Method of holding animals in test chamber: Snout only
- Source and rate of air: A supply of clean dried air was connected to the aerosol generator and the supply pressure was adjusted to give a flow rate of 15 litres/minute measured at the outlet of the generator. An in-line flow meter was used to monitor airflow throughout the exposure.
- Method of conditioning air and generate aerosols: A syringe filled with the test substance was fitted to a syringe pump and connected to a generator via Teflon tubing. To generate a chamber concentration at a target of 5 mL/L of air an initial feed rate of 1.4 mL/minute was selected.
- Method of particle size determination: Two air samples were taken during the exposure at a sampling rate of 2 L/minute using a Marple cascade impactor (Model 296, Graseby Andersen Inc., Atlanta, USA) to determine particle size distribution.
- Temperature, humidity, pressure in air chamber: Mean temp: 20.9°C (control group) and 21.1°C (test group), relative humidity: 41% (control group) and 98% (test group).

TEST ATMOSPHERE
- Brief description of analytical method used: Six air samples were taken during exposure. Chamber air was drawn at a measured rate of 2 L/minute, through a pre- weighed glass fibre filter (Whatman GF/A) mounted in an open face filter holder. Filters were dried for 20 minutes in an oven at 35-40°C, and then re-weighed for gravimetric analysis of the test aerosol.

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: 85% respirable (< 7 µm in aerodynamic diameter)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): MMAD: 3.1µm. GSD: 2.19
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
>= 4 h
Concentrations:
4.95 mg/L
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations for clinical signs of effect: At the end of the chamber equilibration period, at 0.25, 0.5 and 1.0 hours then at hourly intervals during the exposure, immediately following completion of the exposure and then at 1.0 and 2.0 hours post-exposure. Subsequent, daily observations: Body weight: Twice during the week prior to exposure, immediately before exposure (day 0) and weekly during the observation period.
- Necropsy of survivors performed: yes
- Other examinations performed: Organ weights: The lungs, liver and kidneys of each animal were weighed.
Statistics:
Not performed.

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 4.95 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality.
Clinical signs:
other: During exposure exaggerated breathing was first noted in test rats after 15 minutes of exposure, and after 2 hours of exposure in all test rats. The exaggerated breathing persisted in 1 test female up to 2 hours post exposure. The fur of all test rats wa
Body weight:
The mean bodyweight gain for male and female test rats were 81 g and 20 g compared with 95 g and 23 g for control male and female rats during the 14-days observation period respectively.
Gross pathology:
No abnormalities.

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
There were no unscheduled deaths or evidence of a toxic response following exposure of rats for 4 hours to a droplet aerosol generated from Xylanase, PPQ 6460 at a chamber concentration of 4.95 mg/L in air.
Executive summary:

The present study was performed in rats, in accordance with GLP and in compliance with EEC, OECD and US EPA (Health Effects Test Guidelines, OPPTS 870, 1300, Acute Inhalation Toxicity, 5 August 1998) and JMAFF test guidelines for acute inhalation studies. One control group and one test group each consisting of 5 females and 5 males were included.

The animals in the test group were exposed by snout-only exposure for 4 hours to air containing a liquid droplet aerosol generated from the test substance, Xylanase, PPQ 6460, at a concentration of 4.95 mg/L. In total 85% of the particles of Xylanase, PPQ 6460, were < 7 µm and had a mass median aerodynamic diameter of 3.1 µm.

The animals were observed during exposure, for two hours after the exposure and daily during the 14-day observation period. After the observation period, the animals were sacrificed and examined pathologically.

During exposure exaggerated breathing was noted in all animals. The exaggerated breathing persisted in 1 test female up to 2 hours post exposure. Fur/skin soiled with excreta was observed in all test and control rats immediately following exposure, persisting in 1 control male up to 2 hours post exposure. Wet fur (whole body) was noted in a proportion of control and test rats immediately following exposure, persisting up to 1-hour post exposure. Brown staining on the head was seen on a proportion of control and test rats up to 2 hours after exposure. These were temporary signs and were considered to be associated with the tube restraint for inhalation exposure.

The mean bodyweight gain for male and female test rats were 81 g and 20 g compared with 95 g and 23 g for control male and female rats during the 14-days observation period respectively.

In conclusion, as there were no unscheduled deaths or evidence of a toxic response following exposure of rats for 4 hours to a droplet aerosol generated from Xylanase, PPQ 6460 at a chamber concentration of 4.95 mg/L in air, the LC50 for Xylanase is in excess of 4.95 mg/L based on test material.