Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 25 September 2015 and 26 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes

Test material

Constituent 1
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals and Animal Husbandry
Male and female RccHan™ : WIST strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. After an acclimatization period of at least five days the animals were given a number unique within the study by ear punching and a number written on a color coded cage card. At the start of the study the animals were approximately eight to twelve weeks old and within the weight range of 200g to 350g. The females were nulliparous and non-pregnant.

The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK) and provided with environmental enrichment items: wooden chew blocks and cardboard “fun tunnels” (Datesand Ltd., Cheshire, UK). With the exception of the exposure period, free access to mains drinking water and food (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water, bedding, chew blocks and cardboard “fun tunnels” are routinely analyzed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness. The animals were retained in this accommodation at all times except during the exposure period.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Inhalation Exposure
Atmosphere Generation
A dust atmosphere was produced from the test item using a SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany) located adjacent to the exposure chamber. The SAG 410 was connected to a metered compressed air supply.

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 SAG 410.

The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high). The concentration within the chamber was controlled by adjusting the test item feed rate from the SAG 410. 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.
Homogeneity of the test atmosphere within the chamber was not specifically determined during this study. Chambers of the same design (ADG Developments Ltd, Hitchin, Herts, UK) have been fully validated and shown to produce evenly distributed atmospheres in the animals’ breathing zone with a wide variety of test items (Green J D et al, 1984).

Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterization period test item input rates and grinding times were varied in an attempt to achieve the required atmospheric conditions.


Exposure Procedure
One day 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.

Following an appropriate equilibration period a single group of ten rats (five males and five females) was exposed to an atmosphere of the test item for a period of four hours. A target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 101 % of target and no deaths occurred, no further levels were required.


Exposure Chamber Temperature and Relative Humidity
The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every thirty minutes throughout the four-hour exposure period.


Exposure Chamber Oxygen Concentration
Oxygen levels within the exposure chamber were measured by an electronic oxygen analyzer (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the four-hour exposure period.


Exposure Chamber Atmosphere Concentration
During the characterization phase of the study the test atmosphere was sampled twice and filter samples were then submitted for chemical analysis to determine if the original test item was similar to the composition of the airborne test item.
The actual chamber concentration was measured at regular intervals during the exposure period. The gravimetric method used glass fiber filters placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through the filter using a vacuum pump.

Each filter was weighed before and after sampling in order to calculate the weight of collected test item. The difference in the two weights, divided by the volume of atmosphere sampled, gave the actual chamber concentration.

The nominal chamber concentration was calculated by dividing the mass of test item used by the total volume of air passed through the chamber.

The nominal concentration was 198 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was relatively straightforward.


Particle Size Distribution
The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 µm cut points) with stainless steel collection substrates and a backup glass fiber filter, housed in an aluminum sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump.

The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference.

The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 µm was calculated.

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.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
See below
Duration of exposure:
4 h
Concentrations:
5.0 mg/L
No. of animals per sex per dose:
Five males and five females
Control animals:
no
Details on study design:
Observations
Clinical Signs
All animals were observed for 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 fourteen days. Any evidence of overt toxicity was recorded at each observation.

Body Weight
Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14.

Necropsy
At the end of the fourteen day observation period the animals were killed by intravenous overdose of sodium pentobarbitone. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.
Statistics:
No data

Results and discussion

Preliminary study:
No data
Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.03 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
There were no deaths
Clinical signs:
other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-Hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. Generalized fur sta
Body weight:
All animals exhibited body weight losses or showed no body weight gains on the first day post-exposure. With the exception of three female animals which showed further body weight losses from Days 1 to 3 post-exposure, all animals exhibited body weight gains during the remainder of the recovery period.
Gross pathology:
No macroscopic abnormalities were detected amongst animals at necropsy.
Other findings:
Exposure Chamber Concentration
The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.
The theoretical chamber equilibration time (T99) was 3 minutes* (Silver, 1946).
* Test atmospheres were generated for a total of 14 minutes prior to animal insertion to ensure the target test item concentration was being achieved.

