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

Endpoint summary

Description of key information

Skin irritation/corrosion - in vivo

SH-0850 is a "non-irritant".

Eye irritation - in vitro

The test item is not classified as a severe irritant and not classified as non-irritant. No conclusion of in vivo significance can be made from the adherence of the test item to the cornea, since in vivo eye lids may clear the surface, but abrasion may occur. It is concluded that an in vivo study is required for classification. 

Eye irritation - in vivo

SH-0850 does not require classification as an eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 February 2014 to 14 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Species and strain: New Zealand White rabbits
Source: S&K-LAP Kft. 2173 Kartal, Császár út 135, HUNGARY
Justification of strain: The New Zealand White albino rabbit is one of the standard strains used for acute irritation toxicity studies.
Animal health: Only animals in acceptable health condition were used for the test.
Number of animals: 3
Age of animals at treatment: 11 weeks old
Sex: Male
Body weight range at the
beginning of the life phase: 2720-2865 g
at the end of the life phase: 2761-2892 g
Date of receipt: 05 February 2014
Acclimation time: 6 days
Animal identification: The animals were identified by engraved ear tags. The cages were marked with individual identity cards with information about study code, sex, cage number, dose group and individual animal number.

Husbandry
Number of animal room: 620
Lighting periods: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature range during the study: 20 ± 3 °C
Relative humidity during the study: 24- 48%
Housing/Enrichment: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity values were measured continuously. The measured range was checked at least daily during the acclimatisation and experimental phases.

Food and Feeding
Animals received UNI diet for rabbits produced by AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi út, Hungary, ad libitum. Animals were provided with the following batches:
 0381 12 13, expiry date: 14 February 2014
 0391 12 13, expiry date: 19 March 2014
The details of the diets used will be archived with the raw data and are not reported.

Water Supply
The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system.

Water Analysis
The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The 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). Copies of the relevant Certificates of Analysis are retained in the archives at CiToxLAB Hungary Ltd.

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Single dose of 0.5 g of SH-0850, applied to the test area.

Duration of treatment / exposure:

Duration of exposure: 4 hours.

Observation period:

72 hours after patch removal.

Number of animals:

3 animsl

Details on study design:

Dosage
The test item was used as supplied, as a single dose of 0.5 g of SH-0850, applied to the test area. The untreated skin of each animal served as a control.

Application of the Test Item
Patch testing was used to detect primary irritating effects of the test item. Three male animals in acceptable health condition were selected for this test.
Approximately 24 hours prior to the test, the hair was clipped from the back and flanks of the animals. Removal of hair was performed in two steps. The majority of hair was clipped with an electronic hair clipper and the remaining hair was moistened with water and shaved with a razor.
The test item was applied to an approximately 6 cm² area of intact skin as follows:
• A single layer of a fine medical gauze (open-weave with large holes) of approximately 5x5 cm was placed over the application area,
• The appropriate amount of test item was carefully spread over the application area (the gauze helped maintain the test item in place),
• Three more layers of gauze were placed over the test item,
• These gauze patches were kept in contact with the skin by a patch of clear plastic with a surrounding adhesive hypoallergenic plaster to ensure continued good contact between the moistened test item and the shaved skin.
• The entire trunks of the animals were wrapped with plastic wrap for 4 hours.
• Medical elastic tubing was placed over the plastic to keep it in place.
An initial test was performed using one animal. One hour after application of the test item, the application site was examined. No severe irritation or corrosive effect was found in the initial test, therefore the bandage was replaced and the exposure continued for a further 3 hours (a total 4 hours exposure). Two additional animals were then included in the study.

Duration of Exposure
Duration of exposure: 4 hours. After the treatment period, the test item was removed with water at body temperature.

OBSERVATIONS AND SCORING
Clinical Observations
Animals were examined for signs of erythema and oedema, and the responses scored at 60 minutes and then at 24, 48 and 72 hours after patch removal.

