Disodium dihydrogenpyrophosphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Remarks:

Study was performed prior to the update of the REACH Regulation and prior to the validation of in vitro test methods

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 27 April 2010 and 26 May 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Principles of method if other than guideline:

.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 15 September 2009 Date of Signature on GLP certificate: 26 November 2009

Test material

Test material information

Constituent 1

Reference substance name:

Disodium dihydrogenpyrophosphate

EC Number:

231-835-0

EC Name:

Disodium dihydrogenpyrophosphate

Cas Number:

7758-16-9

Molecular formula:

H4O7P2.2Na

IUPAC Name:

Sodium acid pyrophosphate

Details on test material:

Sponsor's identification: Disodium dihydrogenpyrophosphate
Description : white solid
Batch number : 447/09
Date received : 18 September 2009
Expiry date : 31 August 2011
Storage conditions: room temperature in the dark over silica gel

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source:
Harlan Laboratories U.K. Ltd., Loughborough, UK

- Age at study initiation:
Twelve to twenty weeks old

- Weight at study initiation:
2.0 to 3.5 kg

- Housing:
The animals were individually housed in suspended cages. 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.

- Diet (e.g. ad libitum):
ad libitum (2030 Teklad Global Rabbit diet supplied by Harlan Laboratories U.K. Ltd., Oxon, UK)

- Water (e.g. ad libitum):
ad libitum.

- Acclimation period:
At least five days


ENVIRONMENTAL CONDITIONS

- Temperature (°C):
17 to 23°C

- Humidity (%):
30 to 70%

- Air changes (per hr):
At least fifteen changes per hour

- Photoperiod (hrs dark / hrs light):
Twelve hours continuous light (06:00 to 18:00) and twelve hours darkness


IN-LIFE DATES:
From: day 1 To:day 3

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye remained untreated and was used for control purposes.

Amount / concentration applied:

TEST MATERIAL

- Amount(s) applied (volume or weight with unit):
A volume of 0.1 ml of the test material was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball.

- Concentration:
Used as supplied

VEHICLE

- Amount(s) applied:
Not applicable

- Concentration:
Not applicable

- Lot/batch no.:
Not applicable

- Purity:
Not reported

Duration of treatment / exposure:

up to 1 hour

Observation period (in vivo):

Approximately 1 hour and 24, 48 and 72 hours following treatment. Additional observations were made on Days 7 and 14 to assess the reversibility of the ocular effects.

Number of animals or in vitro replicates:

2 animals were tested in total. (After consideration of the ocular responses produced in the first treated animal, one additional animal was treated. )

Details on study design:

REMOVAL OF TEST SUBSTANCE

- Washing:
No

- Time after start of exposure:
Not applicable


SCORING SYSTEM:
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation given in Appendix 2, (from Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49). Additional observations were made on Days 7 and 14 to assess the reversibility of the ocular effects.


TOOL USED TO ASSESS SCORE:

Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal: 69148 Male

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks:

7 days

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal: 69202 Male

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 days

Results 3

Irritation parameter:

iris score

Basis:

animal: 69148 Male

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Results 4

Irritation parameter:

iris score

Basis:

animal: 69202 Male

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Results 5

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 69148 Male

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Small areas of haemorrhage scattered over the upper and lower conjunctival membrane and nictitating membrane

Results 6

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 69202 Male

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 14 days

Results 7

Irritation parameter:

chemosis score

Basis:

animal: 69148 Male

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Results 8

Irritation parameter:

chemosis score

Basis:

animal: 69202 Male

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritant / corrosive response data:

Ocular Reactions
Individual and group mean scores for ocular irritation are given in Table 1 and Table 2.
Scattered or diffuse corneal opacity was noted in both treated eyes at the 24, 48 and 72 Hour observations and in one treated eye at the 7-Day observation.
Iridial inflammation was noted in both treated eyes one hour after treatment and at the 24, 48 and 72-Hour observations.
Moderate conjunctival irritation was noted in both treated eyes one hour after treatment and at the 24, 48 and 72-Hour observations. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 7 Day observation.
Small areas of haemorrhage scattered over the upper and lower conjunctival membrane and nictitating membrane were noted in one treated eye at the 24, 48 and 72 Hour observations.
Both treated eyes appeared normal at the 14-Day observation.

Other effects:

Body weight
Both animals showed expected gain in bodyweight during the study.

Any other information on results incl. tables

Interpretation of Results

The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:

Score for conjunctivae  =         (A + B + C) x 2
Score for iris                            =         D x 5
Score for cornea                     =         (E x F) x 5

Using the numerical data obtained a modified version of the system ( Modified Kay and Calandra Interpretation of Eye Irritation Test was used to classify the ocular irritancy potential of the test material. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test material.

If evidence of irreversible ocular damage is noted, the test material will be classified as corrosive to the eye.

Table 1              Individua lScores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	69148 Male						69202 Male
	IPR= 3						IPR = 0+
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	14 Days	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	14 Days
CORNEA
E = Degree of Opacity	0	1	1	1	0	0	0	1	1	1	1	0
F = Area of Cornea Involved	0	4	4	4	0	0	0	4	4	4	2	0
Score (E x F) x 5	0	20	20	20	0	0	0	20	20	20	10	0
IRIS
D	1	1	1	1	0	0	1	1	1	1	0	0
Score (D x 5)	5	5	5	5	0	0	5	5	5	5	0	0
CONJUNCTIVAE
A = Redness	2	2H	2H	2H	1	0	2	2	2	2	2	0
B = Chemosis	2	2	2	2	1	0	2	2	2	3	1	0
C = Discharge	2	3	3	2	0	0	2	3	3	3	1	0
Score (A + B + C) x 2	12	14	14	12	4	0	12	14	14	16	8	0
Total Score	17	39	39	37	4	0	17	39	39	41	18	0

IPR=  Initial pain reaction

+=     One drop of local anaesthetic instilled into both eyes 1 to 2 minutes before treatment

H =      Small areas of haemorrhage scattered over the upper and lower conjunctival membrane and nictitating membrane

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	7 Days	14 Days
69148Male	17	39	39	37	4	0
69202Male	17	39	39	41	18	0
Group Total	34	78	78	78	22	0
Group Mean Score	17.0	39.0	39.0	39.0	11.0	0

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The test material produced a maximum group mean score of 39.0 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The data are sufficient to classify the material as irritating to eyes (category 2), in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Executive summary:

Introduction. The study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit. The method was designed to meet the requirements of the following:

 OECD Guidelines for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 24 April 2002)

 Method B5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No. 440/2008

Result. A single application of the test material to the non-irrigated eye of two rabbits produced scattered or diffuse corneal opacity, iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 14-Day observation.

Conclusion. The test material produced a maximum group mean score of 39.0 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.