Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met. Minor deviation: relative humidity not considered to affect the study reliability.
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:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
relative humidity was lower than targetted (actual 20-30 %). This was considered to be unavoidable and is considered not to have affected the purpose or validity of the study.
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
yes
Remarks:
See above
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2017; signature: November 2017
Test type:
standard acute method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Liquid
- Storage condition of test material: Approximately 4°C in the dark
- Other: Colourless

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan : WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- 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% targeted (20-30 % achieved - not considered to impact study integrity).
- Air changes (per hr): > 15 air changes per hour
- Photoperiod: 12 h light / 12 h dark

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.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The test item was aerosolized using a metal 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 40 L/min providing 80 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: metal concentric jet nebulizer; the concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump and air flow settings into the chamber. The chamber flow rate was maintained 40 L/min providing 80 air changes per hour.
- 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 20 °C and 30 to 70% humidity. (Chamber temperature actual was: 19 to 20 °C ; Chamber relative humidity actual was: 20 – 30% during exposure).

TEST ATMOSPHERE
- Brief description of analytical method used: The test atmosphere was sampled nine times during the exposure period. The sampling procedure involved a known volume of test atmosphere being drawn through a glass fibre filter. Each filter was then submitted for chemical analysis by gas chromatography (GC).

The test filter samples received were extracted with methanol to achieve the working concentration. Blank filters were accurately fortified with known amounts of test item and either analysed without further procedure or purged with nitrogen prior to analysis. A range of standard solutions were prepared in methanol from a stock solution of 1.024 mg/mL by serial dilution covering the concentration range 0.0517 to 0.1551 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 filters were found to have a recovery value of ± 10% of the fortification. The results indicate the accurate use of the test item and filters 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 a single group of ten rats (five males and five females), was subjected to a single exposure to the test item for a period of four hours. Based on the results of the sighting test (no mortality and/or observations at mean achieved atmosphere 2.05 mg/L), a target concentration of 5.0 mg/L was used for the definitive test exposure. As the mean achieved concentration was 103% of target and no mortalities occurred, no further exposure levels were required. 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, body weight,organ weights, and any other relevant toxicological effects were reported.
Statistics:
Using the mortality data obtained, where applicable: an estimate of the acute inhalation median lethal concentration (LC50) would be calculated using validated data analysis software which utilized Log-Normal (Probit) regression models in order to calculate the LC50 values and associated 95% confidence limits for males and females separately.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.14 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: Effect levels based on analytically confirmed: mean achieved concentrations for each exposure/sex.
Mortality:
No mortalities during the study.
Clinical signs:
other: In group 1: 5.14 mg/L: During exposure, all exhibited decreased respiratory rate. On removal from the chamber all animals exhibited decreased respiratory rate and ataxia, one hour post-exposure no change was noted. At Day 1 post-exposure, all exhibited l
Body weight:
In group 1: 5.14 mg/L: All exhibited body weight losses on the first day post-exposure. Except for three males and one female which exhibited body weight losses from Days 1 to 3 post-exposure, body weight gains were noted for all during the remainder of the recovery period.
Gross pathology:
In group 1: 5.14 mg/L: Dark and/or pale patches on the lungs and/or pale lungs were noted amongst eight of ten at necropsy. No macroscopic abnormalities were detected in two males.
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

1.69

83.6

2.43

 

Table 2. Mean achieved atmosphere concentrations:

Group Number

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

1

5.14

0.22

15.25

 

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

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: greater than 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. Following a sighting test, one group of ten RccHanTM : WIST strain rats (five males and five females) were exposed to an aerosol atmosphere of the test item. The groups were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period. The mean achieved atmosphere concentrations was 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: 3.48 μm and 83.6%. The Geometric Standard Deviation was 2.43. There was no mortalities in Group 1. Common abnormalities noted during the study included decreased respiratory rate, ataxia, lethargy, noisy respiration, hunched posture, pilo-erection, sneezing, wet fur and fur stained by clear sticky test item. All animals recovered to appear normal from Days 8 to 11 post-exposure. All animals exhibited body weight losses on the first day post-exposure. Except for three males and one female which exhibited body weight losses from Days 1 to 3 post-exposure, body weight gains were noted for all animals during the remainder of the recovery period. Dark and/or pale patches on the lungs and/or pale lungs were noted amongst eight animals at necropsy. No macroscopic abnormalities were detected in two males. Under the conditions of this study, the 4-hour inhalation LC50 (male/female) was greater than 5.14 mg/L within the RCCHan WIST rat.