Diphosphoric acid, copper salt

Endpoint summary

Description of key information

Skin: not corrosive in vitro (OECD 431, GLP); irritating in vitro (OECD 439, GLP); not irritating in vivo (OECD 404, GLP)
Eye: not corrosive ex vivo (OECD 437, GLP); irritating in vivo (OECD 405, GLP)
Respiratory tract: no study available

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-07-07 - 2014-08-02

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): diphosphoric acid, copper salt
- Physical state: light blue, solid powder
- Analytical purity: > 80% (Cu content > 34%)
- Impurities (identity and concentrations): water < 18%, free orthophosphate < 2%
- Lot/batch No.: 9000025486
- Storage condition of test material: 15-25 °C
- Sterility: unsterile

Species:

rabbit

Strain:

other: Zika

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kaninchenbetrieb Kock, Warnkenhagen, Germany
- Age at study initiation: 14-15 weeks
- Weight at study initiation: 3.0 - 3.1 kg
- Housing: Test animals were hold single caged
- Diet: conventional laboratory diet (a half-and-half blend of "Holstenstolz Kaninchenverbrauchsfutter 2, Type038" [Wilhelm Stroeh jr. GmbH & Co. KG, Germany] and "Rabbit maintenance, MuesliMash", [ssniff Spezialdiäten GmbH, Germany], ad libitum
- Water: tap water (drinking quality) ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

other: moistened with 500 µL water

Controls:

other: untreated skin areals of the same animal served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g

Duration of treatment / exposure:

Animal 1: 3 min, 1 and 4 h (on different patches, respectively)
Animals 2 and 3: 4 h

Observation period:

Up to 8 days
Scoring time points:
Animal 1: 3 min, 1 and 4 h immediately after exposure
Animals 1-3: 1, 24, 48 and 72 h after patch removal

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 6 cm²
- Type of wrap if used: gauze patch

REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test substance was removed with water
- Time after start of exposure: 4 h

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicabe

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicabe

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicabe

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicabe

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicabe

Irritant / corrosive response data:

There were no erythema and edema observed at any given timepoint.

Other effects:

Pretest with one animal was performed to determine the corrosive properties of the test material. Therefore the exposure was terminated after 3 minutes, 1 and 4 hours and scored, respectively. Every scored timepoint for erythema and edema was 0.

No further local or systemic effects were observed during the study period.
No effects on body weight (gain) were noted.

Table 1: Scoring of skin lesions

 

Skin reaction	Observation time (following patch removal after 4 h exposure, if not otherwise indicated)	Individual scores – Rabbit number and sex
		2014-55 female	2013-63 female	2013-64 female
Erythema/Eschar formation	Immediately after 3 minutes exposure	0	-	-
	Immediately after 1 hour exposure	0	-	-
	Immedieately after 4 hours exposure	0	-	-
	1 hr	0	0	0
	24 hrs	0	0	0
	48 hrs	0	0	0
	72 hrs	0	0	0
	Mean score (24-72 hrs)	0	0	0
Oedema formation	Immediately after 3 minutes exposure	0	-	-
	Immediately after 1 hour exposure	0	-	-
	Immediately after 4 hours exposure	0	-	-
	1 hr	0	0	0
	24 hrs	0	0	0
	48 hrs	0	0	0
	72 hrs	0	0	0
	Mean score (24-72 hrs)	0	0	0

Table 2: Bodyweight and weight gain in kilograms

Rabbit number and sex	Individual bodyweight (kg)		Bodyweight change (kg)
	Day 0	Day 3 and 8
2014-55 female	3.02	3.19	+ 0.17
2014-63 female	3.00	3.06	+ 0.06
2014-64 female	3.10	3.17	+ 0.07

Interpretation of results:

GHS criteria not met

Conclusions:

The test material caused under the conditions of the test of "Acute dermal irritation/corrosion" (OECD 404) no symptoms of irritation/corrosion.

