Phosphoric acid C12-alkyl and C18-alkyl esters, potassium salts

Endpoint summary

Description of key information

Skin: Based on a weight of evidence-apporoach, the test item is considered to be irritating to skin.

Eyes: The substance was classified as corrosive to the eyes (cat. 1) as a worst-case-approach, as the available BCOP-study did not allow for predicting the corneal irritation and damage potential of the test item.

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:

key study

Study period:

16. Dec 1986 - 06. Jan 1987

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:

yes

Remarks:

test item was applied as 77% paste in water

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst Ag, Kastengrund, conventional breed
- Age at study initiation: 3-5 months
- Weight at study initiation: 2,3 - 3,4 kg
- Housing: single cages
- Diet (e.g. ad libitum): Altromin 2123 (Altromin GmbH, Lage/Lippe, Germany) ad libitum, 15 g hay/day
- Water (e.g. ad libitum): water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3°C
- Humidity (%): 50 +/-20 %
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: 16. Dec. 1986 To: 06. Jan. 1987

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

not required

Amount / concentration applied:

500 mg of 77% test substance in water

Duration of treatment / exposure:

4 h

Observation period:

a. 30-60 min post applicationem
b. 24 h post applicationem
c. 48 h post applicationem
d. 72 h post applicationem
e. 7 d post applicationem
f. 14 d post applicationem
g. 21 d post applicationem

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 2.5 x 2.5 cm
- % coverage: 100 %
- Type of wrap if used: The substance was applied over the whole surface of a 2.5 x 2.5 cm cellulose patch on a piece of surgical plaster. The plaster was fixed to the prepared skinarea and covered with a semiocclusive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with warm water
- Time after start of exposure: 4 h

SCORING SYSTEM:

Erythema and eschar formation
No erythema.......................................................................................................................0
very slight erythema (barely perceptible)......................................................................1
well-defined erythema......................................................................................................2
moderate to severe erythema..........................................................................................3
severe erythema (beet redness) to slight eschar formation (lesion in depth).........4

Oedema
No oedema...................................................................................................................................0
very slight oedema (barely perceptible)..................................................................................1
slight oedema (edges of area well defined by definite raising)............................................2
moderate oedema (raised approximately 1mm)....................................................................3
severe oedema (raised more than 1mm and extending beyond area of exposure.........4

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24, 48, 72 h

Score:

2.89

Max. score:

4

Reversibility:

fully reversible within: 21 d

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24, 48, 72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

fully reversible within: 21 d

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 d

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.6

Max. score:

4

Reversibility:

fully reversible

Remarks:

7 d

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.3

Max. score:

4

Reversibility:

fully reversible within: 14d

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.6

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 d

Other effects:

Treated skin areas were mostly dry and rough.

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

With reference the reported scores and the reversibility of the observed effects the test item has to be classified as irritant to the skin (H315 - causes skin irritation) according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).

Executive summary:

The test item was tested for its skin irritant properties in 3 New Zealand White rabbits. The study was performed according to OECD Guideline 404. The only deviation was that the test item was applied as 77% paste in water, but since according to the guideline dry powders have to be moistened, the testing conditions and results are considered to be the same. Effects on the skin (erythema grades up to 4 and edema scores up to 3) were observed in all animals 24 hours after application. These signs were reversible within the 21 days observation period. With reference the reported scores and the reversibility of the observed effects the test item has to be classified as irritant to the skin (H315 - causes skin irritation, skin irritant category 2) according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

test item was applied as 77% paste in water

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24, 48, 72 h

Score:

2.89

Max. score:

4

Reversibility:

fully reversible within: 21 d

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24, 48, 72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

fully reversible within: 21 d

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 d

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.6

Max. score:

4

Reversibility:

fully reversible within: 7 d

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.3

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.6

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 d

Interpretation of results:

irritating

Conclusions:

With reference the reported scores and the reversibility of the observed effects the read across item has to be classified as irritant to the skin (H315 - causes skin irritation) according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).

Executive summary:

The read across-item was tested for its skin irritant properties in 3 New Zealand White rabbits. The study was performed according to OECD Guideline 404. The only deviation was that the test item was applied as 77% paste in water, but since according to the guideline dry powders have to be moistened, the testing conditions and results are considered to be the same. Effects on the skin (erythema grades up to 4 and edema scores up to 3) were observed in all animals 24 hours after application. These signs were reversible within the 21 days observation period. With reference the reported scores and the reversibility of the observed effects the test item has to be classified as irritant to the skin (H315 - causes skin irritation, skin irritant category 2) according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (EC) No 1272/2008.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 March 2018 - 06 April 2018

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)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40Bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SCT)
- Tissue batch number: 25892

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsing with sterile PBS (filling and emptying of insert 20 times in a constant soft stream of PBS)
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: plate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The tissue viability was within the acceptable range.
- Barrier function: The barrier function was within the acceptable range.
- Morphology: The morphology was within the acceptable range.
- Contamination: No contaminations were determined.