Scoring and Assessment of Local Reactions
The dermal irritation scores were evaluated according to the scoring system by Draize (1959). The animals were observed for 72 hours and the duration of the study was sufficient to evaluate fully the reversibility or irreversibility of the effects observed.

Measurement of Body Weight
Body weights were recorded at the beginning and at the end of experiment.

Termination
At the end of the observation period, euthanasia of the animal was by intramuscular injections of CP-Ketamin 10 % and CP-Xylazin 2 % followed by i.v. RELEASE 300 mg/ml inj. A.U.V. anaesthesia (see details in 3.1.2.). Death was verified by checking pupil and cornea reflex, absence of respiration and pulse.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

At observation one, 24, 48 and 72 hours after patch removal, there were no observed clinical signs noted on the skin of the treated animals.
As no clinical signs were observed the study was terminated after the 72 hours observation.
The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema and oedema were 0.00, 0.00 and 0.00 respectively.

Other effects:

MORTALITY
There was no mortality observed during the study.

BODY WEIGHTS
There was no effect of treatment on body weight.

CLINICAL OBSERVATION
General Daily Examination
There were no treatment-related clinical signs noted.

SCORING OF ERYTHEMA FORMATION

Animal No./Sex	Body weight (g)		1 h	24 h	48 h	72 h
	At the beginning of the experiment	At the end of the experiment
00722/M	2720	2761	0	0	0	0
00723/M	2764	2789	0	0	0	0
00713/M	2865	2892	0	0	0	0
TOTAL	-	-	0	0	0	0

 

SCORING OF OEDEMA FORMATION

Animal No./Sex	Body weight (g)		1 h	24 h	48 h	72 h
	At the beginning of the experiment	At the end of the experiment
00722/M	2720	2761	0	0	0	0
00723/M	2764	2789	0	0	0	0
00713/M	2865	2892	0	0	0	0
TOTAL	-	-	0	0	0	0

 

M = male

h = hour

 

MEAN VALUES OF SKIN IRRITATION SCORES

(24, 48, 72 hours reading)

Animal Number	Sex	Erythema	Oedema
00722	Male	0.00	0.00
00723	Male	0.00	0.00
00713	Male	0.00	0.00

 

Interpretation of results:

GHS criteria not met

Conclusions:

According to Directive 2001/59/EC, SH-0850 does not require classification as a skin irritant.
According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, SH-0850 does not require classification as a skin irritant.
According to the classification system based on the scheme devised by Draize (1959), SH-0850 is a "non-irritant".

Executive summary:

An acute skin irritation study was performed with SH-0850 in New Zealand White rabbits. Parameters monitored during this study included mortality, body weight measurements and clinical observations. The irritancy of the test item was evaluated according to the Draize method (OECD No.: 404, 2002).

 

A weight of 0.5 g for solid test item was applied to the skin of the experimental animals. The test item was applied as a single dose. Sufficient water to damp the material was used to ensure good contact with the skin and an adhesive clear plastic patch was applied. The trunk was wrapped in clear plastic with medical tubing used to hold the patch in place. The untreated skin of each animal served as control.

 

After 4 hours, the remaining test item was removed with water at body temperature.

 

To assess skin irritation, animals were examined at 1, 24, 48 and 72 hours after the patch removal. Additional general examinations were performed daily.

 

There was no mortality or systemic clinical changes related to SH-0850 administration.

 

There was no effect of treatment on body weight.

 

At observation one, 24, 48 and 72 hours after patch removal, there were no observed clinical signs noted on the skin of the treated animals.

 

As no clinical signs were observed the study was terminated after the 72 hours observation.

 

The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema and oedema were 0.00, 0.00 and 0.00 respectively.

 

According to Directive 2001/59/EC, SH-0850 does not require classification as a skin irritant.

 

According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, SH-0850 does not require classification as a skin irritant.

 

According to the classification system based on the scheme devised by Draize (1959), SH-0850 is a "non-irritant".

