Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-12-08 to 1998-02-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
1981
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
traditional method
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent
- Age at study initiation: eight to ten weeks old
- Weight at study initiation: 253 to 313 g (males), 219 to 253 g (females)
- Housing: in groups of five by sex in sold-floor polypropylene cages with stainless stell lids, furnished with softwood flakes
- Diet: ad libitum (with exception of exposure period)
- Water: ad libitum (with exception of exposure period)
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: cylindrical exposure chamber
- Exposure chamber volume: 30 L
- Method of holding animals in test chamber: Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of exposure chamber and sealed by means of a rubber 'O' ring.
- Source and rate of air: Compressed air was supplied by means of an oil free compressor and was passed through a water trap and respiratory quality filters before it was introduced to the dust feed.
- Method of conditioning air: Oxygen levels within the exposure chamber were measured by an electronic oxygen analyzer located in the animals’ breathing zone. The test atmosphere was generated to contain at least 19% oxygen.
- Method of particle size determination: Marple Cascade Impactor was used for determination. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone. Exposure chamber air was drawn through the Cascade Impactor using a vacuum pump for a suitable time period. The collection substrates and back up filter were weight before and after sampling and the weight of test material, collected at each stage, calculated by difference.
- Treatment of exhaust air: The concentration within the exposure chamber was controlled by adjusting the rate of the motor and the air flow rate through the chamber. The extract from the exposure chamber passed through a "scrubber" trap and was connected with a hight efficientcy filter to a metered exhaust system.
- Temperature, humidity, pressure in air chamber: 19 - 21°C, 35 - 58%, negative pressure

TEST ATMOSPHERE
- Brief description of analytical method used: The gravimetric method used employed glass fibre filters (Gelman type A/E 25 mm) placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals’ breathing zone. Exposure air was drawn through the filter at a measured rate using a vacuum pump for a suitable time period. Each filter was weighed before and after sampling in order to calculate the weight of collected test material. The difference in the two weights divided by the volume of atmosphere sampled was the chamber concentration.
- Samples taken from breathing zone: yes
- Particle size distribution: From the results obtained the distribution of particles in the size range > 10 µm, 10 to 6 µm, 6 to 3.5 µm, 3.5 to 1.6 µm, 1.6 to 0.9 µm and 0.9 to < 0.5 µm was calculated.



Analytical verification of test atmosphere concentrations:
yes
Remarks:
control by adjusting the rate of the motor and the air flow rate through the chamber
Duration of exposure:
> 3.9 - < 4.1 h
Concentrations:
Mean achieved atmosphere concentration (mg/L):
Dose Group 3: 5.09 mg/L
Dose Group 2: 3.78 mg/L
Dose Group 1: 0.98 mg/L
No. of animals per sex per dose:
10 (5 male and 5 female) per dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Day 0, 7 and 14/or death
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other:
Statistics:
Calculation of the acute inhalation median lethal concentration (LC50) and 95 % conficence limits based on the method of Thompson W R (1947). LC50 and 95 % confidence limits were calculated for males and females separately.

Results and discussion

Preliminary study:
During characterisation two rats (one male and one female) were exposed to an atmosphere of the test material at a concentration of approximately 5 mg/L for a period of 75 minutes. Severe signs of toxicity were observed and included lethargy, ataxia and laboured respiration. The animals were killed in extremis one hour after removal from the chamber and a gross necropsy was performed. Lung changes were noted and included pallor, generalised speckled appearance and dark foci. Both animals shoed pale kidneys and gaseous distension in the stomach. There were also congestion in the large intestine of the female.
Effect levelsopen allclose all
Sex:
male/female
Dose descriptor:
LC50
Effect level:
>= 1.85 - <= 4.07 mg/L air
Based on:
test mat.
95% CL:
> 94 - < 96
Exp. duration:
4 h
Sex:
male
Dose descriptor:
LC50
Effect level:
>= 1.37 - <= 3.08 mg/L air
Based on:
test mat.
95% CL:
> 94 - < 96
Exp. duration:
4 h
Key result
Sex:
female
Dose descriptor:
LC50
Effect level:
>= 1.55 - <= 12.7 mg/L air
Based on:
test mat.
95% CL:
> 94 - < 96
Exp. duration:
4 h
Mortality:
One male was dead on removal from the chamber following exposure to 5.09 mg/L, a second male was found dead 32 minutes after completion of exposure and a third male was killed in extremis one hour after completion of exposure. The follwoing day one male and one female in this dose group were found dead and three animals were killed in extremis. Two females survived.

Following exposure to 3.78 mg/L one male was found dead and two males were killed in extremis on Day 1 following exposure and one female was killed in extremis on Day 12. Two males and four females survived.

No deaths occurred in a group of animals exposed to 0.98 mg/mL.
Clinical signs:
During exposure to 5.09 mg/L wet fur and decreased respiratory rate were commonly observed and several animals showed laboured respiration. On removal from the chamber two surviving males showed extreme lethargy. The other animals were also lethargic and all showed pilo-erection and laboured respiration. Signs of hunched posture, ptosis, increased respiratory rate and pallor of the extremities were noted and there were occasional or isolated incidents of ataxia, gasping or noisy respiration and test material staining around the snout. One hour after completion of exposure similar signs of toxicity were observed in surviving animals. On Day 1 following decreased respiratory rate continued to be noted together with incidents of ptosis, laboured, gasping and/or noisy respiration, pallor of the extremities and red/brown staining around the eyes and/or snout. On Day 2 only two females remained and, although no longer lethargic, both showed decreased respiratory rate, occasional sneezing and red/brown staining around eyes were also noted in one animal. Abnormalities persisted for several days but these two animals recovered to appear normal seven to ten days after exposure.

