Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-08-26 to 2003-03-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS

- Source: Charles River Wiga GmbH, Sulzfeld, Germany

- Age at study initiation: 8 - 9 weeks (Group A and C); 6 - 7 weeks (Group B)

- Weight at study initiation: The mean body weights of the rats on day 0 were 225 g and 166 g (Group A), 165 gand 141 g (Group B) and 247 g and 163 g (Group C) for male and female animals, respectively.

- Housing: During exposure the animals were housed individually in the holders. After exposure, the animals were returned to their living
cages, held for an observation period of 14 days and sacrific.ed. The animals were housed in animal room 6.0.04, 5 males or 5 females to a cage.

- Diet: (Rat & Mouse No. 3 Breeding Diet RM3) from SDS Special Diets Services, Witham, England. Each batch of this diet is analysed by SDS for nutrients and contaminants. The certificates of analysis pertaining to the batches used (Batch nos. 2205 and 2305) will be kept in the archives of the Institute., ad libitum, except during exposure

- Water: Tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EEC Council Directive 98/831EEC) was supplied by N.V. Hydron Midden-Nederland, ad libitum, except during exposure

- Acclimation period: The duration of the acclimatization periods in the animal room was 18, 6 and 23 days for groups A, Band C, respectively


ENVIRONMENTAL CONDITIONS

- Temperature (°C): average 21.3 (+/- 0.2)°C

- Humidity (%): average 58 (+/- 2)%

- Air changes (per hr): 10

- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
other: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

Animals were exposed to the test atmosphere in a nose-only inhalation chamber, a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom. The inhalation chamber consisted of a cylindrical aluminium column, surrounded by a transparent cylinder. The column had a volume of ca. 50 l and consisted of a top assembly with two mixing chambers, underneath a rodent tube section and the exhaust section at the bottom. The rodent tube section had 20 ports for animal exposure. Several empty ports were used for test atmosphere sampling, particle size analysis, measurement of oxygen concentration, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Male and female rats of each group were placed in alternating order. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column.
In the study labs experience, the animal's body does not exactly fit in the animal holder which always results in some leak from high to low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose and dilution of test atmosphere at the nose of the animals is prevented.


TEST ATMOSPHERE

The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. The test atmosphere was generated by passing test material using a syringe pump (World Precision Instruments, Sarasota FL, USA, type SP220i) to a glass column evaporator (Institute's design). Dry air controlled by a mass flow control unit, entered the evaporator at the bottom and the liquid test material was introduced about half way the evaporator. The evaporator was filled with glass beads· to increase the surface area and surrounded by a water mantle that was kept at 30°C. The resulting test atmosphere was directed to the inlet of the exposure unit, and from there, directed downward towards the animal noses.
For exposures B and C, humidified air (the amount controlled by a second mass flow controller) was added at the inlet of the exposure unit and the flow through the evaporator was reduced accordingly. At the bottom ofthe unit the test atmosphere was exhausted.
The setting of the pump and the read-out of the mass flow controller were recorded at regular intervals (approximately each halfhour) during the generation of the test atmosphere. The (mean) flow of air through the exposure unit was 16.3, 16.8 and 16.8 l/min for exposures A, B and C, respectively.
The animals were placed in the exposure unit after stabilization of the test atmosphere. The period between the start of the generation of the test atmosphere and the start of exposure of the animals was 79 minutes for group A, 116 minutes for group B and 100 minutes for group C. These relatively long periods were necessary to reach a steady state, although equilibration is expected to be accomplished in less than 15 minutes.



- Rationale for the selection of the starting concentration: In a preliminary exposure of two animals during 4 hours (one male, one female rat; data not shown, but kept with the raw data) at the technically maximally attainable vapour concentration of 3.7 g/m3 mortality occurred (the male animal died during exposure, the female animal was found dead 3 days later). Thus, the main study was started with one group (Group A) consisting of 5 male and 5 female rats, which was exposed for 4 hours to a target limit concentration of 2 g/m3.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
The actual concentration of the test material in the test atmosphere was monitored with a total carbon analyser (TCA, Bernath Atomic, Wennigsen, Germany).
Duration of exposure:
4 h
Remarks on duration:
Two groups (A and B) were exposed during a single period of four hours. A third exposure was performed (group C) during 4 hours, 2 hours, 1 hour, 30 minutes and 15 minutes, respectively.
Concentrations:
2.04 ± 0.11 g/m3 (N=48) analytical for Group A
0.52 ± 0.01 g/m3 (N=40) analytical for Group B
0.26 ± 0.00 g/m3 (N=48) analytical for Group C