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

OECD Guidelines for Testing of Chemicals 438 (26th July 2013)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

EU Commission Regulation (EC) No 1152/2010 (8th December 2010) amending, Regulation (EC) No 440/2008: Method B 48.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

OPPTS 870.2400 (EPA 712-C-98-195) August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

Strain of chicken: COBB 500
Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út. 129.
Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test item was applied in an amount of 30 mg
Positive control were treated with 30 mg powdered Imidazole.
Negative control was treated with 30μL of Saline (Salsol solution, NaCl 0.9% w/v).

Duration of treatment / exposure:

exposure period of 10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

One eye was treated with isotonic saline, three eyes with the test item and another three ones with Imidazole.

Details on study design:

Treatment
After the zero reference measurements, the eye (in its retainer) was removed from the chamber, and placed on a layer of tissue with the cornea facing upwards. The eyes were held in horizontal position, while the test item was applied onto the cornea. The test item was applied in an amount of 30 mg onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance, taking care not to damage or touch the cornea.
The positive control eyes were treated in a similar way with 30 mg powdered Imidazole. The negative control eye was treated with 30μL of Saline (Salsol solution, NaCl 0.9% w/v).
One eye was treated with isotonic saline, three eyes with the test item and another three ones with Imidazole.

Test item removal
The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL isotonic saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test item if possible.
The test item and the Imidazole were stuck on the corneas’ surface after the post-treatment rinse. Gentle rinsing with 20 mL saline was performed and the rate of saline-drops was increased at each observation time point.
The test item treated cornea surfaces were cleared 75 minutes (1/3) or 120 minutes (1/3) or 240 minutes (1/3) after the post-treatment rinse.
The Imidazole treated cornea surfaces were not cleared 240 minutes after the post-treatment rinse.

Observation and assessment of corneal effects
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

Irritation parameter:

percent corneal swelling

Run / experiment:

Test item up to 75 min

Value:

1.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0

Positive controls validity:

valid

Remarks:

2.7

Irritation parameter:

percent corneal swelling

Run / experiment:

Test item up to 240 min

Value:

1.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0

Positive controls validity:

valid

Remarks:

8.6

Irritation parameter:

cornea opacity score

Run / experiment:

Test item

Value:

0.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.00

Positive controls validity:

valid

Remarks:

3.83

Irritation parameter:

fluorescein retention score

Run / experiment:

Test item

Value:

0.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0

Positive controls validity:

valid

Remarks:

3.00

Other effects / acceptance of results:

Based on this in vitro eye irritation assay in the isolated chicken eyes test with SH-0850, the test item is not classified as a severe irritant and not classified as non-irritant. No conclusion of in vivo significance can be made from the adherence of the test item to the cornea, since in vivo eye lids may clear the surface, but abrasion may occur. It is concluded that an in vivo study is required for classification.

 Test Item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	1.4%	I
Mean maximum corneal swelling at up to 240 min	1.4%	I
Mean maximum corneal opacity	0.33	I
Mean fluorescein retention	0.33	I
Other observations	Test item was stuck on the cornea surface after the post-treatment rinse. The cornea surfaces were cleared 75 minutes (1/3) or 120 minutes (1/3) or 240 minutes (1/3) after the post-treatment rinse.
Overall ICE class	3xI

Based on this in vitro eye irritation assay in the isolated chicken eyes test with SH-0850, the test item is not classified as a severe irritant and not classified as non-irritant. No conclusion of in vivo significance can be made from the adherence of the test item to the cornea, since in vivo eye lids may clear the surface, but abrasion may occur. It is concluded that an in vivo study is required for classification.

 

Positive Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	2.7%	I
Mean maximum corneal swelling at up to 240 min	8.6%	II
Mean maximum corneal opacity	3.83	IV
Mean fluorescein retention	3.00	IV
Other observations	Imidazole stuck on the cornea surface after the post-treatment rinse. The cornea surfaces were not cleared 240 minutes after the post-treatment rinse.
Overall ICE class	1Xii 2Xiv

The positive control Imidazole was classified as severely irritating, UN GHS Classification: Category 1.

