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Description of key information

Skin irritation / corrosion
A number of in vivo studies were available for phosphoric acid but these studies were not performed according to current guidelines or under the conditions of GLP. For the purpose of classification and labelling, in vitro corrosivity studies were performed to an appropriate and validated test method under the conditions of GLP on solutions of phosphoric acid. These are presented as a weight of evidence.
Eye irritation:
No reliable data are available for the eye irritation endpoint. Nevertheless as the substance is classified as corrosive to the skin, a classification for eye damage is also required. This is conclusion is in accordance with the results of the supporting studies presented under this endpoint.
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Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 04 February to 06 February
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conclusive, done to a valid guideline (OECD TG 431, adopted 26 July 2013) and was conducted under GLP conditions. No deviations from the test methods were noted.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of Inspection: 10 July 2012 Date of Signature on Certificate: 30 November 2012
Species:
other: human
Strain:
other: EpiSkin Reconstructed Human Epidermis Model
Details on test animals or test system and environmental conditions:
Test System: EpiSkinTM Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date Received:04 February 2014
EpiSkinTM tissues (0.38 cm2) lot number: 14-EKIN-003
Maintenance medium lot number: 14-MAIN3-00
Assay medium lot number: 14-ESSC-004

Description: The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Adaption to cell culture conditions (pre-incubation): 2.0 mL of maintenance medium, warmed to approximately 37 ºC, was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for each test item, control and time point.

Incubation Conditions:

Temperature: 37 °C
Atmosphere: 5% CO2 in air overnight.
Type of coverage:
other: open in vitro system
Preparation of test site:
other: intact reconstructed human epidermis
Vehicle:
water
Controls:
other: not applicable - positive and negative control items are used
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1.00 g of test item + 7.5 g deionised water
- Concentration (if solution): 10% w/w

NEGATIVE CONTROL
Identification: 0.9% w/v sodium chloride solution
Purity: 0.9%
The negative control item was used as supplied.
Amount applied: 50 μL

POSITIVE CONTROL
Identification: Glacial acetic acid
Purity: >99.7%
The positive control item was used as supplied.
Amount applied: 50 μL
Duration of treatment / exposure:
3, 60 and 240 minutes
Observation period:
Not applicable. The tissues were transferred into the MTT filled wells and incubated for 3 hours ± 5 minutes in a humidified atmosphere at 37°C, 5% CO2 in air.
Number of animals:
Not applicable. The test was performed in triplicates for each treatment and control group.
Details on study design:
REMOVAL OF TEST SUBSTACE
- Washing: At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (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 and 240 minutes.

CELL VIABILITY MEASUREMENTS
-Method: MTT Reduction Assay
-Principles of method: Cell viability and thus toxicity is determined by the metabolic conversion (reduction) of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells, relative to the negative control. After a 3-hr incubation period with MTT, each tissue is placed on absorbent paper to dry. A sample of tissue is placed in a tube with 500 μL of acidified isopropanol in order to extract the formazan crystals. At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. The optical density of the extracts is measured at 562 nm using an Anthos 2001 microplate reader. Acidified isopropanol alone was added to the two wells designated as ‘blanks’.
-Time after exposure: Optical measurements: day 2

INTERPRETATION OF RESULTS
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-Minute exposure periods, 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 as follows:
Relative mean viability (%) = (mean OD562 of test item / mean OD562 of negative control) x 100

Classification of corrosivity potential was based on relative viabilities for each exposure time according to the following prediction model:

-Treatment time = 3 mins
Relative mean tissue viability < 35%: Corrosive, Category 1A/H314 according to Regulation (EC) No. 1272/2008 and UN GHS. UN Packing Group I.

- Treatment time = 3 / 60 mins
Relative mean tissue viability ≥35% / <35%: Corrosive, Category 1B or 1C /H314 according to Regulation (EC) No. 1272/2008 and UN GHS. UN Packing Group II or III.
-Treatment time = 60 / 240 mins
Relative mean tissue viability ≥35% / <35%: Corrosive, Category 1B or 1C /H314 according to Regulation (EC) No. 1272/2008 and UN GHS. UN Packing Group II or III.

-Treatment time = 240 mins
Relative mean tissue viability ≥35%: Non-Corrosive, Not classified according to Regulation (EC) No. 1272/2008 and UN GHS.


QUALITY CRITERIA

The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean optical density for the negative control treated tissues is ≥ 0.600 and ≤ 1.500.

Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was 0 to 20% relative to the negative control treated tissues following the 240-Minute exposure period.

