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

Skin irritation / corrosion

Administrative data

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

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 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal 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

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.

Test system

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.

Results and discussion

In vivo

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Results 5

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

Results 6

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.

Any other information on results incl. tables

            

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

Applicant's summary and conclusion

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.