Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
between 23 January 1995 and 6 March 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no/or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.1150 (Acute inhalation toxicity)
Deviations:
not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 31st January 1994 Date of Signature:16th March 1994
Test type:
other:
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent.

- Age at study initiation: ten to twelve weeks old

- Weight at study initiation:
Males 285g to 294g.
Females 214g - 251g.

- Fasting period before study: Not applicable

- Housing: polypropylene grid-floor cages over trays lined with absorbant paper.

- Diet: ad libitum (Rat and Mouse Expanded Pelleted Diet No. 1, Special Diets Services Limited, Witham, Essex, UK)

- Water: ad libitum (Mains drinking water)

- Acclimation period: at least seven days


ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23 deg C

- Humidity: 45 to 56%

- Air changes (per hr): Approximately 15 changes per hour.

- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness


IN-LIFE DATES: From: Day 1 To: Day 14

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
A glass concentric jet nebuliser (Radleys, Saffron Walden, Essex, UK) located at the top of the exposure chamber/ The nebuliser was connected to a glass syringe attached to a modified infusion pump, which provided a continuous supply of test material under pressure, and to a metered compressed air supply.

- Exposure chamber volume:
approximately 30 litres

- Method of holding animals in test chamber:
Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.

- Source and rate of air:
Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters to removed particulate material above 0.005 microM before it was introduced to the glass concentric jet nebuliser. Chamber air flow rates were maintained at 16 l/m during each concentration, providing 32 air changes per hour.

- Method of conditioning air:
water trap and respiratory quality filters

- System of generating particulates:
A glass concentric jet nebuliser

- Method of particle size determination:
Marple Personal Cascade Impactor. This device consisted of six impactor stages with stainless steel collection substrates (10, 6, 3.5, 1.6, 0.9 and 0.5 micro/M cut off points) and a back up glass fibre filter housed in an aluminium sampler.

- Treatment of exhaust air:
filtered

- Temperature, humidity, pressure in air chamber:
Temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Kane-May Ltd., Welwyn Garden City, Hertfordshire, UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every thirty minutes throughout the four-hour exposure period. The pressure in the air chamber was kept at negative pressure throughout the study.


TEST ATMOSPHERE
- Brief description of analytical method used:
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 and a suitable known volume of exposure chamber air was drawn through the filter using a vacuum pump (Gravimetric). Each filter was weighed before and after sampling in order to calculate the weight of collected test material.

- Samples taken from breathing zone:
yes


VEHICLE
- Composition of vehicle (if applicable): Not applicable
- Concentration of test material in vehicle: Not applicable
- Justification of choice of vehicle: Not applicable
- Lot/batch no. (if required): Not applicable
- Purity: Not applicable


TEST ATMOSPHERE (if not tabulated)
- Particle size distribution:
Determined three or four times during each exposure period.
Please see Table 2 for a summary of the results

- MMAD (Mass median aerodynamic diameter):
Group 1 - 1.7
Group 2 - 1.8
Group 3 - 1.7


CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration:
During the characterisation phase two animals (one male one female) were introduced to an atmosphere of the test material at a concentration of approximately 2.5 mg/l for 168 minutes as a preliminary screen. Since no adverse effects were noted and after consultation with the study sponser, the study was started at the limit test concentration (5 mg/l).
Analytical verification of test atmosphere concentrations:
no
Remarks:
Gravimetric only
Duration of exposure:
4 h
Concentrations:
Characterisation phase:
approximately 2.5 mg/l

Main study
Group 1 Males and Females:
Target concentration - 5 mg/l
Mean achieved - 5.06 mg/l
Nominal concentration - 19.1 mg/l

Group 2 Females
Target concentration - not recorded
Mean achieved - 2.57 mg/l
Nominal concentration - 9.9 mg/l

Group 3 Females
Target concentration - not recorded
Mean achieved - 1.19 mg/l
Nominal concentration - 4.5 mg/l
No. of animals per sex per dose:
Characterisation phase:
1 male, 1 female

Main study
Group 1:
5 males and 5 females
Group 2:
5 females
Group 3:
5 females
Control animals:
no
Details on study design:
- Duration of observation period following administration:
14 days.

- Frequency of observations and weighing:
All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for fourteen days. Individual bodyweights were recorded prior to treatment on the day of exposure and on Days 7 and 14 or at death.

- Necropsy of survivors performed:
All animales, including those that died during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

- Other examinations performed:
The respiratory tract was subjected to a detailed macroscopic examination for signs of irritatancy or local toxicity.
The lungs *, larynx, pharynx, trachea, kidneys, liver, nasal cavities and sinuses were retained from all animals together with tissues showing macroscopic abnormalities and preserved in buffered 10% formalin.
* Lungs were perfused with buffered 10% formalin.
Statistics:
Data evaluations included the relationship, if any, between the animals’ exposure to the test material and the incidence and severity of all abnormalities including behavioural and clinical observations, necropsy findings, bodyweight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) of the test material was made.

