N,N'-bis(5,5-dimethyl-1,3,2-dioxaphosphinane-2-oxide-2-yl)ethane-1,2-diamine

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2015 to 02 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

OECD guideline 403 (07 September 2009)

Deviations:

yes

Remarks:

See "Any other information" for details

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Version / remarks:

Council Regulation (EC) No 440/2008, Annex Part B, B.2: "Acute Toxicity (Inhalation)", Official Journal of the European Union No. L 142, dated May 31st, 2008

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-({2-[(5,5-dimethyl-2-oxo-1,3,2λ⁵-dioxaphosphinan-2-yl)amino]ethyl}amino)-5,5-dimethyl-1,3,2λ⁵-dioxaphosphinan-2-one

EC Number:

835-272-7

Cas Number:

256374-76-2

Molecular formula:

C12H26N2O6P2

IUPAC Name:

2-({2-[(5,5-dimethyl-2-oxo-1,3,2λ⁵-dioxaphosphinan-2-yl)amino]ethyl}amino)-5,5-dimethyl-1,3,2λ⁵-dioxaphosphinan-2-one

Test material form:

solid: particulate/powder

Details on test material:

Name
Name: Reaction products of ethane-1,2-diamine, phosphoryl=trichloride and 2,2-dimethylpropane-1,3-diol which makes N,N'-bis(5,5-dimethyl-1,3,2-dioxaphosphinane=2-oxide-2-yl)ethane-1,2-diamine as a main component
Other name: SH-0850
CAS number: 256374-76-2 (main component)

Structural formula
Molecular formula: C12H26N2O6P2 (main component)
Molecular weight: 356.29 (main component)

Provided sample
Purity of the test substance: 100%
Lot number: SK-241002

Physical-chemical properties
Solubility in water: Less than 0.03% (w/w) by visual observation
Melting point: 277 °C
Appearance at normal temperatures: White powder

Storage condition
The test substance was stored in a dark place at room temperature.

Precaution for handling
Protective gloves, mask, glasses and clothes were put on in order to avoid contacts with skin or eyes and inhalation.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Specification
Species and strain: CRL:(WI) rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Justification of strain: Recognized by international guidelines as a recommended test system.
Number of animals: 12
Sex: 6 male and 6 female rats, the females were nulliparous and non-pregnant.
Age and body weight (at dosing): Sighting exposures: 8 weeks old, 334 g (male) and 220 g (female)
Main study: 11 weeks old, 374-398 g (males) and 226-246 g (females).
Randomization: Selected based on bodyweight prior to the exposure.
Acclimatization period: 10 days (Sighting group), 28 days (Main group)

Husbandry
Animal health: Only healthy animals were used for the test. The health status was certified by the veterinarian.
Animal room: 245/7
Housing: Group of 5 (by sex) for main study; individually for sighting
Cage type: Type III solid floor cages with stainless steel mesh lids
Bedding: Lignocel Bedding for Laboratory Animals was available to animals during the study.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.4 - 25.5°C
Relative humidity: 41 – 73%
Ventilation: 15-20 air exchanges/hour
Enrichment: Rodents were housed with deep wood sawdust bedding to allow digging and other normal rodent activities.

Diet and Water
The animals were provided with ssniff SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, D-59494 Soest Germany; batch: 814 3108; expiry: Aug 2015) and tap water fit for human consumption, ad libitum.
The diet and drinking water are routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates of Analysis are retained in the archive of CiToxLAB Hungary Ltd.
Water quality control analysis is performed once every 3 months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36, Hungary). The quality control results are retained in the archive of CiToxLAB Hungary Ltd.

Identification
Each animal was identified by a unique number marked on the tail. The animal number was assigned on the basis of the CiToxLAB Hungary Ltd. master file.
Cages were identified by cage card, giving details of study code, sex, dose-group, cage number and individual animal numbers.

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.92 - <= 3.15 µm

Geometric standard deviation (GSD):

>= 2.94 - <= 2.97

Remark on MMAD/GSD:

The particle size of the test atmosphere was determined three times during the exposure period using a 7-stage impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles in the discrete aerodynamic size ranges. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone).
The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by this difference.
The total amount collected 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 0.55, 0.96, 1.55, 2.11, 3.56, 6.66 and 10.55 μm was calculated.
From these data, using software supplied with the impactor (TSE Systems GmbH, Bad Homburg, Germany), the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated. In addition, the proportion (%) of aerosol less than 4μm (considered to be the inhalable portion) was determined.