 

Negative Control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other observations	None
Overall ICE class	3xI

The negative control Saline (Salsol solution, NACl 0.9% w/v) was classified as non-irritating, UN GHS Classification: Non-classified.

 

Historical Control data (n=17, data from 2013):

 

Negative Control: Saline (Salsol solution, NaCl 0.9% w/v)

Observation	Min. Value	Max. Value
Maximum corneal swelling up to 75 min	0.0%	1.2%
Maximum corneal swelling up to 240 min	0.0%	1.2%
Maximum corneal opacity change	0.00	0.00
Fluorescein retention	0.00	0.00

Positive Control: Saline (Imidazole)

Observation	Min. Value	Max. Value
Maximum corneal swelling up to 75 min	1.1%	5.9%
Maximum corneal swelling up to 240 min	4.6%	10.6%
Maximum corneal opacity change	3.50	4.00
Fluorescein retention	2.00	3.00

 

Table of individual data (SH-0850)

Chamber number ↓	Corneal thickness (instrument units)										Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	Change	30	75	Max change up to 75	120	180	240	Max change up to 240	0	30	75	120	180	240	Max ∆ Opac	0	30	∆ Flu ret
1	74	73	-1.4%	74	73	1.4%	72	72	71	1.4%	0	0	0	0	0	0	0.0	0	0.5	0.5
2	74	74	0.0%	75	75	1.4%	73	71	71	1.4%	0	0	0	0	0	0.5	0.5	0	0.5	0.5
4	74	74	0.0%	75	75	1.4%	75	74	74	1.4%	0	0	0	0	0	0.5	0.5	0	0	0.0
Mean values:						1.4%				1.4%							0.33			0.33

Comment: The Test Item was stuck on the cornea surface after the post-treatment rinse. The cornea surfaces were cleared 75 minutes (1/3) or 120 minutes (1/3) or 240 minutes (1/3) after the post-treatment rinse.

 

Table of individual data (Imidazole)

Chamber number ↓	Corneal thickness (instrument units)										Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	Change	30	75	Max change up to 75	120	180	240	Max change up to 240	0	30	75	120	180	240	Max ∆ Opac	0	30	∆ Flu ret
5	74	74	0.0%	76	76	2.7%	78	80	80	8.1%	0.5	4	4	4	4	4	3.5	0	3	3.0
7	74	73	-1.4%	74	75	2.7%	76	78	79	8.2%	0	4	4	4	4	4	4.0	0	3	3.0
8	72	74	2.8%	75	76	2.7%	78	78	81	9.5%	0	4	4	4	4	4	4.0	0	3	3.0
Mean values:						2.7%				8.6%							3.83	3.00		0.33

Comment: The Imidzole was stuck on the cornea surface after the post-treatment rinse. The cornea surfaces was not cleared 240 minutes after the post-treatment rinse.

 

Table of individual data (Saline (Solsol solution, NaCl 0.9% w/v))

Chamber number ↓	Corneal thickness (instrument units)										Corneal opacity score							Fluorescein retention
Relative observation time (min) →	-45	0	Change	30	75	Max change up to 75	120	180	240	Max change up to 240	0	30	75	120	180	240	Max ∆ Opac	0	30	∆ Flu ret
9	75	73	-2.7%	73	73	0.0%	73	73	73	0.0%	0	0	0	0	0	0	0.0	0	0	0.0

 

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on this in vitro eye irritation in the isolated chicken eyes test with SH-0850, the test item is not classified as a severe irritant and not classified as non-irritant. No conclusion of in vivo significance can be made from the adherence of the test item to the cornea, since in vivo eye lids may clear the surface, but abrasion may occur. It is concluded that an in vivo study is required for classification.

Executive summary:

An in vitro eye irritation study of the test item SH-0850 was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No.: 438 (26th July 2013).

