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

Endpoint summary

Description of key information

Acute toxicity: oral

LD50 value of SH-0850 was found to be above 2000 mg/kg bw in female RccHan:WIST rats.

Acute toxicity: inhalation

4hr LC50 of SH-0850, in CRL: (WI) Wistar strain rats, was therefore considered to be greater than 5.07 mg/L.

Acute toxicity: dermal

LD50 of SH-0850 after a single dermal administration was found to be greater than 2000 mg/kg bw in male and female Crl:WI rats.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 February 2014 to 26 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 423. Acute Oral Toxicity – Acute Toxic Class Method. Adopted: 17 December 2001

Deviations:

yes

Remarks:

The test item was not ranked into categories of Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423 according to the Sponsor request. This deviation has no impact on the outcome or integrity of the study .

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

Commission Regulation (EC) NO 440/2008 of 30 May 2008, B.1.Tris

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

EPA Health Effects Test Guidelines (OPPTS 870.1100), United States, EPA 712-C-98-190 (1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Species and strain: RccHan:WIST rats
Source: Harlan Laboratories S.r.l. S.Pietro al Natisone (UD), Zona Industriale Azzida, 57 33040, Italy
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Number of animals: 6 animals
Sex: Female, nulliparous and non-pregnant.
Age of animals at dosing: Young healthy adult rats, ~9 weeks old
Date of receipt: 29 January 2014
Body weight at treatment: 160 – 184 g
Acclimation period: at least 13 days

Husbandry
Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
Number of animal room: 522/4
Housing: 3 animals / group
Cage type: Type II polypropylene/polycarbonate
Bedding: Lignocel Bedding for Laboratory Animals was available to animals during the study. A copy of the Certificate of Analysis is retained in the archive at CiToxLAB Hungary Ltd.
Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: 15-20 air exchanges/hour
Enrichment: Animals were housed by group to allow social interaction and with deep wood sawdust bedding to allow digging and other normal rodent activities.
The temperature and relative humidity were recorded twice daily during the study.

Food and Water Supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from the municipal supply, as for human consumption from 500 ml bottle ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The batch of feed employed in the study was as follows:
 186 0298, expiry date: May 2014
The supplier provided an analytical certificate for the batch used. Copy of the certificate will be archived with the raw data.
Water quality control analysis is performed once every three 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 archives at CiToxLAB Hungary Ltd.

Animal Identification
Animals were individually identified using numbers written on the tail with an indelible marker pen. The numbers were given on the basis of CiToxLAB Hungary Ltd.' s Master File, for each animal allocated to the treatment groups. The cages were identified by cards, with information about study code, sex, dose group, cage number and individual animal numbers.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Test item was freshly formulated at a concentration of 200 mg/mL in the vehicle, in the Central Dispensary Unit of CiToxLAB Hungary Ltd. on the day of administration. The formulation container was stirred continuously during administration with the objective of ensuring that the syringe was filled from a suspension.
Vehicle Distilled water
Batch No. 0790713
Expiry Date 31 July 2016
Dose volume 10 mL/kg bw

The initial dose level was selected by the study director to be that which is most likely to produce mortality in some of the dosed animals. In the lack of any preliminary toxicological information, 2000 mg/kg bw was selected to be the starting dose.

Doses:

single oral gavage administration

No. of animals per sex per dose:

Initially, three females (Group 1)
Confirmatory group (Group 2), three animals

Control animals:

no

Details on study design:

Initially, three females (Group 1) were treated at a dose level of 2000 mg/kg bw of SH-0850. The test item did not cause mortality in this group; therefore a confirmatory group (Group 2) was treated at the same dose level. The test item did not cause mortality in the confirmatory group, so no further testing was required according to OECD 423 and Commission Regulation (EC) NO 440/2008 of 30 May 2008, B.1.Tris.

Procedure
A single oral gavage administration was followed by a fourteen-day observation period. On the day before treatment, the animals were fasted. The food but not water was withheld during an overnight period. Animals were weighed just before treatment. The test item was administered by oral gavage in the morning. The food was returned 3 hours after the treatment.

OBSERVATIONS
Clinical Observations
Clinical observations were performed on all animals at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Individual observations were performed on the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body Weight Measurement
The body weight was recorded on the day before treatment (Day -1), on the day of the treatment (Day 0) and weekly thereafter.

NECROPSY
Macroscopic examination was performed on all animals. The surviving animals were sacrificed by exsanguination under pentobarbital anaesthesia. After examination of the external appearance, the cranial, thoracic and the abdominal cavities were opened and the organs and the tissues were observed. Macroscopic abnormalities were recorded.

Statistics:

The method used was not intended to allow the calculation of a precise LD50 value.
Clinical signs, body weight, body weight gain and gross macroscopic data were tabulated.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

SH-0850 did not caused mortality at the dose level of 2000 mg/kg bw.