Details on inhalation exposure:

INHALATION EXPOSURE
Technical Trials
Prior to animal exposures, test material atmospheres were generated within the exposure chamber. During these technical trials, air-flow settings and test material input rates were varied to achieve the required atmospheric characteristics.

Atmosphere Generation
The test item was aerosolised using two Wright’s Dust Feed Systems (TSE Systems GmbH, Bad Homburg, Germany; Serial Number: 040804-59) located at the top of the exposure chamber. Compressed air was supplied by means of an oil-free compressor and passed through a suitable filter system prior to introduction to the dust generator.

Animal Exposure System
The animals were exposed, nose-only, to an atmosphere of the test item using a TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany). This system comprises of two, concentric anodised aluminium chambers and a computer control system incorporating pressure detectors and mass flow controllers.
Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where, under positive pressure, it was distributed to the individual exposure ports. The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animal’s nares to enter the exposure port. After passing through the animal’s breathing zone, used aerosol entered the outer cylinder from where it was exhausted through a suitable filter system. Atmosphere generation was therefore dynamic.
Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposure port (breathing zone). The flow of air through each port was at least 0.7 L/min. This flow rate was considered adequate to minimise re-breathing of the test atmosphere as it is about twice the respiratory minute volume of a rat.
Homogeneity of the test atmosphere within the test chamber and amongst the exposure ports was not specifically determined during this study. However, chambers of this design have been fully validated and have shown to produce evenly distributed atmospheres in the animals’ breathing zones.

Sighting Exposure
Sighting exposures were performed in order to estimate the test item’s inhalation toxicity, identify sex differences in susceptibility and assist in selecting exposure concentration levels for the main study.

Exposure Procedure
Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber. Only the nose of each animal was exposed to the test atmosphere.
Following an equilibration period of at least the theoretical chamber equilibration time (T99), a group of ten rats (five male and five female) was exposed to an atmosphere of the test material for a period of four hours. Five mg/L as the target concentration was used for the main group. As no death occurred at 5.07 mg/L concentration in the main group, no further data were required.

EXPOSURE MONITORING
Test Atmosphere Concentrations
The test atmosphere was sampled at regular intervals during each exposure period. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone) by pulling a suitable, known volume of test atmosphere through weighed GF10 glass fibre filters (Whatman GmbH, Hahnestraße 3 – D-37586 Dassel, Germany). The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.
The nominal concentration was calculated by dividing the mass of test material disseminated into the chamber by the total volume of air that went through the chamber during the same period.

Analytical verification of test atmosphere concentrations:

no

Duration of exposure:

4 h

Concentrations:

A sighting exposure was performed first: 5.05 mg/L was tested on single animals of both sexes (Group 0.1).
Based on the lack of lethality at this concentration, the main study group, was exposed to the target concentration of 5.07 mg/L SH-0850.

No. of animals per sex per dose:

10 (5 male and 5 female) CRL: (WI) Wistar strain rats

Control animals:

no

Details on study design:

Morbidity/Mortality
Animals were checked hourly during exposure, one hour after exposure and twice daily (early and late in the working day) during the 14-day observation period for morbidity and/or mortality.

Clinical Signs
All animals were observed for clinical signs at hourly intervals during exposure, as soon as practically possible following removal from restraint at the end of exposure, one hour after exposure and subsequently once daily for fourteen days.

Bodyweight
Individual bodyweights were recorded prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.

Necropsy
At the end of the fourteen day observation period, the animals were euthanised by exsanguination under anaesthesia (intra-peritoneal injection of pentobarbital solution – Euthanimal 40%; Lot No.: 1409236-06; Expiry: 09-2017; Produced by Alfasan Nederland BV, Kulpersweg 9, Woerden, Netherlands) and gross macroscopic examination was performed. All animals were subject to a gross necropsy which included a detailed examination of the abdominal and thoracic cavities. Special attention was given to the respiratory tract for macroscopic signs of irritancy or local toxicity.

Statistics:

Not specified

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.07 mg/L air (nominal)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality

Clinical signs:

other:

Body weight:

The exposure procedure caused slight bodyweight loss in 9/10 animals. By the second part of the observation period the bodyweight was normalized in most of the animals.

Gross pathology:

A single four hour nose-only exposure of SH-0850 to Crl:WI rats at a concentration of 5.05 mg/L during Sighting exposure or exposed to a concentration of 5.07 mg/L in Main study, was not associated with any gross changes.