 

After the zero reference measurements, the eye was held in horizontal position and 30 mg of SH-0850 was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated with 30 mg Imidazole. The negative control eye was treated with 30 μL of Saline (Salsol solution, NaCl 0.9% w/v).

 

Based on this in vitro eye irritation in the isolated chicken eyes test with SH-0850, the test item is not classified as a severe irritant and not classified as non-irritant. No conclusion of in vivo significance can be made from the adherence of the test item to the cornea, since in vivo eye lids may clear the surface, but abrasion may occur. It is concluded that an in vivo study is required for classification. 

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 April 2014 to 13 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species and strain: New Zealand White rabbits
Source: S&K-LAP Kft. 2173 Kartal, Császár út 135, Hungary
Justification of strain: The New Zealand White rabbit is one of the standard strains used for acute irritation toxicity studies.
Animal health: Only animals in acceptable health condition were used for the test. Both eyes of each animal provisionally selected for testing were examined prior to starting the study. Animals showing eye irritation, ocular defects or pre-existing corneal injury were not used.
Number of animals: 3 animals
Age of animals at treatment: ~11 weeks old (adult)
Sex: Male
Body weight range on the day of treatment: 2737 g and 3100 g
before euthanasia: 2860 g and 3159 g
Date of receipt: 16 April, 04 June 2014
Acclimatization time: at least 6 days
Animal identification: The individual identification was by engraved ear tag. The cages were marked with individual identity cards with information about study code, sex, dose, cage number and individual animal number.

HUSBANDRY
Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
Number of animal room: 620, 619
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m. (and during of the analgesic/anaesthetic treatment)
Temperature during the study: 20 ± 3°C
Relative humidity during the study: 32 - 84 %
Housing/Enrichment: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity values were measured continuously. The measured range was checked at least daily during the acclimatisation and experimental phases.

FOOD AND FEEDING
Animals received UNI diet for rabbits produced by AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi út, Hungary, ad libitum. Animals were provided with the following batches:
 0410 03 14, expiry date: 03 June 2014
 0460 05 14, expiry date: 05 August 2014
The details of the diets used will be archived with the raw data and are not reported.

WATER SUPPLY AND QUALITY CONTROL OF WATER
The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system. The 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 of CiToxLAB Hungary Ltd.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

single dose of 0.1 g of test item

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

Three male animals in acceptable health condition were selected for the test.

Details on study design:

Identification of pH
The pH of the test item was determined. The pH was found to be 5.0, so the test item is permitted for use in animal studies.

Pre-study examination
Three male animals in acceptable health condition were selected for the test. Care was taken to select only those animals that had a normal eye condition and any with ocular lesions were rejected.

Chronology of animal use
Initially only one rabbit was treated with test item. As there was no severe effect was observed then one further rabbit was treated after 48 hour observation of the first rabbit. Then as the results in the second rabbit were not severe, a third rabbit was treated as for determined the classification.

Analgesic and anaesthetic treatment
Sixty minutes (60 ±10 min) prior to test substance application, a systemic opiate analgesic was administered subcutaneous injection (SC) under direct Veterinary supervision.
Five minutes (5 ±1.5 min) prior to test substance application, a topical ocular anaesthetic was applied to each eye (including the control eye to ensure direct comparison of any ocular observations).
Eight hours (8 to 9 hr) after test substance application, a systemic opiate analgesic and a nonsteroidal anti-inflammatory drug (NSAID) were administered subcutaneous injection (SC) under direct Veterinary supervision. The systemic opiate analgesic was injected ~12 hours, until the ocular lesions were resolved and no clinical signs of pain or distress were present.
Systemic opiate analgesic: Buprenorphine 0.01 mg/kg.
Topical ocular anaesthetic: Humacain (oxybuprocaine) one-two drops per eye.
Nonsteroidal anti-inflammatory drug: Meloxicam 0.5 mg/kg.

ADMINISTRATION OF THE TEST ITEM
Dosage
A single dose of 0.1 g of test item SH-0850 was administered to the animal.