2.98 g/m3 nominal for Group A, indicating a generation efficiency of 69%
0.57 g/m3 nominal for Group B, indicating a generation efficiency of 91%
0.29 g/m3 nominal for Group C, indicating a generation efficiency of 88%
No. of animals per sex per dose:
5M/ 5F (Group A and B)

Five pairs of animals (1 male and 1 female, each) exposed during 4 hours, 2 hours, 1 hour, 30 minutes and 15 minutes, respectively (Group C)
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: Behaviour, clinical signs and mortality - The rats were visually inspected just before exposure, for reactions to treatment during the exposure, shortly after exposure, and at least once daily during the observation period. Body weights of the animals were recorded just prior to exposure (day 0), on day 7 and on day 14, and at intercurrent death

- Necropsy of survivors performed: yes

- Other examinations performed: All rats were necropsied and examined for gross pathological changes as soon after death as
Statistics:
The actual concentration of the test material during exposure was calculated along with the standard deviation.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 260 - < 520 mg/m³ air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
None of the animals of groups A and B survived the 14-day observation period. The animals of group C all survived the observation period
Clinical signs:
other: Although observation of the rats was limited during exposure due to the stay in restraining tubes, a decreased breathing rate and laboured breathing was seen in a concentration related fashion. Hence the gravity of the decreased breathing rate in group A
Body weight:
The animals of groups A and B had lost weight at death, especially the females surviving until day 3. With respect to group C, in the first week
weight loss or decreased weight gain was noted in the pair of animals, that was exposed during 4 hours. In the next week and in the other animals of group C
during both weeks of the observation period, weight gain was within the limits expected in animals of this strain and age.
Gross pathology:
At necropsy, (dark) red discoloured or haemorrhagic lungs and a hydro- or haemothorax were almost invariably found in the animals of group A.
In the animals of group B, (dark) red discoloured or haemorrhagic lungs were also almost invariably seen, but a hydrothorax only incidentally. In addition,
nasal and/or mouth encrustations or haemorrhages were frequently observed. In the animals of group C, only minor fmdings were noted such as one or two
petechiae on a lobe of the lungs and a discoloured area on one lobe of the lung. Such findings are occasionally also seen in untreated animals (Slaoui et aI, 1998) and did not occur in the animals exposed for the longest duration and were therefore not considered to be related to treatment.
Other findings:
No other effects reported.

Any other information on results incl. tables

.

Applicant's summary and conclusion

Interpretation of results:
very toxic
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Because mortality was 100% in animals exposed to concentrations of 2.04 or 0.52 g/m3, the 4-hour LC50 of inhalatory exposure to vapour of the test material was lower than 0.52 g/m3. Additional exploration at 0.26 g/m3, using exposure durations ranging from 4 hours to 15 minutes, suggested that the 4-hour LC50 was higher than 0.26 g/m3, hence between 0.26 and 0.52 g/m3.
Executive summary:

Two groups (groups A and B) of 5 male and 5 female rats were exposed during a single period of four hours to a test atmosphere

containing the test material at the limit concentrations of 2.04 and 0.52 g/m3, respectively. To explore the toxicity below the limit concentration of 0.5 g/m3, a third exposure at 0.26 g/m3 was performed with five pairs of animals exposed during 4 hours, 2 hours, lhour, 30 minutes and 15 minutes, respectively.

The salient effects of the study were the 100% mortality in groups A and B. In contrast the animals of group C all survived. Treatment-related effects observed during exposure consisted of breathing abnormalities that were related to concentration and duration. Shortly after exposure especially for the animals of group B an extensive list of clinical signs was noted. In the animals of group C exposed during 2 or 4 hours breathing abnormalities were seen on day 1 and day 2, but not thereafter. Effects on body weight gain during the fust week were apparant in the animals of group C exposed during 4 hours, but not in the other animals of that group. Also, treatment related macroscopic findings were not found in the animals of group C, although red or dark red discoloured or haemorrhagic lungs

were found in the animals of groups A and B that were found dead in the first few days after exposure.

Because mortality was 100% in animals exposed to concentrations of 2.04 or 0.52 g/m3, the 4-hour LC50 of inhalatory exposure to vapour of the test material was lower than 0.52 g/m3. Additional exploration at 0.26 g/m3, using exposure durations ranging from 4 hours to 15 minutes, suggested that the 4-hour LC50 was higher than 0.26 g/m3, hence between 0.26 and 0.52 g/m3.