Any other information on results incl. tables

Test Atmosphere Concentrations – Group 0.1

Exposure Duration (minutes)	Sample Volume (L)	Atmospheric Concentration of SH-0850 (mg/L)
0	2.0	5.22
12	2.0	5.41
26	2.0	5.36
39	2.0	53.0
49	2.0	4.82
63	2.0	5.11
77	2.0	4.93
94	2.0	4.61
108	2.0	4.99
123	2.0	4.72
138	2.0	5.30
154	2.0	5.26
168	2.0	5.07
183	2.0	4.82
198	2.0	5.22
214	2.0	4.95
228	2.0	4.81

Maximum attainable Atmosphere Concentration = 5.05 mg/L

Standard Deviation = 0.24

 

Nominal Concentration:

Amount of Test Item Used (g): 97.58

Total Volume of Air Used (L): 7500

Nominal Concentration = 13.01 mg/L

 

Test Atmosphere Concentrations – Group 1

Exposure Duration (minutes)	Sample Volume (L)	Atmospheric Concentration of SH-0850 (mg/L)
0	2.0	4.94
12	2.0	5.32
24	2.0	5.15
36	2.0	5.24
48	2.0	5.43
65	2.0	4.65
76	2.0	4.59
93	2.0	4.73
109	2.0	5.29
124	2.0	4.75
138	2.0	5.37
153	2.0	5.28
168	2.0	5.45
183	2.0	5.04
200	2.0	4.86
213	2.0	4.92
228	2.0	5.11

Maximum attainable Atmosphere Concentration = 5.057 mg/L

Standard Deviation = 0.28

 

Nominal Concentration:

Amount of Test Item Used (g): 91.42

Total Volume of Air Used (L): 7560

Nominal Concentration = 12.09 mg/L

 

Test Atmosphere Particle Size Distribution Data – Group 0.1

Stage Number	Cut Point (µm)	Amount Collected (mg)			Total Collected per Stage (mg)
		Sample 1	Sample 2	Sample 3
1	10.55	0.64	0.72	0.50	1.86
2	6.66	0.68	0.54	0.85	2.07
3	3.56	1.14	1.26	1.38	3.78
4	2.11	1.22	1.20	1.33	3.75
5	1.55	0.60	0.53	0.65	1.78
6	0.96	0.45	0.45	0.49	1.39
7	0.55	0.26	0.26	0.27	0.79
Filter	<0.55	0.40	0.38	0.53	1.31
Total Amount Collected (mg)					16.73
Size Range (µm)		Total Mass/Stage (mg)		Cumulative Mass (%)
<0.55		1.31		7.83
0.55 – 0.96		0.79		15.55
0.96 – 1.55		1.39		20.86
1.55 – 2.11		1.78		31.50
2.11 – 3.56		3.75		53.92
3.56 – 6.66		3.78		76.51
6.66 – 10.55		2.07		88.88
>10.55		1.86		100.00

Maximum attainable Atmosphere Concentration = 5.05 mg/L

Mean Mass Median Aerodynamic Diameter (MMAD) – 3.15 µm

Geometric Standard Deviation (GSD) = 2.94

Inhalable Fraction (% <4 µm) = 58.8%

 

Test Atmosphere Particle Size Distribution Data – Group 1

Stage Number	Cut Point (µm)	Amount Collected (mg)			Total Collected per Stage (mg)
		Sample 1	Sample 2	Sample 3
1	10.55	0.53	0.58	0.62	1.73
2	6.66	0.85	0.65	0.64	2.14
3	3.56	1.33	1.16	1.12	3.61
4	2.11	1.39	1.30	1.31	4.00
5	1.55	0.62	0.67	0.59	1.88
6	0.96	0.55	0.53	0.43	1.51
7	0.55	0.40	0.33	0.28	1.01
Filter	<0.55	0.63	0.49	0.42	1.54
Total Amount Collected (mg)					17.42
Size Range (µm)		Total Mass/Stage (mg)		Cumulative Mass (%)
<0.55		1.54		8.84
0.55 – 0.96		1.01		14.64
0.96 – 1.55		1.51		23.31
1.55 – 2.11		1.88		34.10
2.11 – 3.56		4.00		57.06
3.56 – 6.66		3.61		77.78
6.66 – 10.55		2.14		90.07
>10.55		1.73		100.00

