Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Nov - 24 Nov 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
(MMAD was slightly higher than recommeded. However, extensive measures were taken to maximize both the test concentration as well as the respirability of dust)
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Version / remarks:
adopted 1998
Deviations:
yes
Remarks:
(MMAD was slightly higher than recommeded. However, extensive measures were taken to maximize both the test concentration as well as the respirability of dust)
GLP compliance:
yes
Test type:
traditional method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
FORM AS APPLIED IN THE TEST (if different from that of starting material): aerosol (dust)

Test animals

Species:
rat
Strain:
Wistar
Remarks:
(HsdCpb:Wu)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 2 months
- Weight at study initiation: males: 166 - 186 g; females: 156 - 183 g
- Housing: individually in Macrolon Type III H cages
- Diet: KLIBA 3883 = NAFAG 9441 pellets maintenance diet for rats and mice; PROVlMl KLIBA SA, Kaiseraugst, Switzerland (ad libitum)
- Water: tap water (ad libitum)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40 - 60
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 04 Nov 2003 To: 24 Nov 2003

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks:
(compressed air, freed from water, dust and oil)
Mass median aerodynamic diameter (MMAD):
5.93 µm
Geometric standard deviation (GSD):
1.88
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: aluminum inhalation chamber with the following dimensions (inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm)
- Exposure chamber volume: internal volume about 3.8.L
- Method of holding animals in test chamber: tubes were chosen that accommodated the animals' size.
- Source and rate of air: compressed air supplied by Boge compressors, inlet air flow: 28 L/min
- Method of conditioning air: compressed air was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer; adequate control devices were employed to control supply pressure.
- System of generating particulates/aerosols: under dynamic conditions the test substance was fed into the intake of the cylindrical inhalation chamber; for the generation of test atmosphere an EXACTOMAT 4200 (TSE, Bad Homburg, Germany) was used. For powder dispersion, conditioned compressed air (28 L/min; continuous operation) was used.
- Method of particle size determination: the particle-size distribution was analyzed using an ANDERSEN cascade impactor; the individual impactor stages were subjected to gravimetric analysis; adhesive stage coating (silicone spray) was used to prevent particle bounce and reentrainment due to the adsorptive capacity of the filter; gravimetric analyses were made using a digital balance.
- Treatment of exhaust air: exhaust air was purified via cotton-wool/HEPA filters
- Temperature and humidity in air chamber: mean temperature: 22.8°C, relative humidity: 33.8 %

TEST ATMOSPHERE
- Brief description of analytical method used: the test-substance concentration was determined by gravimetric analysis (filter: glass-fiber filter, Sartorius, Göttingen, Germany; electronic balance)
- Samples taken from breathing zone: yes ("in the vicinity of the breathing zone")


TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: The samples for the analysis of the particle-size distribution were taken in the vicinity of the breathing zone. During each exposure two samples were taken.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 5.93 µm / 1.88

Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
5030 mg/m3 (gravimetric concentration), maximum technically attainable concentration
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes
Remarks:
(no concurrent control but periodic in-house control group, utilized for several studies)
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: clinical signs were recorded several times on the day of exposure and at least once daily thereafter; body weights were determined before exposure, on days 3 and 7 and weekly thereafter
- Necropsy of survivors performed: yes
- Other examinations performed: rectal temperatures were measured shortly after cessation of exposure (approximately within 0.5 hour after the end of exposure) using a digital thermometer with a rectal probe for rats; a battery of reflex measurements was made on the first post-exposure day
Statistics:
Necropsy findings: Specific findings from the respiratory tract of surviving rats were evaluated using the pair-wise Fisher test after the R x C chisquared test. The Fisher test was only performed if differences occurred between groups in the R x C chi-squared test or if a frequency value of < 5 was calculated. This procedure was performed in accordance with Gad and Weil (1982). For calculation of the unilateral p value a symmetrical distribution was assumed (p unilateral = (p bilateral)/2).
Body weights: Means and single standard deviations of body weights are calculated. Since in acute studies individual group means may differ prior to commencement of the first exposure, the body weight gain was statistically evaluated for each group. For these evaluations a one-way ANOVA (vide infra) is used.
Physiological data: Data of rectal temperature are evaluated using the ANOVA procedure (vide infra).
Calculation of the LC50: not applicale as no mortalities occurred
Analysis of variance (ANOVA): This parametric method checks for normal distribution of data by comparing the median and mean. The groups are compared at a confidence level of (I-a) = 95% (p = 0.05). The test for the between-group homogeneity of the variance employed Box's test if more than 2 study groups were compared with each other. If the above F-test shows that the intra-group variability is greater than the inter-group variability, this is shown in the Appendix as ''no statistical difference between the groups". If a difference is found then a paitwise post-hoc comparison is conducted (1- and 2-sided) using the Games and Howell modification of the Tukey-Kramer significance test. This program was originally obtained from BCTIC.

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5 000 mg/m³ air (analytical)
Based on:
test mat.
Remarks:
(maximum attainable concentration)
Exp. duration:
4 h
Remarks on result:
other: No mortality occured in the course of the study.
Mortality:
No mortality occurred during the study.
Clinical signs:
other: Clinical signs were noted in all five animals exposed to the test item. These signs consisted of ungroomed hair / coat and piloerection and were noted on days 1 and 2 (males) or 1 to 3 (females), respectively.
Body weight:
Comparisons between the control and exposure group did not reveal any statistically significant and toxicologically relevant changes in body weights.
Gross pathology:
No macroscopic changes attributable to exposure of the test item were noted during scheduled necropsy.
Other findings:
- Other observations: Statistical comparisons between the control and test item-exposed group revealed that the body temperature was mildly, although statistically significantly decreased.
In comparison to the rats of the control group, none of the rats exposed to the test item experienced any changes in their reflex behavior.

Any other information on results incl. tables

Table 1: Mean rectal temperature

Group/sex

Target concentration (mg/m3)

Mean rectal temperature (°C)

1/m

0

37.8

2/m

5000

35.4*

1/f

0

38.1

2/f

5000

36.1**

m = males, f = females, *= p < 0.05, ** = p < 0.01

Applicant's summary and conclusion

Interpretation of results:
other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
CLP: not classified, an LD50 > 2000 mg/m3 was determined