Results and discussion

Effect levelsopen allclose all
Sex:
female
Dose descriptor:
LC50
Effect level:
1.71 mg/L air
Exp. duration:
4 h
Sex:
male
Dose descriptor:
LC50
Effect level:
> 5.06 mg/L air
Exp. duration:
4 h
Mortality:
The mortality data is summarised in Table 1.

All animals that died during the study were found dead on day one following exposure.
Clinical signs:
other: During exposure wet fur and increased respiratory rate were commonly noted. On removal from the chamber hunched posture, pilo-erection and incidents of ptosis were also noted in all dose groups and in the females exposed to 1.19 mg/l there were signs of l
Body weight:
Surviving animals showed normal bodyweight gain during the study.
Gross pathology:
At necropsy animals that died during the study commonly showed swollen and abnormally dark lungs and dark liver whilst several showed reddening of the small intestine. Dark kidneys were noted in one male exposed to 5.06 mg/l and pale kidney were noted in one of the females exposed to 2.57 mg/l. No abnormalities were detected in surviving animals at necropsy.
Other findings:
Not applicable.

Any other information on results incl. tables

Table 1       Mortality Data

Group number Mean Achieved Atmosphere Concentration (mg/L) Sex Deaths During Exposure Deaths Post Exposure (1 Hour) Deaths During Day of Observation Total Deaths
1 2 3 4 5 6 7  8 - 14
1 5.04 Male 0 0 1 0 0 0 0 0 0 0  5/10
Female 0 0 4 0 0 0 0 0 0 0
2 2.57 Female 0 0 3 0 0 0 0 0 0 0  3/5 
3 1.19 Female 0 0 2 0 0 0 0 0 0 0  2/5

Table 2 Summary of Particle Size Distribution

Group Number Mean Achieved Atmosphere Concentration (mg/L) Mean Mass Median Aerodynamic Diameter (microM) Inspirable fraction (% <4 microM) Geometric Standard Deviation (microM)
1 5.04 1.7 82.0 0.40
2 2.57 1.8 85.2 0.46
3 1.19 1.7 86.2 0.45

Table 3 Individual Clinical Observations - Dose Group 1

Mean Achieved Atmospheric Concentration (mg/l) Animal Number and sex Effects Noted during Exposure (hour) Effects noted Post Exposure (hour) Effects Noted during day of observation
1 2 3 4* 1 1 2 3 4 5 6 7  8 - 14
5.06 1 MALE Ri Ri Ri WfRi HP HPRi HLP Ri HPRi H Ri Ri Ri Ri Ri 0
2 MALE 0 0 Ri WfRi HP HPRiPt HPRi HPRi HPRi HPRi HPRi Ri Ri 0
3 MALE Ri Ri WfRi WfRi HP HPRi x  
4 MALE 0 Ri Ri WfRd PtHP HPRdPt HLP Rd HPRd HPRi Ri Ri Ri Ri 0
5 MALE WfRi WfRi WfRi WfRi PtHP HPRiPt HLP Ri HPRi Ri HPRi HPRi HPRi Ri Ri 0
6 FEMALE 0 Ri Ri WfHP Ri HPRiPt x  
7 FEMALE 0 WfRi WfRi WfHP Ri HPRiPt x  
8 FEMALE 0 WfRi WfRi WfHP Ri HPRdPt x  
9 FEMALE 0 WfRi WfRi WfHP RiPt HPRiPt x  
10 FEMALE 0 WfRi WfRi WfHP Ri HPRiPt HLP RiPt HLP Ri HPRi HPRi HPRi HPRi HPRi HPRi*

* = immediately after removal of animals from restraining tube

HPRi* = hunched posture day 8 only, pilo-erection and increased respiratory rate days 8 and 9 only.

Table 4 Individual Clinical Observations - Dose Group 2

Mean Achieved Atmospheric Concentration (mg/l) Animal Number and sex Effects Noted during Exposure (hour) Effects noted Post Exposure (hour) Effects Noted during day of observation
1 2 3 4* 1 1 2 3 4 5 6 7  8 - 14
2.57 11 FEMALE 0 Ri Ri WfHP Ri HPRiPt HPRi HPRi HPRi HPRi Hri Ri Ri Ri*
12 FEMALE 0 0 0 WfHP RiPt HPRiPt x  
13 FEMALE 0 Ri WfRi WfHP Ri WfHP RiPt x  
14 FEMALE Wf WfRi WfRi WfHP RiPt HPRiPt HPRi HPRi HPRi HPRi Ri Ri Ri Ri*
15 FEMALE WfRi WfRi WfRi WfHP RiPt HPRiPt x  

* = immediately after removal of animals from restraining tube

Ri* = increased respiratory rate day 8 only.