Maximum attainable Atmosphere Concentration = 5.07 mg/L

Mean Mass Median Aerodynamic Diameter (MMAD) – 2.92 µm

Geometric Standard Deviation (GSD) = 2.97

Inhalable Fraction (% <4 µm) = 61.4%

 

Test Chamber Environmental and Equilibration Data – Group 0.1

Measurement	Mean Value	Minimum	Maximum
Air Flow In (Inner Plenum) (L/min)	30.4	29.6	31.4
Air Flow Out (Outer Cylinder) (L/min)	24.0	23.7	24.2
Temperature (°C)	25.1	24.8	25.2
Relative Humidity (%)	30.4	23.0	39.0
Oxygen Concentration (%)	20.9	20.8	21.1
Carbon Dioxide (%)	0.0	0.0	0.1

Theoretical Chamber Equilibration Time (T₉₉):

T₉₉= (4.605 x (^{Chamber Volume}/_{Chamber Flow rate})

Chamber volume (inner plenum) = 3.85L

T₉₉(Minimum Acceptable Equilibration Time) = 1 minute

Actual equilibration time allowed = 11 minutes

 

Test Chamber Environmental and Equilibration Data – Group 1

Measurement	Mean Value	Minimum	Maximum
Air Flow In (Inner Plenum) (L/min)	30.5	29.4	31.3
Air Flow Out (Outer Cylinder) (L/min)	24.0	23.7	24.3
Temperature (°C)	24.0	23.0	24.5
Relative Humidity (%)	14.4	6.0	35.0
Oxygen Concentration (%)	21.2	21.1	21.2
Carbon Dioxide (%)	0.0	0.0	0.1

Theoretical Chamber Equilibration Time (T₉₉):

T₉₉= (4.605 x (^{Chamber Volume}/_{Chamber Flow rate})

Chamber volume (inner plenum) = 3.85L

T₉₉(Minimum Acceptable Equilibration Time) = 1 minute

Actual equilibration time allowed = 13 minutes

 

Mortality Data

Day Number	Number of Deaths
	Group 0.1 (5.05 mg/L)		Group 1 (5.07 mg/L)
	Male	Female	Male	Female
0 (During Exposure)	0	0	0	0
0 (After Exposure)	0	0	0	0
1	0	0	0	0
2	0	0	0	0
3	0	0	0	0
4	0	0	0	0
5	0	0	0	0
6	0	0	0	0
7	0	0	0	0
8 – 14	0	0	0	0
Total Deaths	0/1	0/1	0/5	0/5
Grand Total Deaths	0/2		0/10

 

Individual Clinical Observations – Group 0.1

SIGHTING EXPOSURE

DOSE GROUP: 0.1

CONCENTRATION: 5.05 mg/L                     SEX: MALE/FEMALE

Animal number

Observations

Days of study

Frequency

0 (exposure)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

During

After

1h

2h

3h

4h

5h

3696

Normal Laboured Respiration – Slight Wet fur – On/In restraining apparatus Wet fur – While body Fur staining by test item – First third of animal

- + - - -

- + - - -

- + + - -

- + - + +

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

15/19 4/19 1/19 1/19 1/19

3711

Normal Laboured Respiration – Slight Wet fur – On/In restraining apparatus Wet fur – Whole body Red-brown staining – nose Fur staining by test item – First third of animal

- + - - - -

- + - - - -

- + + - - -

- + - + + +

+ - - - - -

+ - - - - -

+ - - - - -

+ - - - - -

+ - - - - -

+ - - - - -

+ - - - - -

+ - - - - -

+ - - - - -

+ - - - - -

+ - - - - -

+ - - - - -

+ - - - - -

+ - - - - -

+ - - - - -

15/19 4/19 1/19 1/19 1/19 1/19

COMMENT:

+ = Present

- = Absent

 

Individual Clinical Observations – Group 1

MAIN STUDY

DOSE GROUP: 1

CONCENTRATION: 5.07 mg/L                     SEX: MALE

Animal number

Observations

Days of study

Frequency

0 (exposure)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

During

After

1h

2h

3h

4h

5h

3496

Normal Laboured Respiration – Slight Red-brown staining - Nose Fur staining by test item – First third of animal

+ - - -

+ - - -

- + - -

- + - +

- - + -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

16/19 2/19 1/19 1/19

3499

Normal Laboured Respiration – Slight Wet fur – On/In restraining apparatus Wet fur – Whole body Fur staining by test item – First third of animal