Executive summary:

The test material caused under the conditions of the test of "Acute dermal irritation/corrosion" (OECD 404) no symptoms of irritation/corrosion.

The individual mean values of the erythema/eschar and edema from gradings at 24, 48 and 72 hours were 0 in all three animals. Therefore, the test material is "not classified" according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-11-06 to 2012-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP Inspection: 10 July 2012 Date of Signature on Certificate: 30 November 2012

Specific details on test material used for the study:

- Name of test material (as cited in study report): Copper (II)-pyrophosphate
- Physical description: pale blue powder
- Analytical purity: >80% (based on Cu content >34%)
- Impurities (identity and concentrations): <18% water, <2% free orthophosphate, pyrophosphate
- Purity test date: 19 September 2012
- Lot/batch No.: MV500
- Date Received: 24 September 2012
- Expiration date of the lot/batch: 12 February 2015
- Storage condition of test material: room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

other: reconstructed human epidermis model, not further detailed

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SITE
- 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. Approximately 10 mg of the test item was applied to the epidermis surface (26.3 mg/cm2). The epidermis surface had previously been moistened with 5 µL of sterile distilled water to improve contact between the solid test item and the epidermis.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tisses were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

- Time after start of exposure: 15 minutes

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was used as supplied.

Amount(s) applied (volume or weight with unit):
Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test item and the epidermis.

Duration of treatment / exposure:

15 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

day 6

Value:

21.2

Direct MTT Reduction

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Test Item, Positive Control Item and Negative Control Item

The mean OD₅₄₀ values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1. The relative mean viability of the test item treated tissues was 21.2% after a 15-minute exposure period.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 5.4% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 2.2%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₄₀ for the negative control treated tissues was 0.697 and the standard deviation value of the percentage viability was 8.2%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 0.3%. The test item acceptance criterion was therefore satisfied.

Table 1: Mean OD₅₄₀ Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD540of tissues	Mean OD540of triplicate tissues	± SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative Mean Viability (%)
Negative Control Item	0.728	0.697	0.057	10.4	100*	8.2
	0.631			90.5
	0.732			105.0
Positive Control Item	0.032	0.038	0.015	4.6	5.4	2.2
	0.055			7.9
	0.026			3.7
Test Item	0.148	0.148	0.002	21.2	21.2	0.3
	0.149			21.4
	0.146			20.9

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item was concluded as irritant to the skin.

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-hour post-exposure incubation period is also determined for test items which are found to be borderline nonirritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result. This method was designed to be compatible with the following:

 OECD Guideline for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010)

 Method B.46 of Commission Regulation (EC) No. 440/2008

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 21.2% after the 15-Minute exposure period.

Quality Criteria

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was classified as irritant to the skin. The following classification criteria were met:

EU DSD (Council Directive 67/548/EEC) requires symbol “Xi” risk phrase R38 “Irritating to Skin”.

EU CLP Regulation (EC) No 1272/2008 and UN GHS Hazard statement H315 “Causes Skin Irritation” Category 2.

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-07-27 to 2010-07-29.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 19-21 July 2011, Date of signature 31 August 2011.

Specific details on test material used for the study:

Sponsor's identification: Copper (II) Pyrophosphate
Description: pale blue powder
Batch number: A
Date received: 28 August 2009
Expiry date: not supplied
Storage conditions: room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

other: reconstituted human epidermis model

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST MATERIAL

- The test item was applied neat.

- Amount(s) applied (volume or weight with unit):
20 mg of the solid test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. 100 µl of 0.9% w/v sodium chloride solution was added for wetting of the test item.


TEST SITE
- Area of exposure:
20 mg of the solid test material was applied. 100 µl of 0.9 % w/v sodium chloride solution was added for wetting of the test material.

- % coverage:
The test item was applied topically to the corresponding tissues ensuring uniform covering.

