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

Endpoint summary

Description of key information

The key in vivo skin irritation study for HEBMP-xNa, conducted according to OECD Test Guideline 404 and in compliance with GLP, concluded that the test material was not irritating to skin (Harlan Laboratories, 2012c). 

The key in vivo eye irritation study for HEBMP-xNa, conducted according to OECD Test Guideline 405 and in compliance with GLP, concluded that the test material was not irritating to eyes (Harlan Laboratories, 2012d). 

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:

The study was performed between 22 May 2012 and 25 May 2012

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:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

no

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

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Three 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 2.62 to 2.80 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.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

For the purpose of the study the test item was used as supplied.
At each test site a quantity of 0.5 ml of the test item was introduced.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3

Details on study design:

PROCEDURE:

On the day before the test each of a group of three rabbits was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.

On the day of the test a suitable test site was selected on the back of each rabbit. At each test site a quantity of 0.5 ml of the test item was introduced under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period.

Four hours after application the corset and patches were removed from each animal and any residual test item removed by gentle swabbing with cotton wool soaked in distilled water.

SCORING SYSTEM:
Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the following scale:

EVALUATION OF SKIN REACTIONS
Erythema and Eschar Formation
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema.: 4

Oedema Formation
 No oedema: 0
Very slight oedema (barely perceptible): 1
Slight oedema (edges of area well-defined by definite raising): 2
Moderate oedema (raised approximately 1 millimetre): 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure): 4

Any other skin reactions and 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.

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

(animal - 72039 male)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

(Animal - 72040 Male)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

( Animal - 72041 male)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

(animal - 72039 male)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

(Animal - 72040 male)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

(Animal - 72041 male)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The individual scores for erythema/eschar and oedema are given in Table 1.
No evidence of skin irritation was noted during the study.

Other effects:

Individual bodyweights and bodyweight changes are given in Table 2.
All animals showed expected gain in bodyweight during the study.

            

Skin Reactions

The individual and individual mean scores for erythema and oedema required for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals were as follows:

Skin Reaction	Reading (Hours)	Individual Scores – Rabbit Number and Sex
		72039Male	72040Male	72041Male
Erythema/Eschar Formation	24	0	0	0
	48	0	0	0
	72	0	0	0
Total		0	0	0
Mean Score		0.0	0.0	0.0
Oedema Formation	24	0	0	0
	48	0	0	0
	72	0	0	0
Total		0	0	0
Mean Score		0.0	0.0	0.0

Table 1              Individual Skin Reactions

Skin Reaction	Observation Time (following patch removal)	Individual Scores – Rabbit Number and Sex			Total
		72039Male	72040Male	72041Male
Erythema/Eschar Formation	Immediately	0	0	0	(0 )
	1 Hour	0	0	0	( 0 )
	24 Hours	0	0	0	0
	48 Hours	0	0	0	( 0 )
	72 Hours	0	0	0	0
Oedema Formation	Immediately	0	0	0	( 0 )
	1 Hour	0	0	0	( 0 )
	24 Hours	0	0	0	0
	48 Hours	0	0	0	( 0 )
	72 Hours	0	0	0	0
Sum of 24 and 72‑Hour(S)    :          0
Primary Irritation Index (S/6)                  :          0/6 = 0.0
Classification                                       :          NON‑IRRITANT

(   ) = Total values not used for calculation of primary irritation index

Table 2              Individual Bodyweights and Bodyweight Changes

Rabbit Number and Sex	Individual Bodyweight (kg)		Bodyweight Change (kg)
	Day 0	Day 3
72039Male	2.62	2.67	0.05
72040Male	2.77	2.96	0.19
72041Male	2.80	2.89	0.09

Interpretation of results:

GHS criteria not met

Conclusions:

The key in vivo skin irritation study for HEBMP-xNa, conducted according to OECD Test Guideline 404 and in compliance with GLP, concluded the test material to be not irritating to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

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

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

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.

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

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

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

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

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

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

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

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

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

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

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

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The key in vivo skin irritation study for HEBMP-xNa, conducted according to OECD Test Guideline 404 and in compliance with GLP, concluded that the test material was not irritating to skin (Harlan Laboratories, 2012c). In the study, 0.5 ml of the undiluted test item were applied onto the skin of 3 rabbits and kept in contact for 4-hours under semi-occlusive dressing. At the end of the exposure period, the corset and patches were removed from each animal and any residual test item removed by gentle swabbing with cotton wool soaked in distilled water. Erythema and oedema skin reactions were assessed at 24, 48 and 72 hours after test item removal. Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period. No evidence of skin irritation was noted during the study. All animals showed expected gain in bodyweight during the study.

The key in vivo eye irritation study for HEBMP-xNa, conducted according to OECD Test Guideline 405 and in compliance with GLP, concluded that the test material was not irritating to eyes (Harlan Laboratories, 2012d). In the study, a volume of 0.1 ml of the test item was placed into the conjunctival sac of the right eye of 3 rabbits, 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. Eye irritation was assessed at 24, 48 and 72 hours post-instillation. Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period. 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. All animals showed expected gain in bodyweight during the study.

Justification for classification or non-classification

Based on the available information, no classification is required for skin or eye irritation of HEBMP-xNa according to Regulation (EC) No 1272/2008.