Any other information on results incl. tables

Exposure Chamber Concentration

The test atmosphere was sampled seventeen times during the exposure period and the actual concentration of the test item calculated. The mean values obtained were:

 

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

5.03

0.12

9.96

Particle Size Distribution

The particle size analysis of the atmosphere drawn from the animals’ breathing zone, was as follows:

 

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (µm)

Inhalable Fraction

(% <4 µm)

Geometric Standard Deviation

5.03

3.66

54.1

2.36

Exposure Chamber Atmosphere Concentrations

Duration of Exposure (minutes)

Net Weight of Sample (mg)

Volume of Air Sampled (L)

Chamber Flow Rate (L/min)

Atmosphere Concentration (mg/L)

5

10.26

2

60

5.13

15

10.11

2

60

5.06

30

9.51

2

60

4.76

45

9.75

2

60

4.88

60

10.26

2

60

5.13

75

10.00

2

60

5.00

90

10.29

2

60

5.15

105

10.22

2

60

5.11

120

9.72

2

60

4.86

135

10.32

2

60

5.16

150

10.16

2

60

5.08

165

10.12

2

60

5.06

180

9.90

2

60

4.95

195

10.05

2

60

5.03

210

9.89

2

60

4.95

225

10.22

2

60

5.11

235

10.21

2

60

5.11

Mean achieved atmosphere concentration (mg/L) =5.03

Standard deviation =0.12

 

Nominal concentration:

Test item used (g)

152

Air Flow (L/min)

60

Total Generation Time (mins)

254[1]

Nominal Concentration (mg/L)

9.96

[1]= Test atmospheres were generated for a total of 14 minutes prior to animal insertion to ensure the target test item concentration was being achieved.

Particle Size Distribution

Cascade Impactor Data 

Impactor Stage Number

Cut Point

(µm)

Amount Collected (mg) per Sample Number

Mean Amount Collected (mg)

1

2

3

3

8.4

0.35

0.29

0.33

0.32

4

7.3

0.36

0.32

0.34

0.34

5

3.6

0.66

0.53

0.62

0.60

6

1.3

0.75

0.73

0.65

0.71

7

0.94

0.22

0.25

0.22

0.23

8

0.43

0.11

0.16

0.15

0.14

Back-up Filter

<0.43

0.01

0.01

0.01

0.01

Total Mean Amount of Test Item Collected

2.35

 

Calculation 

Cut Point

(µm)

Log10

Cut Point

Mean Cumulative Amount Less Than Cut Point

(mg)

(%)

Probit

8.4

0.924

2.03

86.4

6.10

7.3

0.863

1.69

71.9

5.58

3.6

0.556

1.09

46.4

4.91

1.3

0.114

0.38

16.2

4.01

0.94

-0.027

0.15

6.38

3.48

0.43

-0.367

0.01

0.43

2.37

Results 

Mean Mass Median Aerodynamic Diameter (MMAD) =3.66µm

Geometric Standard Deviation (GSD) =2.36

Predicted amount less than 4 µm =54.1%

 

Mortality Data

MeanAchievedAtmosphere Concentration (mg/L)

Sex

Deaths During Exposure

Deaths Post Exposure

(1 Hour)

Deaths During Day of Observation

Total Deaths

1

2

3

4

5

6

7

8-14

5.03

Male

0

0

0

0

0

0

0

0

0

0

0/10

Female

0

0

0

0

0

0

0

0

0

0

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
No deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.03 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of FAT 40871/A TE, in the RccHanTM : WIST strain rat, was greater than 5.03 mg/L.
Executive summary:

Introduction

A study was performed to assess the acute inhalation toxicity of the test item.The method used was designed to be compatible with that described in the OECD Guideline for Testing of Chemicals No. 403 “Acute Inhalation Toxicity” (2009) and Method B2. Acute Inhalation Toxicity, 2014, of Commission Regulation (EC) No. 440/2008.

 

Methods.......

A group of ten RccHan™ : WIST strain rats (five males and five females) was exposed to a dust atmosphere. The animals were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period.

 

Results…..

The mean achieved atmosphere concentration was as follows: 

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

5.03

0.12

9.96

 

The characteristics of the achieved atmosphere were as follows: 

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (µm)

Inhalable Fraction

(% <4 µm)

Geometric Standard Deviation

5.03

3.66

54.1

2.36

 

The mortality data were summarized as follows: 

Mean Achieved Atmosphere Concentration (mg/L)

Deaths

Male

Female

Total

5.03

0/5

0/5

0/10

 

Clinical Observations

Common abnormalities noted during the study included decreased respiratory rate, hunched posture, pilo-erection, generalized orange fur staining caused by the test item and wet fur. Occasional instances of increased respiration were also noted. All animals recovered such that no significant observations were apparent from Days 2 to 3 post-exposure.

 

Body Weight

All animals exhibited body weight losses or showed no body weight gains on the first day post-exposure. With the exception of three female animals which showed further body weight losses from Days 1 to 3 post-exposure, all animals exhibited body weight gains during the remainder of the recovery period. 

 

Necropsy

No macroscopic abnormalities were detected amongst animals at necropsy.

 

Conclusion

No deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.03mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of FAT40871/A TE, in the RccHanTM: WIST strain rat, was greater than 5.03mg/L.