Clinical signs:

other: Treatment with SH-0850 did not cause any test item related effect at a dose level of 2000 mg/kg bw.

Gross pathology:

There was no evidence of the macroscopic observations at a dose level of 2000 mg/kg bw.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the acute oral LD50 value of the test item SH-0850 was found to be above 2000 mg/kg bw in female RccHan:WIST rats.

Executive summary:

The single-dose oral toxicity of SH-0850 was performed according to the acute toxic class method (OECD 423 and Commission Regulation (EC) NO 440/2008 of 30 May 2008, B.1.Tris) in RccHan:WIST rats.

 

Two groups of three female RccHan:WIST rats were treated with the test item at a dose level of 2000 mg/kg bw (Group 1 and Group 2).

 

Initially, three females (Group 1) were treated at a dose level of 2000 mg/kg bw SH-0850. The test item did not cause mortality in this group; therefore, a confirmatory group (Group 2) was treated at the same dose level. The test item did not cause mortality in the confirmatory group, so no further testing was required according to OECD 423 and Commission Regulation (EC) NO 440/2008 of 30 May 2008, B.1.Tris.

 

A single oral treatment was carried out by gavage for each animal after an overnight food withdrawal. Food was made available again 3 hours after the treatment. The test item was administered formulated in distilled water at a concentration of 200 mg/mL at a dosing volume of 10 mL/kg bw.

 

Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Body weight was measured on Days -1, 0 and 7 and Day 14 before necropsy. All animals were subjected to a necropsy and a macroscopic examination.

Results

 

Mortality

SH-0850 did not caused mortality at the dose level of 2000 mg/kg bw.

 

Clinical observations

Treatment with SH-0850 did not cause any test item related effect at a dose level of 2000 mg/kg bw.

 

Body weight and body weight gain

Body weight gains of SH-0850 treated animals during the study showed no indication of a treatment-related effect.

 

Macroscopic Findings

There was no evidence of the macroscopic observations at a dose level of 2000 mg/kg bw.

 

Conclusion:

Under the conditions of this study, the acute oral LD50 value of the test item SH-0850 was found to be above 2000 mg/kg bw in female RccHan:WIST rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

K1

Acute toxicity: via inhalation route

Link to relevant study records

Reference

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

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

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

Specific details on test material used for the study:

No further details specified in the study report.

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.

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

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.

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

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+ - - - -

+ - - - -

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 = +

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

5 070 mg/m³ air

Quality of whole database:

K1

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2015 to 02 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Section 4, Number 402 "Acute Dermal Toxicity", adopted February 24, 1987.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Version / remarks:

Commission Regulation (EC) No 440/2008, B.3 (L 142, 30 May 2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
Species and strain: Crl:WI rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany
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: 5 animals / sex
Sex: Male and female, female rats were nulliparous and non-pregnant.
Age of animals at dosing: Young adult rats
Body weight range at dosing: Males: 242-262 g; Females: 216-237 g
Age at dosing: Males: 7 weeks; Females: 8-9 weeks
Acclimatization time: 7 days

Husbandry
Animal health: Only healthy animals were used for the study. The veterinarian certified the health status.
Room: 242/6
Housing: Individual caging
Cage type: Type II. polypropylene/polycarbonate
Bedding: “Grade 5” Bedding for Laboratory Animals was available to animals during the study. A copy of the Certificate of Analysis is retained in the archives at CiToxLAB Hungary Ltd.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.0 – 23.4 °C
Relative humidity: 31 – 51 %
Ventilation: 15-20 air exchanges per hour
Enrichment: Rodents are housed with deep wood sawdust bedding to allow digging and other normal rodent activities.
The temperature and relative humidity were recorded twice daily during the study.

Food and water supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494, Soest, Germany (Batch no.: 680 2237, Expiry date: March 2015 or Batch no.: 814 3108, Expiry date: August 2015), ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottles, ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Water quality control analysis is performed once every three 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 archives at CiToxLAB Hungary Ltd.

Identification
Animals were identified by numbers written on the tail with an indelible marker. The numbers were given on the basis of the CiToxLAB Hungary Ltd. master file, for each animal allocated to the study.
The boxes were identified by cards holding information on the study code, the sex of animals, the dose group, the cage number and the individual animal number.