Variation: In the range 20-100% viability and for ODs ≥ 0.3, difference of viability between the two tissue replicates of each treatment group should not exceed 30%.
Irritation / corrosion parameter:
other: other: Relative mean tissue viability (%)
Value:
104.4
Remarks on result:
other:
Remarks:
Basis: other: mean value test item. Time point: 3 minutes. Max. score: 100.0. Reversibility: other: reversibility not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: Relative mean tissue viability (%)
Value:
47.4
Remarks on result:
other:
Remarks:
Basis: other: mean value test item. Time point: 60 minutes. Max. score: 100.0. Reversibility: other: reversibility not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: Relative mean tissue viability (%)
Value:
6.4
Remarks on result:
other:
Remarks:
Basis: other: mean value test item. Time point: 240 minutes. Max. score: 100.0. Reversibility: other: reversibility not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: Relative mean tissue viability (%)
Value:
3.5
Remarks on result:
other:
Remarks:
Basis: other: mean value positive control item. Time point: 240 minutes. Max. score: 100.0. Reversibility: other: reversibility not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: Relative mean tissue viability (%)
Value:
100
Remarks on result:
other:
Remarks:
Basis: other: mean value negative control item. Time point: 240 minutes. Max. score: 100.0. Reversibility: other: reversibility not applicable. Remarks: The mean value of the negative control tissues is set at 100%. (migrated information)
Other effects / acceptance of results:
The individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viability values 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 6.4% after a 240-minute exposure period.

Table 1: Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

Exposure Period

Mean OD562 of individual tissues

Mean OD562 of duplicate tissues

Relative mean viability (%)

Negative control item

240 minutes

0.946

0.954

100

0.961

Positive control item

240 minutes

0.034

0.033

3.5

0.032

 

 

Test Item

240 minutes

0.046

0.061

6.4

0.075

60 minutes

0.470

0.452

47.4

0.433

3 minutes

1.003

0.996

104.4

0.989

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 3.5% relative to the negative control treated tissues following the 240 -minute exposure period. The positive acceptance criterion was therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.954. The negative control acceptance criterion was therefore satisfied.

The difference of viability between the two tissue replicated of each treated group did not exceed 30% and therefore the variation acceptance criterion was therefore satisfied.

Interpretation of results:
Category 1B (corrosive)
Conclusions:
The test item was classified as corrosive to the skin. The following classification criteria apply:

EU CLP Regulation (EC) No 1272/2008 and UN GHS Hazard Statement H314 "Causes severe skin burns and eye damage" Category 1B or 1C.

UN Packing Group II or III "Substance Presenting Minor or Medium Danger".
Executive summary:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKINTMin vitro Reconstructed human epidermis (RHE) model after treatment periods of 3, 60 and 240 minutes.

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. 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 (OD) was measured at 562 nm (OD562).

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 viabilities of the test item treated tissues were:

240 minutes exposure: 6.4%

60 minutes exposure: 47.4%

3 minutes exposure: 104.4%

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

The test item was classified as corrosive to the skin. The following classification criteria apply: EU DSD (67/548/EEC) Corrosive requires symbol "C" risk phrase P34 "CAUSES BURNS" EU CLP Regulation (EC) No 1272/2008 and UN GHS Hazard Statement H314 "Causes severe skin burns and eye damage" Category 1B or 1C. UN Packing Group II or III "Substance Presenting Minor or Medium Danger".

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
The study that gives rise to the greatest concern is selected. Study is performed according to an appropriate guideline and under the conditions of GLP (Klimisch 1).

Justification for selection of eye irritation endpoint:
Not applicable - endpoint conclusion based on classification for skin corrosive effects and harmonised classification for eye damage.

Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: corrosive

Justification for classification or non-classification

Skin irritation:

 

According to harmonised classification of orthophosphoric acid as reported in Annex VI of Regulation (EC) No. 1272/2008 the substance is classified as corrosive to skin, category 1B with the following concentration limits:

Skin Irrit. 2; H315: 10% ≤ C < 25%

Skin Corr. 1B; H314: C ≥ 25%

 

The registrant has performed additional in vitro corrosivity studies which support this classification of category 1B but suggests skin corrosion occurs at ≥ 10%.

 

Eye irritation:

According to harmonised classification of orthophosphoric acid as reported in Annex VI of Regulation (EC) No. 1272/2008 the substance is considered to be an eye irritant, category 2 with a concentration limit of 10% ≥ C < 25%

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