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

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test guideline 3

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

835-272-7

Cas Number:

256374-76-2

Molecular formula:

C12H26N2O6P2

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

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

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

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

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

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

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

Specific details on test material used for the study:

No further details specified in the study report.

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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.

Any other information on results incl. tables

 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

 

Applicant's summary and conclusion

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.