+ - - - -

- + + - -

- + + - -

- + - + +

- - - + -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

+ - - - -

15/19 3/19 2/19 2/19 1/19

3500

Normal Laboured Respiration – Slight Red-brown staining - Nose Fur staining by test item – First third of animal

+ - - -

+ - - -

- + - -

- + + +

- - + -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

16/19 2/19 2/19 1/19

3501

Normal Laboured Respiration – Slight Fur staining by test item – First third of animal

+ - -

+ - -

- + -

- + +

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

17/19 2/19 1/19

3504

Normal Laboured Respiration – Slight Fur staining by test item – First third of animal

+ - -

- + -

- + -

- + +

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

16/19 3/19 1/19

COMMENT:

+ = Present

- = Absent

 

MAIN STUDY

DOSE GROUP: 1

CONCENTRATION: 5.07 mg/L                     SEX: FEMALE

Animal number

Observations

Days of study

Frequency

0 (exposure)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

During

After

1h

2h

3h

4h

5h

3512

Normal Laboured Respiration – Slight Fur staining by test item – First third of animal

+ - -

+ - -

- + -

- + +

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

17/19 2/19 1/19

3517

Normal Laboured Respiration – Slight Fur staining by test item – First third of animal

+ - -

+ - -

- + -

- + +

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

+ - -

17/19 2/19 1/19

3519

Normal Laboured Respiration – Slight Red-brown staining - Nose Fur staining by test item – First third of animal

+ - - -

+ - - -

- + - -

- + + +

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

17/19 2/19 1/19 1/19

3523

Normal Laboured Respiration – Slight Red-brown staining - Nose Fur staining by test item – First third of animal

+ - - -

+ - - -

- + - -

- + + +

- - + -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

16/19 2/19 2/19 1/19

3524

Normal Laboured Respiration – Slight Red-brown staining – Nose Fur staining by test item – First third of animal

+ - - -

+ - - -

- + - -

- + + +

- - + -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

+ - - -

16/19 2/19 2/19 1/19

COMMENT:

+ = Present

- = Absent

 

Individual Bodyweight Data – Groups 0.1 and 1

SIGHTING EXPOSURE

DOSE GROUP: 0.1

CONCENTRATION: 5.05 mg/L                     SEX: MALE/FEMALE

Animal Number	Body weight (g) on days					Body weight (g) between days
	0	1	3	7	14	0-1	1-3	3-7	7-14	0-14
3969	334	325	344	362	405	-9	19	18	43	71
3711	220	214	220	229	241	-6	6	9	12	21

 

MAIN STUDY

DOSE GROUP: 1

CONCENTRATION: 5.07 mg/L                     SEX: MALE

Animal Number	Body weight (g) on days					Body weight (g) between days
	0	1	3	7	14	0-1	1-3	3-7	7-14	0-14
3496	374	366	357	391	409	-8	-9	34	18	35
3499	388	382	368	397	417	-6	-14	29	20	29
3500	398	392	383	404	423	-6	-9	21	19	25
3501	386	384	375	405	426	-2	-9	30	21	40
3504	393	395	386	420	441	2	-9	34	21	48

DOSE GROUP: 1

CONCENTRATION: 5.07 mg/L                     SEX: FEMALE

Animal Number	Body weight (g) on days					Body weight (g) between days
	0	1	3	7	14	0-1	1-3	3-7	7-14	0-14
3512	246	240	246	250	246	-6	6	4	-4	0
3517	244	239	244	250	243	-5	5	6	-7	-1
3519	230	241	244	255	258	11	3	11	3	28
3523	243	235	238	242	242	-8	3	4	0	-1
3524	226	225	222	229	234	-1	-3	7	5	8

 

Individual Necropsy Findings – Groups 0.1 and 1

 

SIGHTING EXPOSURE

DOSE GROUP: 0.1

CONCENTRATION: 5.05 mg/L                     SEX: MALE/FEMALE

NECROPSY FINDINGS	Animal numbers
	3696	3711
NO INTERNAL OBSERVATION RECORDED	+	+
NO EXTERNAL OBSERVATION RECORDED	+	+
STUDY DAYS	14	14
DATE OF NECROPSY	26 June 2015

 

