Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-07-2014 to 24-11-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: March 2014; signature: May 2014
Test type:
traditional method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: In refrigerator 4°C in the dark, under nitrogen
- Other: clear colourless

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: 8 - 12 weeks; females were nulliparous and non pregnant.
- Weight at study initiation: 200 - 350 g
- Fasting period before study: None.
- Housing: Housed in groups of up to five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes provided with environmental enrichment items.
- Diet (e.g. ad libitum): Certified diet from recognised supplier, provided ad libitum (except for exposure period period)
- Water (e.g. ad libitum): ad libitum (except for exposure period period)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70%
- Air changes (per hr): > 15 air changes per hour
- Photoperiod: 12 h light / 12 h dark

IN-LIFE DATES: 17-07-2014 to 17-09-2014

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks:
Oxygen concentration (%) was monitored during the study by an electronic oxygen analyzer. The test atmospheres were generated to contain at least 19% oxygen.
Mass median aerodynamic diameter (MMAD):
1.85 µm
Geometric standard deviation (GSD):
2.78
Remark on MMAD/GSD:
MMAD and GSD associated with the highest concentration level: 5.14 mg/L
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The test item was aerosolized using a glass concentric jet nebulizer located at the top of the exposure chamber. The nebulizer was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and to a metered compressed air supply.
- Exposure chamber volume: approximately 30 litres (dimensions: 28 cm diameter x 50 cm high)
- Method of holding animals in test chamber: Prior to the day of exposure each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. During the exposure period, 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: filtered air; chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.
- Method of conditioning air: Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the nebulizer.
- System of generating particulates/aerosols: glass concentric jet nebulizer; the concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump.
- Method of particle size determination: Particle size was determined using a cascade impactor. The device consisted of six impactors stages. The mean amount for each stage was used to determine the cumulative amount below each cut-off point size to calculate the proportional (%) aerosol of defined size ranges. The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (%) of aerosol less than 4 μm (considered to be the inhalable fraction) was determined.
- Treatment of exhaust air: The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure. A schematic of the dynamic (continuous flow) system is presented in the full study report.
- Temperature, humidity, in air chamber: The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter: 19 to 21 °C and 30 to 70% humidity.

TEST ATMOSPHERE
- Brief description of analytical method used: The test atmosphere was sampled nine times during each exposure period. The sampling procedure involved two liters of test atmosphere being drawn through a glass fiber filter. The samples were then submitted for chemical analysis by gas chromatography (GC). A range of standard solutions were prepared in acetonitrile from a stock solution of 1.00 mg/mL by serial dilution covering the concentration range 0 to 0.165 mg/mL. Full details of the analytical method are provided in the full study report. The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. The regression coefficients calculated were found to be > 0.994. The fortified samples of impingers were found to have a recovery value of ± 10% of the fortification chromatographic run. In conclusion, the results indicate the accurate use of the test item and impingers during this study. Working solutions of the test item were shown to be sufficiently stable to complete the analysis without deterioration of the analyte. Full details of the analytical method are provided in the full study report.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- Particle size distribution: The particle size of the generated atmosphere inside the exposure chamber was determined three times during each exposure period using a cascade impactor. The particle size distribution for each group is reported in table 1.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The Mass Median Aerodynamic Diameter (MMAD) was determined and is reported for each group in table 1.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Following an appropriate equilibration period three groups were subjected to a single exposure to the test item for a period of four hours. A target concentration of 5.0 mg/L was used for the first exposure. Further concentrations were to be selected after consideration of the results of the previous exposure. Full details are provided in table 2.
No. of animals per sex per dose:
5 per sex per dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: 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 up to fourteen days. Any evidence of mortality or overt toxicity was recorded at each observation. Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14 or after mortality.
- Necropsy of survivors performed: yes (and in the event of any mortalities)
- Other examinations performed: clinical signs, bodyweight, organ weights, and any other relevant toxicological effects were reported.
Statistics:
Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) was calculated using validated data analysis software which utilized Log-Normal (Probit) regression models in order to calculate the LC50 values. LC50 values and 95% confidence limits were calculated for males and females separately. Where appropriate.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.14 mg/L air
Based on:
test mat.
Remarks:
mean achieved concentration
Exp. duration:
4 h
Remarks on result:
other: Effect levels based on analytically confirmed: mean achieved concentrations for each exposure/sex groups
Mortality:
There were no mortalities during the study as reported in table 3.
Clinical signs:
Common abnormalities noted during the study included Increased respiratory rate, hunched posture, pilo-erection and wet fur were noted in all animals but which recovered to appear normal on Day 5 post-exposure.
In group 1, 5.14 mg/L: During exposure, on removal from the chamber and one hour post-exposure: increased respiratory rate. One day after exposure, increased respiratory rate and hunched posture only. Observations gradually receded over the recovery period and all appeared normal on Day 5 post-exposure.
Body weight:
In group 1, 5.14 mg/L: Three male animals and four females exhibited body weight losses or showed no body weight gain on the first day post-exposure. One male and two female animals showed no body weight gains from Days 1 to 3 post-exposure and two females showed slight body weight losses from Days 3 to 7 post-exposure. Reasonable body weight gains were noted in all animals during the final week of recovery, with the exception of one male animal which exhibited a slight body weight loss.
Gross pathology:
In group 1, 5.14 mg/L: No abnormalities were noted at necropsy.
Other findings:
- Other observations: The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity during necropsy.

Any other information on results incl. tables

Table 1. Characteristics of the achieved atmosphere:

Group Number

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (µm)

Inhalable Fraction

(% <4 µm)

Geometric Standard Deviation

1

5.14

1.85

77.7

2.78

 

Table 2. Mean achieved atmosphere concentrations:

Group Number

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

1

5.14

0.23

14.1

 

Table 3. Mortality data summary:

Group Number

Mean Achieved Atmosphere Concentration

(mg/L)

Deaths

Male

Female

Total

1

5.14

0/5

0/5

0/10

The target concentrations were group 1: 5.00 mg/L

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the inhalation LC50 (male/female) was: > 5.14 mg/L within the RCCHan WIST rat.
Executive summary:

The study was performed according to OECD TG 403 and EU Method B.2 in accordance with GLP to assess the acute inhalation toxicity of the test item. A group of ten RccHanTM : WIST strain rats (five males and five females) were exposed to an aerosol atmosphere of the test item. The groups was exposed for four hours using a nose only exposure system, followed by a fourteen day observation period. The mean achieved atmosphere concentrations were as follows: Group 1: 5.14 mg/L. The characteristics of the achieved atmosphere where Mean Mass Median Diameter (particle size) and Inhalable Fraction <4 μm: Group 1: 1.85 μm and 77.7% . The Geometric Standard Deviation was Group 1: 2.78. There were no male or female mortalities. Seven animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. Three animals showed no body weight gains from Days 1 to 3 post-exposure and two animals showed slight body weight losses from Days 3 to 7 post-exposure. Body weight gains were noted in all animals during the final week of recovery, with the exception of one animal which exhibited a slight body weight loss. Increased respiratory rate, hunched posture, pilo-erection and wet fur were noted in all animals. All recovered and appeared normal on Day 5 post-exposure. No macroscopic abnormalities were detected amongst animals at necropsy. Under the conditions of this study, the inhalation LC50 (male/female) was > 5.14 mg/L within the RCCHan WIST rat.