- Type of wrap if used:
None used

REMOVAL OF TEST ITEM
- Washing (if done):
At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item.

- Time after start of exposure:
3, 60 or 240 minutes post exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-minute treatments, compared to the mean of the negative control tissues (n=2) treated with 0.9 % w/v sodium chloride solution. The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of post-treatment incubation (if applicable):

3, 60 & 240 minutes post exposure incubation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure period: 240 minutes

Value:

93.8

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure period: 60 minutes

Value:

119.9

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure period: 3 minutes

Value:

110.6

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability for the positive control treated tissues was 3.1% relative to the negative control treated tissues following the 240-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

RESULTS

The qualitative evaluation of tissue viability is given in Table 2.

Following the 3, 60 and 240-Minute exposure periods the test material treated tissues appeared blue which was considered to be indicative of viable tissue.

 

Table1 :Mean OD₅₄₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

 

Item	Exposure period	Mean OD540of duplicate tissues	Relative mean viability (%)
Negative control**	240 minutes	0.161	100*
Positive control**	240 minutes	0.005	3.1
Test substance	240 minutes	0.151	93.8
	60 minutes	0.193	119.9
	3 minutes	0.178	110.6

 

*=    The mean viability of the negative control tissues is set at 100

** = Control group shared with Harlan Laboratories Ltd Project numbers 2920/0150, 2920/0151, 2920/0152, 2920/0154 and 2920/0155

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

The test item was considered to be non-corrosive to the skin.

In accordance with the testing strategy detailed in Annex VIII, column 1 of Regulation (EC) No. 1907/2006 the assessment of the endpoint ‘skin irritation or skin corrosion’  has been performed following the consecutive steps detailed in the Regulation. As such an in vitro skin corrosion study has been performed. This study is not considered as the key study because it is not sufficient for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

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:

weight of evidence

Study period:

2013-01-14 to 2013-02-04

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Sponsor's identification: IP 37: Copper (II)-pyrophosphate
Description: pale blue powder
Batch number: MV500
Purity: >80% (based on Cu content >34%)
Impurities: <18% water, <2% free orthophosphate, pyrophosphate
Date received: 24 September 2012
Expiry date: 12 February 2015
Storage conditions: room temperature in the dark

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK.
- Age at study initiation: 12-20 weeks
- Weight at study initiation: 2.48 or 2.59 kg
- Housing: animals were individually housed in suspended cages.
- Diet (e.g. ad libitum): ad libitum (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least 15 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml of the test item (weighing approximately 83 mg as measured by gently compacting the required volume into an adapted syringe).

Duration of treatment / exposure:

Animals were observed for 7 days post-dosing.

Observation period (in vivo):

7 Days

Number of animals or in vitro replicates:

2

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

The test material 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.

After consideration of the ocular responses produced in the first treated animal, a second animal was treated. In order to minimise pain on application of the test item, one drop of local anaesthetic (Tetracaine hydrochloride 0.5%, Bausch & Lomb, Surrey, UK) was instilled into both eyes of the second animal 1 to 2 minutes before treatment.

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).

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

ADDITIONAL OBSERVATIONS:
Any clinical signs of toxicity, if present, were also recorded.
An additional observation was made on Day 7 to assess the reversibility of the ocular effects.
Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Irritation parameter:

cornea opacity score

Remarks:

opacity

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable as no effects noted

Remarks on result:

other: IPR = 3

Irritation parameter:

cornea opacity score

Remarks:

opacity

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable as no effects noted

Remarks on result:

other: IPR = 0 (anaesthetic administered)

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.66

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Remarks:

(conjunctivae)

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Remarks:

(conjunctivae)

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Individual scores for ocular irritation are given in Table 1. The individual and mean scores as required for EU and GHS classification and labelling are presented in Table 2.
No corneal effects were noted during the study.
Iridial inflammation was noted in both treated eyes one hour after treatment and at the 24 and 48-Hour observations and in one treated eye at the 72-Hour observation.
Moderate conjunctival irritation was noted in both treated eyes one hour after treatment and at the 24 and 48-Hour observations. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye 72-Hour observation.
Both treated eyes appeared normal at the 7-Day observation.