Application of the Test Item
The test substance was placed in the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for at least one second in order to prevent loss of the material.
The untreated contralateral eye was served as the control.

Duration of Exposure
The treated eye was rinsed with physiological saline solution at the first observation time point in all animals at one hour after the application of test item as the irritation scores were greater than 1 and residual test item was noted on the eye.

OBSERVATIONS AND SCORING
Clinical Observations and Evaluation of Ocular Irritation
The eyes were examined at 1, 24, 48 and 72 hours after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. Any clinical signs of toxicity or signs of ill-health during the study were recorded. At the end of the observation period, the animal was sacrificed by intramuscular injections of CP-Ketamin 10% and CP-Xylazin 2% followed by i.v. Pentobarbital sodium. Death was verified by checking pupil and corneal reflex and the absence of respiration.
All rabbits were examined for distress at least twice daily, with observations at least 6 hours apart. Clinical observations or signs of ill-health were recorded.

Scoring and Assessment of Local Reaction
The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (02 October 2012).

Measurement of Body Weight
Individual body weight was recorded on the day of treatment and before euthanasia.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Remarks:

discharge

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Remarks:

discharge

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Remarks:

Discharge

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

The eyes were examined at 1, 24, 48 and 72 hours after the application.
Initial Pain Reaction (IPR/PR) was not observed.

First animal (No: 01000) clinical observation
At one hour after the application: Conjunctival redness (score 2), chemosis (score 2) and discharge (score 3) were found. Residual test item was noted on the eye.
At 24 hours after the application: Conjunctival redness (score 1), chemosis (score 1) and discharge (score 1) were found.
At 48 and 72 hours after the application There were no clinical signs, and no conjunctival or corneal effects observed. The animal was euthanized after the 72 hours observation.

Second animal (No: 00985) clinical observation
At one hour after the application: Conjunctival redness (score 2), chemosis (score 1) and discharge (score 3) were found. Residual test item was noted on the eye.
At 24 hours after the application: Conjunctival redness (score 1), chemosis (score 1) and discharge (score 1) were found.
At 48 and 72 hours after the application There were no clinical signs, and no conjunctival or corneal effects observed. The animal was euthanized after the 72 hours observation.

Third animal (No: 01647) clinical observation
At one hour after the application: Conjunctival redness (score 2), chemosis (score 2) and discharge (score 3) were found. Residual test item was noted on the eye.
At 24 hours after the application: Conjunctival redness (score 1) and discharge (score 1) were found.
At 48 and 72 hours after the application There were no clinical signs, and no conjunctival or corneal effects observed. The animal was euthanized after the 72 hours observation.

Other effects:

MORTALITY
There was no mortality observed during the study.

BODY WEIGHTS
The body weights of the animals were considered to be within the normal range of variability.

CLINICAL OBSERVATION
General daily examination
There were no clinical signs observed that could be related to treatment.

INDIVIDUAL SCORES FOR OCULAR IRRITATION

Abbreviations:R = Redness              OD = Opacity degree of density

                       CH = Chemosis                     OE = Extent of opaque area

                       D = Discharge            IPR/PR = Initial pain reaction

                       0 = Normal (in case of control eye and other lesions)

 

Animal No.: 01000

Time		Score of irritation							IPR/PR	Other sign
		Conjunctivae			Cornea		Iris	Control eye
		R	CH	D	OD	OE	R
Pre-treatment	0	0	0	0	0	0	0	0	0	0
Post-treatment (h = hour)	1 h	2	2	3	0	0	0	0	0	*
	24 h	1	1	1	0	0	0	0	0	0
	48 h	0	0	0	0	0	0	0	0	0
	72 h	0	0	0	0	0	0	0	0	0

*: Residual test item was noted on the eye

 