Type of coverage:

semiocclusive

Vehicle:

water

Details on dermal exposure:

The test item was administered in a single dose as supplied by the Sponsor, but sufficient water was used to moisten the test material to ensure good contact with the skin.
A single administration was performed by the dermal route and was followed by a fourteen-day observation period. The test item was applied as supplied.
The back of the animals were shorn (approximately 10% area of the total body surface) approximately 24 hours prior to the treatment. Only the animals without injury or irritation on the skin were used in the test.
On the test day (Day 0), the test item was applied as a single dose of 2000 mg/kg bw after the moistening with sufficient water, applied uniformly over the skin by use of a gauze pad (ca. 5 cm x 5 cm), and remained on the skin throughout a 24-hour exposure period. Sterile gauze pads were placed on the skin of rats at the site of application. These gauze pads were kept in contact with the skin by a patch with adhesive hypoallergenic plaster. The entire trunk of the animal was then wrapped with semi occlusive plastic wrap for 24 hours. At the end of the exposure period, residual test item was removed, using body temperature water.

Duration of exposure:

24-hour exposure period.

Doses:

A limit dose of 2000 mg/kg bw was chosen by the Study Director in agreement with the Sponsor.

No. of animals per sex per dose:

10 animals (5 males/5 females)/

Control animals:

no

Details on study design:

Clinical observations
A clinical examination was performed on the day of treatment, at 1 and 5 hours after the application of the test item, and once each day for 14 days thereafter.
Observations included the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous system, and somatomotor activity and behaviour pattern. Particular attention was directed to the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Skin Irritation
Adverse skin reactions at the site of application were recorded daily following the removal of the dressing (for scoring scheme see "Any other information" for details).

Measurement of body weight
The body weight of all animals was recorded on Day 0 (beginning of the experiment) and on Days 7 and 14.

Post Mortem Investigations
All animals were subjected to gross macroscopic examination. All animals were anaesthetised with an injection of pentobarbital solution and exsanguinated. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. Any gross macroscopic findings were recorded.

Statistics:

Not specified

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred after the 24-hour dermal exposure to SH-0850 in Crl:WI rats.

Clinical signs:

other: Systemic Clinical Signs: Each rat was symptom-free during the entire study. Local Dermal Signs: No local dermal signs were recorded after treatment with the test item during the 14 days observation period.

Gross pathology:

No external or internal macroscopic findings were observed at a dose level of 2000 mg/kg bw at necropsy.

Clinical Observations

DOSE LEVEL: 2000 mg/kg bw                                                                              SEX: MALE

Cage No.

Animal No.

Observations

Observations days

Frequency

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

1h

5h

1

2456

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

16/16

2

2457

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

16/16

3

2458

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

16/16

4

2459

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

16/16

5

2460

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

16/16

 

DOSE LEVEL: 2000 mg/kg bw                                                                              SEX: FEMALE

Cage No.

Animal No.

Observations

Observations days

Frequency

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

1h

5h

6

2461

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

16/16

7

2462

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

16/16

8

2463

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

16/16

9

2464

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

16/16

10

2465

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

16/16

Remarks:         + = present

                       h = hour(s)       Treatment day = Day 0

                       Frequency of observation = number of occurrence of observation / total number of observations

 

Body Weight and Body Weight Gain

DOSE LEVEL: 2000 mg/kg bw                                                                  SEX: MALE

Cage No.	Animal No.	Boy weight (g) Days			Body Weight Gain (g)
		0	7	14	0-7	7-14	0-14
1	2456	242	285	325	43	40	83
2	2457	259	297	352	38	55	93
3	2458	252	291	336	39	45	84
4	2459	245	289	335	44	46	90
5	2460	262	332	398	70	66	136
Mean:		252.0	298.8	349.2	46.8	50.4	97.2
Standard deviation:		8.6	19.1	28.9	13.2	10.3	22.1

 

DOSE LEVEL: 2000 mg/kg bw                                                                  SEX: FEMALE

Cage No.	Animal No.	Boy weight (g) Days			Body Weight Gain (g)
		0	7	14	0-7	7-14	0-14
6	2461	225	241	264	16	23	39
7	2462	237	229	238	-8	9	1
8	2463	226	231	237	5	6	11
9	2464	234	252	256	18	4	22
10	2465	216	234	239	18	5	23
Mean:		227.6	237.4	246.8	9.8	9.4	19.2
Standard deviation:		8.3	9.3	12.4	11.3	7.8	14.3

 

Necropsy Findings

DOSE LEVEL: 2000 mg/kg bw                                                                                          SEX: MALE

Cage No.	Animal No.	Necropsy Date/ Study Day	External Observations	Internal Observations	Organ/Tissue
1	2456	02 April 2015 Day 14	No external observations	No internal observations	Not applicable
2	2457	02 April 2015 Day 14	No external observations	No internal observations	Not applicable
3	2458	02 April 2015 Day 14	No external observations	No internal observations	Not applicable
4	2459	02 April 2015 Day 14	No external observations	No internal observations	Not applicable
5	2460	02 April 2015 Day 14	No external observations	No internal observations	Not applicable

 