Other effects:

Individual bodyweights and bodyweight change are given in Table 3.
Both animals showed expected gain in bodyweight during the study.

Table 1. Individual Scores for Ocular Irritation

Rabbit number and Sex	72877 Male					72918 Male
	IPR = 3					IPR = 0+
Time after treatment	1 Hrs	24 Hrs	48 Hrs	72 Hrs	7 days	1 Hrs	24 Hrs	48 Hrs	72 Hrs	7 days
Cornea Degree of opacity	0	0	0	0	0	0	0	0	0	0
Area of cornea involved	0	0	0	0	0	0	0	0	0	0
Iris	1	1	1	0	0	1	1	1	1	0
Conjuctivae Redness	2	2	2	2	0	2	2	2	2	0
Chemosis	3	2	2	1	0	2	2	2	2	0
Discharge	1	2	2	0	0	3	1	1	1	0

 

IPR = Initial pain reaction

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

Table 2. Individual and Mean Scores for Cornea, Iris and Conjunctivae

Rabbit number and sex	Time after treatment	Corneal opacity	Iridial inflammation	Conjuntival redness	Conjunctival chemosis
72877 Male	24 hours 48 hours 72 hours	0 0 0	1 1 0	2 2 2	2 2 1
Mean:		0.0	0.7	2.0	1.7
72918 Male	24 hours 48 hours 72 hours	0 0 0	1 1 1	2 2 2	2 2 2
Mean:		0.0	1.0	2.0	2.0

 

 

Table 3. Individual Bodyweights and Bodyweight Change

Rabbit number and sex	Individual bodyweight (kg)		Bodyweight change (kg)
	Day 0	Day 7
72877 Male	2.48	2.60	0.12
72918 Male	2.59	2.75	0.16

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The test item was classified as irritating to eyes (Category 2) according to Regulation (EC) No 1272/2008.

Executive summary:

The test item was classified as irritating to eyes (Category 2) according to Regulation (EC) No 1272/2008. It is reasonable to assume that the signal word “Warning” and the Hazard Statement “H319: Causes serious eye irritation” are therefore required. This study is considered to be adequate and reliable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP) and as a key study in accordance with Regulation (EC) No. 1907/2006 (REACH).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

A weight of evidence approach was used to evaluate the skin irritation properties of the test substance.

The corrosivity potential of copper pyrophosphate was tested using the EPISKIN™ in vitro Reconstituted Human Epidermis (RHE) Model following OECD Guideline 431 and complying with GLP (Warren, 2011). Duplicate tissues were treated with 20 mg of the test material for 3, 60 and 240 min. Duplicate tissues treated for 240 min with 50 µL 0.9% w/v sodium chloride or glacial acetic acid served as negative and positive controls, respectively. At the end of the exposure period, tissues were rinsed prior to MTT loading. Formazan crystals were extracted from the MTT loaded tissues by acidic isopropanol extraction. The optical density of the extracts was measured at 540 nm. The relative mean viability (MTT reduction to formazan in treated vs. negative control tissues) was calculated as percent mean optical density of the isopropanol extracts from treated tissues relative to the negative control.

The relative mean viability of tissues treated with the test material was 110.6, 119.9 and 93.8% after 3, 60 and 240 min, respectively. The relative mean viability of the positive control treated tissues was 3.1% after 240 min.

Based on the study results, copper pyrophosphate is considered to be non-corrosive to skin.

