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Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From July to December 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study is conducted on a read across test material. The complete read across justification is attached in section 13. The reliability of the original study is 1.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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:
yes

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: Wockhardt Research Centre, Aurangabad, India.
- Age at study initiation: 8 weeks.
- Weight at study initiation: 146 to 211 g.
- Fasting period before study: yes, feeding was suspended during the period of exposure.
- Housing: animals were housed in group of five male and five female in sterilised solid bottom polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilised paddy husk. Cages were suspended on movable stainless steel racks.
- Diet: 'Nutrilab' brand extruded pelleted rat feed manufactured by M/s Provimi Animal Nutrition India Pvt. Ltd., Bangalore, was provided ad libitum.
- Water: potable water, passed through Aqua guard water filter, and subjected to ultra violet irradiation, was provided ad libitum in sterilized bottles with stainless steel sipper tubes, throughout the acclimation and study period, except during the period of exposure.
- Acclimation period: the animals were acclimated for a period of six days in the experimental room before start of the experiment.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Air changes: 10 - 15 per hours.
- Photoperiod: 12 hours light and 12 hours dark.

IN-LIFE DATES: from: 31 July to 20 August, 2010

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Remarks:
only the snouts and nostrils of the animals were exposed to the aerosol.
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: all exposures were conducted in a “Nose-Only Directed-flow Rodent Inhalation Exposure Chamber”. In this chamber, fresh test atmosphere was provided to each animal from the inner plenum and exhausted into the space between the inner and outer plenum.
- Method of holding animals in test chamber: for the inhalation purpose the rats were placed in polycarbonate containment tubes, attached to the exposure ports by friction fit.
- Source and rate of air: the test aerosol was distributed from the inner plenum uniformly to the test animals through multiple trumpets, each connected to one animal containment tube. The amount of air passing through the chamber and the air pressure inside the chamber was regulated by means of a flow control panel, comprising of flow meters and flow controllers. The air flow through the chamber was measured with flometers.
- Method of conditioning air: airflow through the chamber was suitably maintained so as to prevent leakage of the test atmosphere from the system, as well as its dilution with outside air. The exhaust air was decontaminated by subsequent passage through 1 % (w/v, aq.) NaOH solution.
- System of generating particulates/aerosols: the test article is a fine white powder; the test aerosol was generated by employing a rotating brush dust generator at gear ratios set to obtain the desirable concentration and by passing filtered and dry compressed air. The dust feeder opened into the inner plenum of the exposure chamber, and the dust was swept into the chamber by compressed air at rate of 40.8 LPM.
- Aerosol concentration: the aerosol concentration in the chamber was determined gravimetrically five times during the period of exposure, i.e. at initiation (post-equilibration) and after about 1 hour, 2 hours, 3 hours and 4 hours of exposure. Samples of the test atmosphere were passed through a cellulose nitrate filter of 47 mm diameter with a pore size of 0.2 micrometer. The air flow rate for the sample collection was at 1.98 and 2.04 l/min.
- Method of particle size determination: particle size analysis was conducted twice during the exposure, using a seven stage cascade impactor with stainless steel collection substrates. The air flow rate for the measurements was adjusted about to 1.44 l/min by means of an impactor control system In-Tox Products. The amount of particles in the seven size classes was determined gravimetrically.

TEST ATMOSPHERE
- MMAD: 1.70 µm.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
ca. 4 h
Concentrations:
3.30 mg/l
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were individually observed for signs of toxicity and death, immediately after end of exposure period (4h after initiation of exposure), and at 5 after initiation of exposure. Thereafter they were observed once a day for a total of 14 days.
Cageside observation included but were not limited to changes in the skin and fur, eyes, mucous membrane, respiratory, circulatory, autonomic, central nervous system and behaviour pattern. The appearance, progress and disappearance of these signs were recorded.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other: The body weights of rats were individually recorded on days 0, 1, 3, 7 and at termination of the study (day 15).
Statistics:
LC50 value: The inhalation LC50 value could not be estimated as there was no mortality observed at the maximum attainable concentration.
MMAD: The measurements of the aerosol particle size were analysed under the assumption of a log normal size distribution. The mass median aerodynamic diameter (MMAD) was estimated by the probit least square method.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 3.3 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No deaths occurred among the male and female rats on the day of their exposure to the test item at 3.30 mg/l, during or following the 4 hour exposure period. Also, no deaths occurred among these male and female rats during the 14 days observation period.
Clinical signs:
other: Exposure to the test item at 3.30 mg/l for 4 hours induced abnormal, but reversible clinical signs in the male and female rats, as observed at end of the exposure, and during the 14 day observation period thereafter. These included nasal discharge, abdomi
Body weight:
The test item did not adversely affect the body weight gain in the treated male and female rats during the 14 day observation period.
Gross pathology:
No gross pathological alterations were encountered during necropsy examination carried out on all treated animals sacrificed at termination of the study.