DOSE LEVEL: 2000 mg/kg bw                                                                                          SEX: FEMALE

Cage No.	Animal No.	Necropsy Date/ Study Day	External Observations	Internal Observations	Organ/Tissue
6	2461	02 April 2015 Day 14	No external observations	No internal observations	Not applicable
7	2462	02 April 2015 Day 14	No external observations	No internal observations	Not applicable
8	2463	02 April 2015 Day 14	No external observations	No internal observations	Not applicable
9	2464	02 April 2015 Day 14	No external observations	No internal observations	Not applicable
10	2465	02 April 2015 Day 14	No external observations	No internal observations	Not applicable

 

Interpretation of results:

GHS criteria not met

Conclusions:

The median lethal dose (LD50) of SH-0850 after a single dermal administration was found to be greater than 2000 mg/kg bw in male and female Crl:WI rats.

Executive summary:

The purpose of the study was to assess the acute dermal toxicity of SH-0850 when administered to rats by a single semi-occlusive dermal application, followed by an observation period of 14 days. The test item was applied dermally to ten (5 males and 5 females) Crl:WI rats as supplied by the Sponsor. A limit test was carried out at 2000 mg/kg body weight (bw) in both sexes.

 

Clinical observations were performed on all animals at 1 and 5 hours after dosing and daily for 14 days thereafter. Body weight was measured prior to dosing on Day 0 and on Days 7 and 14. Rats were euthanized and subjected to a gross macroscopic examination at the end of the 2-week observation period (Day 14).

 

Results

Mortality

No mortality occurred after the 24-hour dermal exposure to SH-0850 in Crl:WI rats.

Systemic clinical signs

Each rat was symptom-free during the entire study.

Local dermal signs

No local dermal signs were recorded after treatment with the test item during the 14 days observation period.

Body weight

Slight body weight loss was observed in a single female animal between Day 0 and Day 7, but it was considered to be incidental and to have no toxicological relevance.

Necropsy

No external or internal macroscopic findings were noted at a dose level of 2000 mg/kg bw at necropsy.

 

Conclusion

The median lethal dose (LD50) of SH-0850 after a single dermal administration was found to be greater than 2000 mg/kg bw in male and female Crl:WI rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

K1

Additional information

Acute toxicity: oral

The single-dose oral toxicity of SH-0850 was performed according to the acute toxic class method.

Two groups of three female RccHan:WIST rats were treated with the test item at a dose level of 2000 mg/kg bw (Group 1 and Group 2).

Initially, three females (Group 1) were treated at a dose level of 2000 mg/kg bw SH-0850. The test item did not cause mortality in this group; therefore, a confirmatory group (Group 2) was treated at the same dose level. The test item did not cause mortality in the confirmatory group, so no further testing was required.

Results

Mortality: SH-0850 did not caused mortality at the dose level of 2000 mg/kg bw.

Clinical observations: Treatment with SH-0850 did not cause any test item related effect at a dose level of 2000 mg/kg bw.

Body weight and body weight gain: Body weight gains of SH-0850 treated animals during the study showed no indication of a treatment-related effect.

Macroscopic Findings: There was no evidence of the macroscopic observations at a dose level of 2000 mg/kg bw.

 

Conclusion: Under the conditions of this study, the acute oral LD50 value of the test item SH-0850 was found to be above 2000 mg/kg bw in female RccHan:WIST rats.

Acute toxicity: inhalation

The 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

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.

Acute toxicity: dermal

The purpose of the study was to assess the acute dermal toxicity of SH-0850 when administered to rats by a single semi-occlusive dermal application, followed by an observation period of 14 days. The test item was applied dermally to ten (5 males and 5 females) Crl:WI rats as supplied by the Sponsor. A limit test was carried out at 2000 mg/kg body weight (bw) in both sexes.

Clinical observations were performed on all animals at 1 and 5 hours after dosing and daily for 14 days thereafter. Body weight was measured prior to dosing on Day 0 and on Days 7 and 14. Rats were euthanized and subjected to a gross macroscopic examination at the end of the 2-week observation period (Day 14).

 

Results

Mortality: No mortality occurred after the 24-hour dermal exposure to SH-0850 in Crl:WI rats.

Systemic clinical signs: Each rat was symptom-free during the entire study.

Local dermal signs: No local dermal signs were recorded after treatment with the test item during the 14 days observation period.

Body weight: Slight body weight loss was observed in a single female animal between Day 0 and Day 7, but it was considered to be incidental and to have no toxicological relevance.

Necropsy: No external or internal macroscopic findings were noted at a dose level of 2000 mg/kg bw at necropsy.

The median lethal dose (LD50) of SH-0850 after a single dermal administration was found to be greater than 2000 mg/kg bw in male and female Crl:WI rats.