In another GLP-compliant in vitro study, the skin irritation potential of copper pyrophosphate was evaluated using the EPISKIN™ RHE Model according to OECD Guideline 439 and EU Method B.46 (Warren, 2014). Triplicate tissues were treated with 10 mg of the test substance for 15 min, followed by rinsing and a 42 h post-exposure incubation period. Triplicate tissues concurrently treated with 10 µL of Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ or Sodium Dodecyl Sulphate (SDS) 5% w/v served as negative and positive controls, respectively. Following the post-exposure period, MTT tissue loading and determination of relative mean viability was performed as described above.

The relative mean viability of the test substance-treated tissues was 21.2% of the negative control value, while the relative mean tissue viability of the positive control was 5.4%.

Following the cut-off values for the prediction of irritation set out in OECD Guideline 439/EU Method B.46, copper pyrophosphate fulfils the classification criteria for skin irritation Category 2 according to Regulation (EC) No 1272/2008 (CLP) and GHS.

In an in vivo study according to OECD 404 (Prietzsch 2014) the test substance was applied on the skin of rabbits. It was used under semiocclusive conditions. Animal 1 was treated for 3 min, 1 and 4 h on different skin areas. Animals 2 and 3 were treated for 4 h. Animals were observed for up to 8 days. No signs of skin irritation/corrosion were observed and the scores of 24/48/72 h for all animals were 0. Therefore the test substance is considered to be non irritating to skin.

Using the weight of evidence approach all studies are considered together. Each one in vitro study determined irritating and non irritating properties of the test substance. Taking into consideration the available in vivo study in rabbits the overall conclusion can be reached that the test substance is not considered to be skin irritating and no classification is needed.

Eye irritation

A weight of evidence approach was used to evaluate eye irritation potential of the test substance.

A Bovine Corneal Opacity and Permeability (BCOP) Assay was conducted with copper pyrophosphate following OECD Guideline 437 and in accordance with GLP (Warren, 2014). Three corneas were treated with the test substance at 20% w/v in 0.9% w/v sodium chloride solution for 240 min at 32 °C. Two groups of three corneas each treated with 0.9% w/v sodium chloride and 20% w/v imidazole in 0.9% w/v sodium chloride served as negative and positive controls, respectively. Following opacity and permeability measurements, the in vitro irritancy score was calculated.

Negative control corneas were clear post-treatment, while test substance-treated and positive control corneas were slightly cloudy and cloudy post-treatment, respectively. The in vitro irritancy scores of the test substance-treated, negative and positive control corneas were 20.4, 4.2 and 105.5.

The test substance is thus considered not to be an ocular corrosive or severe irritant. Therefore not classified as Cat 1.

Copper pyrophosphate was tested for its irritancy potential to the rabbit eye in a GLP-compliant study conducted according to OECD Guideline 405 (Bradshaw, 2014). Two New Zealand White rabbits were sequentially tested. In each case, a volume of 0.1 mL (corresponding to ca. 83 mg) of the test material was placed into the conjunctival sac of the right eye, the untreated eye serving as control. The treated eyes were not rinsed after exposure, and ocular effects were assessed at 1, 24, 48 and 72 h as well as on Day 7 post-instillation. No corneal effects were noted during the study. Iritis was observed in both treated eyes at 1, 24 and 48 h, persisting in one eye at the 72 h reading. Moderate conjunctival irritation was noted in both treated eyes from 1 to 72 h. The individual mean iris, conjunctival redness and chemosis scores from readings after 24, 48 and 72 h were 0.7/1.0, 2.0/2.0 and 1.7/2.0, respectively. All observed effects were fully reversible within 7 days following instillation.

Copper pyrophosphate thus fulfils the classification criteria for eye irritation Category 2 according to Regulation (EC) No 1272/2008 (CLP) and Category 2B according to GHS.

Respiratory irritation

This information is not available.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

Based on available data on skin irritation the test item is not classified according to Regulation (EC) No 1272/2008 (CLP).

Based on available data on eye irritation the test item is classified into Category 2 as causing eye irritation according to Regulation (EC) No 1272/2008 (CLP).