Any other information on results incl. tables

Clinical signs and mortality

Group: G1; Concentration: 3.30 mg/l

Animal   ID. Incidence of Clinical Signs / Mortality Observed after Exposure
Day 1 Day
4 h 5 h 2 3 4 5 6 7 8 9 10 11 12 13 14 15

MALE RATS

Rf2431 1, 2, 8, 25, 27 1, 2, 8, 25, 27 25 N N N N N N N N N N N N N
Rf2432 1, 2, 8, 25, 27 1, 2, 8, 25, 27 25 N N N N N N N N N N N N N
Rf2433 1, 2, 8, 25, 27 1, 2, 8, 25, 27 25 25 N N N N N N N N N N N N
Rf2434 1, 2, 8, 25, 27 1, 2, 8, 25, 27 25 25 25 N N N N N N N N N N N
Rf2435 1, 2, 8, 25, 27 1, 2, 8, 25, 27 25 25 N N N N N N N N N N N N

FEMALE RATS

Rf2436 1, 2, 8, 25, 27 1, 2, 8, 25, 27 25 25 N N N N N N N N N N N N
Rf2437 1, 2, 8, 25, 27 1, 2, 8, 25, 27 25 25 N N N N N N N N N N N N
Rf2438 1, 2, 8, 25, 27 1, 2, 8, 25, 27 25 N N N N N N N N N N N N N
Rf2439 1, 2, 8, 25, 27 1, 2, 8, 25, 27 25 25 N N N N N N N N N N N N
Rf2440 1, 2, 8, 25, 27 1, 2, 8, 25, 27 25 N N N N N N N N N N N N N

Hours indicate time after initiation of the exposure.  

N - No abnormal clinical signs; 1 – Nasal discharge, 2 – Abdominal breathing; 8 – Tremors; 5 – Hypoactivity; 27 – Ruffled appearance

Individual animal body weight (g)

Group: G1; Concentration: 3.30 mg/l

MALE RATS
Animal ID Day 0 Day 1 Day 3 Change  (1-3) Day 7 Change  (1-3) Day 15 Change  (1-3)
Rf2431 211 213 217 4 223 10 227 14
Rf2432 174 176 181 5 188 12 192 16
Rf2433 193 195 198 3 208 13 210 15
Rf2434 188 191 193 2 198 7 205 14
Rf2435 192 193 196 3 204 11 209 16
Mean 191.6 193.6 197 3.4 204.2 10.6 208.6 15
± SD 13.2 13.2 13.0 1.1 12.9 2.3 12.5 1.0
FEMALE RATS
Animal ID Day 0 Day 1 Day 3 Change  (1-3) Day 7 Change  (1-3) Day 15 Change  (1-3)
Rf2436 149 152 154 2 158 6 165 13
Rf2437 152 155 158 3 162 7 170 15
Rf2438 159 161 163 2 164 3 168 7
Rf2439 153 156 159 3 166 10 172 16
Rf2440 146 148 152 4 156 8 161 13
Mean 151.8 154.4 157.2 2.8 161.2 6.8 167.2 12.8
± SD 4.9 4.8 4.3 0.8 4.1 2.6 4.3 3.5

Particle size distribution data

Group: G1; Concentration: 3.30 mg/l

Sample No: 1
Measured Time: 2 hours

Stage No. Stage ECD (µm) Weight of Material Collected Cumulative Weight
Net (mg) % of Total (%)
1 5.9 0.39 11.96 100
2 4.8 0.62 19.02 88.04
3 3.6 0.73 22.39 69.02
4 2.1 0.54 16.56 46.63
5 1.1 0.45 13.8 30.06
6 0.46 0.36 11.04 16.26
7 0.31 0.17 5.21 5.21
Total = 3.26 mg
ECD = Effective cut-off diameter
            16 % level = 0.66 µm
            50 % level = 1.70 µm (MMAD)
            84 % level = 5.50 µm
Geometric Standard Deviation (GSD)   2.91

Sample No.: 2
Measured Time: 4 hours

Stage No. Stage ECD
(µm)
Weight of Material Collected Cumulative Weight
Net (mg) % of Total (%)
1 5.9 0.42 13.13 100
2 4.8 0.59 18.44 86.87
3 3.6 0.78 24.37 68.44
4 2.1 0.5 15.63 44.06
5 1.1 0.4 12.5 28.44
6 0.46 0.31 9.69 15.94
7 0.31 0.2 6.25 6.25
Total = 3.2 mg
ECD = Effective cut-off diameter
            16 % level = 0.59 µm
            50 % level = 1.70 µm (MMAD)
            84 % level = 5.70 µm
Geometric Standard Deviation (GSD) 3.12

Applicant's summary and conclusion

Interpretation of results:
other: not applicable, according to the CLP Regulation (EC no. 1272/2008)
Conclusions:
LC50 (4h) > 3.30 mg/l
Executive summary:

The test substance was tested for acute inhalation toxicity to rats in compliance with the OECD Guidelines for Testing of Chemicals, Section 4, No. 403 - "Acute Inhalation Toxicity", adopted 07 September, 2009. The study was conducted as a ‘Limit Test’ in which a group of five male and five female rats were subjected to a single exposure of the test article, for a period of 4 hours at the maximum attainable concentration of 3.30 mg/l. Exposed animals were observed for mortality and signs of toxicity periodically after completion of exposure and thereafter for a period of 14 days, and their body weights were recorded weekly. Necropsy was performed on all rats at termination of the study.

None of the exposed rats died during the 4 hours of exposure to the test substance and no deaths occurred during the 14 day observation post-exposure period. Reversible signs of toxicity, such as nasal discharge, abdominal breathing, tremors, hypoactivity and ruffled appearance were observed; nevertheless, all signs were recovered within 5 days after exposure. The substance did not adversely affect the body weight gain in the treated male and female rats during the 14 day observation period. Necropsy findings did not indicate any remarkable and treatment related gross pathological changes.

Conclusion

LC50 (4h) > 3.30 mg/l

According to the CLP Regulation, the higher threshold limit (based on the LD50) to classify a substance (dust/mist) for acute inhalation toxicity is 5.0 mg/l (category 4: 1.0 < ATE ≤ 5.0 mg/l). In the current test, an LD50 was non identified and the highest dose tested (3.30 mg/l) is lower than the limit outlined in the CLP Regulation for classification. Considering the fact that no mortality occurred, a classification category can not be assigned. Thus, a classification according to the CLP Regulation (EC 1272/2008) is not applicable.