Reaction mass of [[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid, sodium salt and 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 30 July 2012 and 09 August 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), Testing Guidelines for Toxicology Studies, 12 NohSan No. 8147, amended 10 December 2002

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 4-(hydrogen phosphonatomethyl)-2-oxo-1,4,2λ⁵-oxazaphosphinan-2-olate hydrogen {[(hydrogen phosphonatomethyl)(2-hydroxyethyl)amino]methyl}phosphonate

EC Number:

939-513-8

Molecular formula:

C4H12NNaO7P2, C4H11NNa2O7P2 and C4H10NNa3O7P2

IUPAC Name:

tetrasodium 4-(hydrogen phosphonatomethyl)-2-oxo-1,4,2λ⁵-oxazaphosphinan-2-olate hydrogen {[(hydrogen phosphonatomethyl)(2-hydroxyethyl)amino]methyl}phosphonate

Test material form:

other: white paste

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Three male New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.17 to 2.64 kg and were twelve to twenty weeks old. After an acclimatisation period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Certified Rabbit diet) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
For the purpose of the study the test item was used as supplied.

A volume of 0.1 ml of the test item was placed into the conjunctival sac of the right eye.

Duration of treatment / exposure:

Single application for 72 hour exposure.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3

Details on study design:

MEASUREMENT OF PH:
The pH of the test item was determined prior to commencement of the study and found to be as follows:
10% w/w aqueous preparation of the test item: pH 4 immediately and after 10 minutes.

PROCEDURE:
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

Initially, a single rabbit was treated. A volume of 0.1 ml of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Appendix 1 (see attached background material: Appendix 1- Initial Pain Reaction).

After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

SCORING SYSTEM:
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical Draize Scale for Scoring Ocular Irritation (see attached background material for Appendix 2 - Draize Scale for Scoring Ocular Irritation).

Any other ocular effects were also noted.

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Other:
Any clinical signs of toxicity, if present, were also recorded.

Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

(Animal 72301)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

(Animal 72340)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

(Animal 72341)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

(Animal 72301)

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

(Animal 72340)

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

(Animal 72341)

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Results 7

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Remarks:

(Animal 72301)

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Remarks on result:

positive indication of irritation

Results 8

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #2

Remarks:

(Animal 72340)

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Results 9

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #3

Remarks:

(Animal 72341)

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Results 10

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

(Animal 72301)

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Results 11

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

(Animal 72340)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Remarks on result:

no indication of irritation

Results 12

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

(Animal 72341)

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.

No corneal or iridial effects were noted during the study.

Moderate conjunctival irritation was noted in all treated eyes one hour after treatment. Minimal conjunctival irritation was noted in all treated eyes at the 24 Hour observation and in one treated eye at the 48 Hour observation.

Two treated eyes appeared normal at the 48 Hour observation and one treated eye appeared normal at the 72 Hour observation.

Other effects:

Bodyweight:
Individual bodyweights and bodyweight changes are given in Table 3.
All animals showed expected gain in bodyweight during the study.

Any other information on results incl. tables

Table1              Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	72301Male				72340Male				72341Male
	IPR= 2				IPR = 1				IPR = 2
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	2	2	1	0	2	1	0	0	2	1	0	0
B = Chemosis	2	1	0	0	1	0	0	0	1	1	0	0
C = Discharge	1	0	0	0	1	0	0	0	1	0	0	0
Score (A + B + C) x 2	10	6	2	0	8	2	0	0	8	4	0	0
Total Score	10	6	2	0	8	2	0	0	8	4	0	0

IPR=  Initial pain reaction

Table 2              Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
72301Male	10	6	2	0
72340Male	8	2	0	0
72341Male	8	4	0	0
Group Total	26	12	2	0
Group Mean Score	8.7	4.0	0.7	0.0

Table 3              Individual Bodyweights and Bodyweight Changes

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
	Day 0	Day 3
72301Male	2.17	2.24	0.07
72340Male	2.49	2.55	0.06
72341Male	2.64	2.75	0.11

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The key in vivo eye irritation study for HEBMP-xNa, conducted according to OECD Test Guideline 405 and in compliance with GLP, concluded the test material to be not irritating to eyes.