During exposure to 3.78 mg/L wet fur was commonly observed together with respiratory abnormalities which included laboured respiration and increased or decreased respiratory rate. On removal from the chamber all animals showed wet fur, hunched posture, lethargy, pilo-erection, ptosis, laboured respiration and pallor of the extremities. Ataxia and decreased respiratory rate were commonly noted and there were isolated incidents of tiptoe gait and red/brown staining around the snout. Similar signs were noted one hour after completion of exposure although two animals showed red/brown staining around the eyes. On Day 1 following exposure wet fur and ataxia were no longer evident and incidents of lethargy, ptosis, laboured respiration, pallor of the extremities, tiptoe gait and red/brown staining gasping
Body weight:
Individual bodyweights were noted.

Incidents of bodyweight loss and reduced bodyweight gain were noted in surviving animals during Week 1 of the study. Normal bodyweight gain was noted during Week 2.
Gross pathology:
Individual necropsy findings were noted.

The animals that died or were killed in extremis during the study commonly showed lung changes at necropsy which included enlargement, haemorrhagic patches, abnormally dark or reddened appearance, pallor and dark patches. Several dark livers were observed and there were incidents of pale kidneys and gaseous distension in the gastro-intestinal tract. The left eye of one female exposed to 3.78 mg/L was opaque and swollen. In surviving animals dark foci on the lungs were noted in one female exposed to 5.09 mg/L and in two animals exposed to 3.78 mg/L. Several surviving animals including all those exposed to 0.98 mg/L showed no abnormalities at the end of the study.

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The acute inhalation median lethal concentration (LC50) and 95% confidence limits of the test material TODI, in the Sprague-Dawley Crl : CD ® BR strain rat, were calculated to be:
All animals: 2.74 (1.85 - 4.07) mg/L
Males only: 2.06 (1.37 -3.08) mg/L
Females only: 4.44 (1.55 - 12.7) mg/L
Executive summary:

1. A study was performed to assess the acute inhalation toxicity of the test material, ground, by exposing groups of ten Sprague-Dawley Crl : CD ® BR strain rats (five males and five females) to various concentrations of a dust atmosphere. The animals were exposed for four hours using a nose only exposure system.

The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 403 "Acute Inhalation Toxicity" referenced as Method B2 in Commission Directive 92/69/EEC "Acute Toxicity - Inhalation" (which constitutes Annex V of Council Directive 67/548/EEC).

2. The mean achieved atmosphere concentrations were as follows:

ATHMOSPHERE CONCENTRATION

GROUP NUMBER

MEAN ACHIEVED mg/L

STANDARD DEVIATION

NOMINAL mg/L

3

5.09

1.07

142.1

2

3.78

0.36

69.9

1

0.98

0.08

16.2

3. The characteristics of the achieved atmospheres were as follows:

GROUP

NUMBER

MEAN ACHIEVED ATMOSPHERE CONCENTRATION (mg/L)

MEAN MASS MEDIAN AERODYNAMIC DIAMETER (μm)

INHALABLE FRACTION

(% <4μm)

GEOMETRIC STANDARD DEVIATION (μm)

3

5.09

6.3

30.2

0.41

2

3.78

5.3

40.0

0.34

1

0.98

3.9

50.6

0.38

4. The mortality data were summarised as follows:

GROUP NUMBER

MEAN ACHIEVED ATMOSPHERE CONCENTRATION(mg/L)

DEATHS

 

MALE

FEMALE

TOTAL

3

5.09

5/5

3/5

8/10

2

3.78

3/5

1/5

4/10

1

0.98

0/5

0/5

0/10

5. Clinical Observations:

Common abnormalities noted during the study were wet fur, hunched posture, lethargy, pilo-erection, ptosis, decreased respiratory rate and laboured and noisy respiration. Incidents of ataxia, gasping respiration, pallor of the extremities and red/brown staining around the eyes and snout were noted and there were occasional or isolated incidents of extreme lethargy, increased respiratory rate, sneezing, vocalisation upon handling, distended abdomen, tiptoe gait, test material staining around the snout and red/brown staining on the head. One female exposed to 3.78 mg/L showed opacity and swelling to the left eye.

6. Bodyweight

Incidents of bodyweight loss and reduced bodyweight gain were noted in surviving animals during Week 1 of the study. Normal bodyweight gain was noted during week 2.

7. Necropsy

The animals that died or were killed in extremis during the study commonly showed lung changes at necropsy which included enlargement, haemorrhagic patches, abnormally dark or reddened appearance, pallor and dark patches. Several dark livers were observed and there were incidents of pale kidneys and gaseous distension in the gastro-intestinal tract. The left eye of one female exposed to 3.78 mg/L was opaque and swollen. In surviving animals dark foci on the lungs were noted in one female exposed to 5.09 mg/L and in two animals exposed to 3.78 mg/L. Several surviving animals including all those exposed to 0.98 mg/L showed no abnormalities at the end of the study.

8. The acute inhalation median lethal concentration (LC50) and 95% confidence limits of the test material TODI, in the Sprague-Dawley Crl : CD ® BR strain rat, were calculated to be:

All animals: 2.74 (1.85 - 4.07) mg/L

Males only: 2.06 (1.37 -3.08) mg/L

Females only: 4.44 (1.55 - 12.7) mg/L