Table 5 Individual Clinical Observations - Dose Group 3

Mean Achieved Atmospheric Concentration (mg/l) Animal Number and sex Effects Noted during Exposure (hour) Effects noted Post Exposure (hour) Effects Noted during day of observation
1 2 3 4* 1 1 2 3 4 5 6 7  8 - 14
1.19 16 FEMALE 0 WfRi WfRi WfHPL RiPtE WfHP RiWtPt HPRi Pt HPRi H Ri Ri Ri Ri Ri Ri*
17 FEMALE 0 Ri Ri WfHP Ri HPRi  HPRi HPRi HPRi Ri Ri Ri Ri Ri*
18 FEMALE 0 Ri Ri WfHP Ri HPRi HPRi HPRi HPRi Ri Ri Ri Ri Ri*
19 FEMALE WfRi WfRi WfRi WfHP RiA HPRi HPRi x  
20 FEMALE Wf  Wf  WfRi WfHPLRi PtEA HPL RiPt x  

* = immediately after removal of animals from restraining tube

Ri* = increased respiratory rate day 8 only.

Table 6 Individual Bodyweights - Dose Group 1

Mean Achieved Atmospheric Concentration (mg/l) Animal Number and Sex Bodyweight (g) on day At death Increment (g) during week
0 7 14 1 2
5.06 1 Male 289 313 348   24 35
2 Male 294 325 370   31 45
3 Male 288  -   -  264  -  - 
4 Male 291 328 374   37 46
5 Male 285 314 360   29 46
6 Female  239  -   -  227  -  - 
7 Female 233  -   - 217  -  - 
8 Female 246  -   - 224  -  -  
9 Female 238  -   - 220  -  -
10 Female 251 260 282   9 22

Table 7 Individual Bodyweights - Dose Group 2

Mean Achieved Atmospheric Concentration (mg/l) Animal Number and Sex Bodyweight (g) on day At death Increment (g) during week
0 7 14 1 2
2.57 11 Female 222 229 237   7 8
12 Female 232  -  - 212  -  -
13 Female 218  -  -  200  -  - 
14 Female 222 228 236   6 8
15 Female 219  -  - 199  -  -

Table 8 Individual Bodyweights - Dose Group 3

Mean Achieved Atmospheric Concentration (mg/l) Animal Number and Sex Bodyweight (g) on day At death Increment (g) during week
0 7 14 1 2
2.57 16 Female 217 230 250   13 20
17 Female 237 253 268   16 15
18 Female 214 237 269   23 32
19 Female 237  -  -   215  -   - 
20 Female 238  -  - 208  -  -

Individual Necropsy findings for Dose groups 1 - 3 are attached (Please see Attachment 1).

Applicant's summary and conclusion

Interpretation of results:
harmful
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute inhalation median lethal concentration (LC50) and 95% confidence limits of the test material ALKANE 2, in the female Sprague-Dawley strain rat, were calculated to be 1.71 (0.49 - 5.99) mg/l.

The LC50 value for the males was estimated to be greater than 5.06 mg/l.
Executive summary:
1. A study was performed to assess the acute inhalation toxicity of the test material, as supplied, by exposing groups of Sprague-Dawley strain rats (five males and/or five females) to various concentrations of an aerosol atmosphere. The animals were exposed for four hours using a nose only exposure system. The method is designed to satisfy the U.S. Environmental Protection Agency (EPA) Health Effects Test Guidelines; 40 CFR Part 798.1150 "Acute Inhalation Toxicity" and the recommendations of the OECD Guidelines for Testing of Chemicals (1981) No 403 "Acute Inhalation Toxicity" referenced as Method B2 in Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC). 2. The mean achieved atmosphere concentrations were as follows:
Atmosphere concentration
Group Number Mean Achieved mg/l Standard Deviation Nominal mg/l
1 5.06 0.19 19.1
2 2.57 0.12 9.9
3 1.19 0.09 4.5

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

Group Number Mean achieved Atmospheric Concentration (mg/l) Mean Mass Medium Aerodynamic Diameter (microM) Inspiable Fraction (% <4 microM) Geometric Standard Deviation (microM)
1 5.06 1.7 82.0 0.40
2 2.57 1.8 85.2 0.46
3 1.19 1.7 86.2 0.45

4. The mortality data were summarised as follows:

Group Number Mean achieved Atmospheric Concentration (mg/l) Deaths
Male Female Total
1 5.06  1 / 5  4 / 5  5 / 10
2 2.57 *  3 / 5  3 / 5
3 1.19 *  2 / 5  2 / 5

* - males were not exposed

5. Clinical Observations: Common abnormalities noted during the study were wet fur, hunched posture, pilo-erection, increased respiratory rate and ptosis. Isolated incidents of lethargy, ataxia, decreased respiratory rate, pallor of the extremities and tiptoe gait were also noted. Surviving animals recovered to appear normal 8 or 10 days after exposure.

6. Bodyweight: Surviving animals showed normal bodyweight gain during the study.

7. Necropsy: Common abnormalities noted at necropsy in animals that died during the study were swollen and abnormally dark lungs, dark liver and reddening of the small intestine. Two animals showed dark or pale kidneys. No abnormalities were detected in surviving animals at necropsy.

8. The acute inhalation median lethal concentration (LC50) and 95% confidence limits of the test material Alkane 2, in the femal Sprague-Dawley strain rat, were calculated to be 1.71 (0,49 - 5.99) mg/l.

The LC50 value for the males was estimated to be greater than 5.06 mg/l.