Registration Dossier

Toxicological information

Acute Toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
other information
Study period:
2000
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Study performed similarly to OECD Guideline 403 with major deviations: exposure duration: 30 min instead of 4 hours; observation period: 30 min; bodyweights and necropsy not followed; no data on housing conditions. The number of groups tested and corresponding concentrations were not reported.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
exposure duration: 30 min instead of 4 hours; observation period: 30 min; bodyweights and necropsy not followed; no data on housing conditions
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Test type:
standard acute method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): S-(-)-limonene
- Analytical purity: 97%
- Source: Fluka Chemie, Switzerland

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Bomholtgard, Denmark
- Weight at study initiation: 27.0 ± 2.0 g
- Housing: Housed in polypropylene cages with sawdust bedding (Lignocel S8, Brogarden, Denmark)
- Diet: Altromin Standard Diet no.1324, Brogarden, Denmark
- Water: Tap water, ad libitum
- Acclimation period: 10-15 min before initiating a 15 min baseline period in the exposure chamber

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
head only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Plethysmograph
- Exposure chamber volume: 2.3 L
- Method of holding animals in test chamber: Mice were placed in separate body plethysmographs and installed head-only into the exposure chamber.
- Source and rate of air: Room air, 17-25 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: Concentrations of the test material were monitored by infrared spectroscopy (Miran 1A, Foxboro).
Analytical verification of test atmosphere concentrations:
yes
Remarks:
Infrared spectroscopy (Miran 1A, Foxboro)
Duration of exposure:
30 min
Concentrations:
Measured concentrations: 316-2421 ppm (mL vapor/cm3 of air)
No. of animals per sex per dose:
Four naive mice
Control animals:
no
Details on study design:
- Duration of observation period following administration: 30 min
- Frequency of observations: Animals were observed for mortality, sensory irritation, airflow limitation and pulmonary irritation at 0-10, 11-20 and 21-30 min
- Concentrations, which cause a 50% decrease in respiratory rate (RD50) and the extrapolated threshold concentration (RD0) were determined.
Statistics:
- Trend over time (time-response relationship) was studied by two-way analysis of variance and regression analysis.
- Both parametric and nonparametric (Spearman's rank correlation) tests were used to evaluate exposure effects on tidal volume.
- Calculations were performed by use of the Minitab Statistical Software, Release 10.51 Xtra (Minitab Inc.). P values less than 0.05 were considered statistically significant.

Results and discussion

Preliminary study:
Not applicable
Effect levelsopen allclose all
Key result
Sex:
male
Dose descriptor:
LC50
Effect level:
>= 2 421 ppm
Based on:
test mat.
Exp. duration:
30 min
Remarks on result:
other: equivalent to 1.68 mg/L/4 h exposure
Sex:
male
Dose descriptor:
other: RD50
Effect level:
1 715 ppm
Based on:
test mat.
95% CL:
>= 1 168 - <= 2 519
Exp. duration:
30 min
Remarks on result:
other: RD50 is concentration causing 50% of decrease of respiratory rate
Sex:
male
Dose descriptor:
other: RD0
Effect level:
199 ppm
Based on:
test mat.
95% CL:
>= 129 - <= 307
Exp. duration:
30 min
Remarks on result:
other: RD50 is concentration causing 50% of decrease of respiratory rate
Mortality:
One mouse died at 1788 and one at 2421 ppm S-(-)-limonene concentrations, after 2 and 4 min, respectively. There was no concentration or
time -dependent effect of death and none of the deaths is considered to be exposure related.
Clinical signs:
other: Sensory irritant: - Respiratory rate: Decreased in a dose-dependent manner; reached plateau within first 10 min and was stable during remaining period - Time of break: Increased in a dose-dependent manner
Body weight:
No data
Gross pathology:
No data
Other findings:
None

Any other information on results incl. tables

Following 30 minutes of exposure, LC50 in mouse was determined to be higher than the highest tested dose, i.e. 2421 ppm, because no treatment-related death was observed at any of the treatment doses. Based on the conversion factor , as 1 ppm corresponds to 5.56 mg/m3 for d-limonene. LC50 is determined to be greater than 8890.44 mg/m3 following 30 minutes of exposure and 1680 mg/m3 (1.68 mg/L) following 4 hours of exposure, based on Haber’s law (Cn x t = k), where n = 1.

Table 1: RD50 and RD0 values at different time periods

 

Period (min)

na

Range of concentrationb(ppm)

Slopec(%/ log concentration)

RD50d(ppm)

RD0d(ppm)

0-10

26

316-2421

61.9 ± 6.3

1467 (902 -2387)

232 (125 -431)

11-20

26

316-2421

53.1 ± 4.0

1734 (1205 -2495)

202 (125 -326)

21-30

26

316-2421

46.3 ± 4.0

1918 (1274 -2887)

159 (90 -281)

0-30

26

316-2421

53.8 ± 3.1

1715 (1168-2519)

199 (129 -307)

aNumber of mice used for construction of the regression line.

bRange of concentration used for construction of the regression line.

cThe ± is the SD value.

dThe 95% confidence intervals are given in brackets.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the test conditions, the acute inhalation LC50 (30 min) value was considered to be greater than 2421 ppm in male mice. LC50 (4 h) was calculated to be > 1.68 mg/L.
Executive summary:

In an acute inhalation toxicity study performed similarly to OECD Guideline 403, groups (4/dose) of male BALB/cA mice were exposed (head only) to vapors of S-(-)-limonene at concentration range of 316 -2421 ppm (measured) for 30 min. Animals were observed for mortality and signs of sensory irritation, airflow limitation and pulmonary irritation during the exposure period.  

 

One mouse died at 1788 and one at 2421 ppm S-(-)-limonene concentrations, after 2 and 4 min, respectively. There was no concentration or time -dependent effect of death and none of the deaths is considered to be exposure related.

A dose-dependent decrease in respiratory rate and increase in time of break indicated the sensory irritant effect of S-(-)-limonene.

The RD50 values at 0-10, 11-20, 21-30 or 0-30 min were estimated to be 1467 (902 -2387), 1734 (1205 -2495), 1918 (1274 -2887) or 1715 (1168 -2519), respectively.

 

Under the test conditions, as no death was considered to be treatment-related, the acute inhalation LC50 (30 min) value was estimated to be greater than 2421 ppm in male mice. LC50 (4 h) was calculated to be > 1.68 mg/L.