Animal No.: 00985

Time		Score of irritation							IPR/PR	Other sign
		Conjunctivae			Cornea		Iris	Control eye
		R	CH	D	OD	OE	R
Pre-treatment	0	0	0	0	0	0	0	0	0	0
Post-treatment (h = hour)	1 h	2	1	3	0	0	0	0	0	*
	24 h	1	1	1	0	0	0	0	0	0
	48 h	0	0	0	0	0	0	0	0	0
	72 h	0	0	0	0	0	0	0	0	0

*: Residual test item was noted on the eye

 

Animal No.: 01647

Time		Score of irritation							IPR/PR	Other sign
		Conjunctivae			Cornea		Iris	Control eye
		R	CH	D	OD	OE	R
Pre-treatment	0	0	0	0	0	0	0	0	0	0
Post-treatment (h = hour)	1 h	2	2	3	0	0	0	0	0	*
	24 h	1	0	1	0	0	0	0	0	0
	48 h	0	0	0	0	0	0	0	0	0
	72 h	0	0	0	0	0	0	0	0	0

 *: Residual test item was noted on the eye

 

MEAN VALUES OF EYE IRRITATION

(24, 48, 72 hour reading)

Animal Number	Sex	Cornea Opacity	Iris	Conjunctivae
				Redness	Chemosis	Discharge
01000	Male	0.00	0.00	0.33	0.33	0.33
00985	Male	0.00	0.00	0.33	0.33	0.33
01647	Male	0.00	0.00	0.33	0.00	0.33

 

BODY WEIGHT DATA

 

Animal No.: 01000

Date of measurement	Body weight (g)	Body weight gain (g)
Before measurement	2883	67
Before euthanasia	2950

 

Animal No.: 00985

Date of measurement	Body weight (g)	Body weight gain (g)
Before measurement	3100	59
Before euthanasia	3159

 

Animal No.: 01647

Date of measurement	Body weight (g)	Body weight gain (g)
Before measurement	2737	123
Before euthanasia	2860

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test item SH-0850, applied to rabbit eye mucosa, caused conjunctival effects in all animals. The effects were fully reversible within 48 hours.
According to Regulation (EC) No 1272/2008, SH-0850 does not require classification as an eye irritant.
According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, SH-0850 does not require classification as an eye irritant.

Executive summary:

An acute eye irritation study of the test item SH-0850 was performed in New Zealand White rabbits. The irritation effects of the test item were evaluated according to the Draize method (OECD No.: 405, 2012). Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Three animals were used to make the classification.

 

The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. An amount of 0.1 g of the test item was administered as a single dose.

 

The eyes were examined at 1, 24, 48 and 72 hours after the application.

 

Initial Pain Reaction (IPR/PR) was not observed.

 

First animal (No: 01000) clinical observation

At one hour after the application: Conjunctival redness (score 2), chemosis (score 2) and discharge (score 3) were found. Residual test item was noted on the eye.

At 24 hours after the application: Conjunctival redness (score 1), chemosis (score 1) and discharge (score 1) were found.

At 48 and 72 hours after the application There were no clinical signs, and no conjunctival or corneal effects observed. The animal was euthanized after the 72 hours observation.

 

Second animal (No: 00985) clinical observation

At one hour after the application: Conjunctival redness (score 2), chemosis (score 1) and discharge (score 3) were found. Residual test item was noted on the eye.

At 24 hours after the application: Conjunctival redness (score 1), chemosis (score 1) and discharge (score 1) were found. 

At 48 and 72 hours after the application There were no clinical signs, and no conjunctival or corneal effects observed. The animal was euthanized after the 72 hours observation.

 

Third animal (No: 01647) clinical observation

At one hour after the application: Conjunctival redness (score 2), chemosis (score 2) and discharge (score 3) were found. Residual test item was noted on the eye.

At 24 hours after the application: Conjunctival redness (score 1) and discharge (score 1) were found.

At 48 and 72 hours after the application There were no clinical signs, and no conjunctival or corneal effects observed. The animal was euthanized after the 72 hours observation.