MAIN STUDY

DOSE GROUP: 1

CONCENTRATION: 5.07 mg/L                     SEX: MALE

NECROPSY FINDINGS	Animal numbers
	3496	3499	3500	3501	3504
NO INTERNAL OBSERVATION RECORDED	+	+	+	+	+
NO EXTERNAL OBSERVATION RECORDED	+	+	+	+	+
STUDY DAYS	14	14	14	14	14
DATE OF NECROPSY	02 July 2015

DOSE GROUP: 1

CONCENTRATION: 5.07 mg/L                     SEX: FEMALE

NECROPSY FINDINGS	Animal numbers
	3512	3517	3519	3523	3524
NO INTERNAL OBSERVATION RECORDED	+	+	+	+	+
NO EXTERNAL OBSERVATION RECORDED	+	+	+	+	+
STUDY DAYS	14	14	14	14	14
DATE OF NECROPSY	02 July 2015

 

COMMENT: NECROPSY FINDINGS PRESENT = +

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, no deaths occurred in a group of ten rats exposed to a concentration of 5.07 mg/L for four hours. The acute inhalation median lethal concentration (4hr LC50) of SH-0850, in CRL: (WI) Wistar strain rats, was therefore considered to be greater than 5.07 mg/L.

Executive summary:

This study was performed to assess the acute inhalation toxicity of SH-0850. The method was designed to meet OECD guideline 403 (07 September 2009), Council Regulation (EC) no. 440/2008, Annex Part B, B.2: "Acute Toxicity (Inhalation)", Official Journal of the European Union No. L 142, dated May 31st, 2008, in line with the Sponsor requirements.

 

Methods

This study was performed to assess the acute inhalation toxicity of SH-0850 following a 4 hours exposure at the target concentration of 5 mg/L to 5 male and 5 female rats.

The study was performed in two phases.

A sighting exposure was performed first: 5.05 mg/L was tested on single animals of both sexes (Group 0.1).

Based on the lack of lethality at this concentration, the main study group, 10 (5 male and 5 female) CRL: (WI) Wistar strain rats, was exposed to the target concentration of 5.07 mg/L SH-0850. The animals were exposed for 4 hours using a nose-only exposure system, followed by a 14-day observation period. The day of exposure was designated Day 0. Aerosol concentrations were measured gravimetrically. The particle size distribution of the test aerosol was determined regularly during the exposure period. Clinical observations and bodyweights were recorded throughout the study and at the end of the scheduled period the animals were euthanized and subjected to a gross examination post mortem.

No control group was exposed in this study.

 

Results

The atmosphere concentration was as follows:

Group Number	Target Concentration (mg/L)	Mean Achieved Concentration (mg/L)	Standard Deviation of Achieved Concentration (mg/L)
0.1	5.00	5.05	0.24
1	5.00	5.07	0.28

 

The characteristics of the test atmosphere were as follows:

Group	Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (MMAD) (µm)	Geometric Standard Deviation (GSD)	Inhalable Fraction (%<4µm)
0.1 Sighting	5.05	3.15	2.94	58.8
1 Main Study	5.07	2.92	2.97	61.4

 

The mortality data were summarised as follows:

Group	Concentration (mg/L)	Male Deaths	Female Deaths	Total Deaths
0.1 Sighting	5.05	0/1	0/1	0/2
1 Main Study	5.07	0/5	0/5	0/10

 

Clinical Observations: Wet fur and fur staining were commonly recorded, mostly on the day of exposure. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not to be biologically significant.

Sighting group (Group 0.1): Slightly laboured respiration was observed during exposure. No abnormalities were detected in either animal from the day following exposure until the end of the observation period.

Main Group (Group 1): Slightly laboured respiration was detected during the exposure. No abnormalities were detected in any animal from the day following exposure until the end of the observation period.

 

Bodyweights: The exposure procedure caused slight bodyweight loss in 9/10 animals. By the second part of the observation period the bodyweight was normalized in most of the animals.

 

Necropsy: A single four hour nose-only exposure of SH-0850 to Crl:WI rats at a concentration of 5.05 mg/L during Sighting exposure or exposed to a concentration of 5.07 mg/L in the Main study, was not associated with any gross changes.

 

Conclusion

Under the experimental conditions of this study, no deaths occurred in a group of ten rats exposed to the concentration of 5.07 mg/L for four hours. The acute inhalation median lethal concentration (4hr LC50) of SH-0850, in CRL: (WI) Wistar strain rats, was therefore considered to be greater than 5.07 mg/L.