 

There was no clinical sign of systemic toxicity observed in the animals during the study and no mortality occurred.

 

During the study, the control eye of each animal was symptom-free.

 

The body weights of the animal were considered to be within the normal range of variability.

 

The animal individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

chemosis: 0.33, 0.33, 0.00

discharge: 0.33, 0.33, 0.33

redness: 0.33, 0.33, 0.33

cornea opacity: 0.00, 0.00, 0.00

iris: 0.00, 0.00, 0.00

 

The test item SH-0850, applied to rabbit eye mucosa, caused conjunctival effects in all animals. The effects were fully reversible within 48 hours.

 

According to Regulation (EC) No 1272/2008, SH-0850 does not require classification as an eye irritant.

 

According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, SH-0850 does not require classification as an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion - in vivo

An acute skin irritation study was performed with SH-0850 in New Zealand White rabbits.

A weight of 0.5 g for solid test item was applied to the skin of the experimental animals. The test item was applied as a single dose. Sufficient water to damp the material was used to ensure good contact with the skin and an adhesive clear plastic patch was applied. The trunk was wrapped in clear plastic with medical tubing used to hold the patch in place. The untreated skin of each animal served as control.

After 4 hours, the remaining test item was removed with water at body temperature.

To assess skin irritation, animals were examined at 1, 24, 48 and 72 hours after the patch removal. Additional general examinations were performed daily.

 

There was no mortality or systemic clinical changes related to SH-0850 administration. There was no effect of treatment on body weight.

At observation one, 24, 48 and 72 hours after patch removal, there were no observed clinical signs noted on the skin of the treated animals. As no clinical signs were observed the study was terminated after the 72 hours observation.

The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema and oedema were 0.00, 0.00 and 0.00 respectively.

SH-0850 is a "non-irritant".

Eye irritation - in vitro

An in vitro eye irritation study of the test item SH-0850 was performed in isolated chicken’s eyes.

After the zero reference measurements, the eye was held in horizontal position and 30 mg of SH-0850 was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated with 30 mg Imidazole. The negative control eye was treated with 30 μL of Saline (Salsol solution, NaCl 0.9% w/v).

Based on the in vitro eye irritation in the isolated chicken eyes test with SH-0850, the test item is not classified as a severe irritant and not classified as non-irritant. No conclusion of in vivo significance can be made from the adherence of the test item to the cornea, since in vivo eye lids may clear the surface, but abrasion may occur. It is concluded that an in vivo study is required for classification. 

Eye irritation - in vivo

An acute eye irritation study of the test item SH-0850 was performed in New Zealand White rabbits. The irritation effects of the test item were evaluated according to the Draize method. Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Three animals were used to make the classification. 

The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. An amount of 0.1 g of the test item was administered as a single dose.

The eyes were examined at 1, 24, 48 and 72 hours after the application.

 

Initial Pain Reaction (IPR/PR) was not observed.

There was no clinical sign of systemic toxicity observed in the animals during the study and no mortality occurred.

During the study, the control eye of each animal was symptom-free. 

The body weights of the animal were considered to be within the normal range of variability.

 

The animal individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

chemosis: 0.33, 0.33, 0.00

discharge: 0.33, 0.33, 0.33

redness: 0.33, 0.33, 0.33

cornea opacity: 0.00, 0.00, 0.00

iris: 0.00, 0.00, 0.00

The test item SH-0850, applied to rabbit eye mucosa, caused conjunctival effects in all animals. The effects were fully reversible within 48 hours.

Justification for classification or non-classification

Skin irritation/corrosion

According to Directive 2001/59/EC, SH-0850 does not require classification as a skin irritant. 

According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, SH-0850 does not require classification as a skin irritant.

According to the classification system based on the scheme devised by Draize (1959), SH-0850 is a "non-irritant".

Eye irritation

According to Regulation (EC) No 1272/2008, SH-0850 does not require classification as an eye irritant.

According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, SH-0850 does not require classification as an eye irritant.