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

60 min

Number of replicates:

2 replicates per test item, 2 replicates negative controls and 2 replicates positive controls

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

1 hour exposure

Value:

90.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

3 minute exposure

Value:

94.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

TABLE 1:  Summary of Optical Density (OD) and Viability (%)

 

3 Minutes Exposure                                                                                   Refer Appendix - 1& 2

Treatment		OD	Viability (%)	Classification
Negative Control (Sterile water)	Mean	1.552	100.0	NC
	±SD	0.034	3.1
	n	2	2
Positive Control (Glacial acetic acid)	Mean	0.065	4.2	C (Category 1A)
	±SD	0.002	0.2
	n	2	2
Test Item [Phosphoric Acid (C12/C18) mono- and/or diester, K Salts (Afilan SPC)]	Mean	1.465	94.4	NC
	±SD	0.022	2.0
	n	2	2

                                           

 

1 Hour Exposure

Treatment		OD	Viability (%)	Classification
Negative Control (Sterile water)	Mean	1.504	100.00	NC
	±SD	0.036	3.3
	n	2	2
Positive Control (Glacial acetic acid)	Mean	0.064	4.3	C (Category 1A)
	±SD	0.000	0.0
	n	2	2
Test Item [Phosphoric Acid (C12/C18) mono- and/or diester, K Salts (Afilan SPC)]	Mean	1.356	90.2	NC
	±SD	0.070	6.6
	n	2	2

NC = Non Corrosive; C = Corrosive; n = No. of tissues; SD = Standard Deviation.

 

Interpretation of results:

other: not Category 1 (corrosive) based on GHS criteria

Conclusions:

Based on the results obtained under the laboratory testing conditions, the test item is categorised as non-corrosive to Reconstructed Human Epidermis (RhE) as the mean percentage tissue viability was greater than 50 % after 3 minutes exposure and greater than 15 % after 1 hour exposure of the negative control.

Executive summary:

The objective of study was to evaluate the in vitro skin corrosion potential the test item by measurement of tissue viability on the Epidermal Model - Epiderm™ (EPI-200-SCT) as per the OECD Guideline for the testing of chemicals No. 431, “In vitro skin corrosion: reconstructed human epidermis (RHE) test method”, adopted on 29th July 2016. The test item did not develop any colour when dissolved in distilled water or isopropanol and was considered not to reduce MTT as no purple colour was developed when mixed and incubated with MTT solution. Tissue inserts were transferred to upper row of 6 well plates prefilled with 0.9 mL of assay medium and incubated in CO2 incubator for 60 minutes. Test items were exposed for 1 hour and 3 minutes separately. All the treatments were maintained in duplicates.For the 3 minutes treatment, a quantity of 50 µL of sterile distilled water (NC) was dispensed into the first insert atop the tissue. After 60 seconds the procedure was repeated with the second tissue and continued for the other tissues. A similar procedure was followed in the same manner until all the tissues were treated. The tissues were treated with 25 mg test item + 25µL distilled waterand 50 µL of positive control (glacial acetic acid). For the 1 hour treatment, a quantity 25 mg test item + 25 µL distilled water, 50 µL of negative control and 50 µL of positive control were dispensed directly atop Epiderm tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 1 hour. At the end of treatment time tissue inserts were rinsed with sterile PBS (fill and empty insert 20 times in a constant soft stream of 1xPBS) to remove any residual test item. The tissues were placed into 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and incubated for 3 hours at 37±1°C and 5±1% CO2. Post incubation, the tissue inserts were removed and blotted onto the tissue paper and transferred to a prelabeled 24-well plate containing 2.0 mL of isopropanol in each designated. The plates were placed on an orbital plate shaker and shaken (̴ 120 rpm/minute) for 5 hours and 19 minutes (for 1 hour exposure) and 4 hours and 44 minutes (for 3 minutes exposure) at room temperature. At the end of the extraction period, the tissue was pierced with an injection needle and allowed the extract to run into the well from which the insert was taken. The punctured inserts were discarded and solution was placed on mixer for 15 minutes until it becomes homogenous. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated. For 3 minutes exposure, percentage viability of negative control, positive control and test item was 100±3.1, 4.2±0.2 and 94.4±2.0 respectively. As the percentage viability of test item was greater than 50% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control, clearly representing the irritation potential of positive control. For the 1 hour exposure, the percentage viability of negative control, positive control and test item was 100±3.3, 4.3±0.0 and 90.2±6.6 respectively. As the percentage viability of test item was greater than 15% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control clearly represents the irritation potential of positive control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-08-06 to 2018-08-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Age at study initiation: at least 9 month old donor cattle

Controls:

yes, concurrent positive control

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 corneae per group (test item, negative control, positive control)

Details on study design:

Three corneas were exposed to each 0.75 mL of a 20% (w/v) suspension of the test item in physiological saline for 240 minutes.
After treatment the test item suspension was rinsed off the corneas and the corneas' opacity was determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.

Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.

For equilibration and prior to application of the test item or controls, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t240).

Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium will be removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values will be determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):
≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)


Criteria for Determination of a Valid Test
The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean IVIS of three runs

Value:

28.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 1: Results

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean
Negative Control	1.00	0.33	0.081	0.070	2.22	1.38	No Category
	0.00		0.061		0.92
	0.00		0.068		1.02
Positive Control	84.67*		0.364*		90.13	98.06	Category 1
	103.67*		0.357*		109.02
	88.67*		0.425*		95.04
Test Item	15.67*		0.732*		26.65	28.35	No prediction can be made
	22.67*		0.659*		32.55
	16.67*		0.613*		25.86

 

Relative to the negative control, the test item did cause a slight increase of the corneal opacity or permeability. The calculated mean IVIS was 28.35 (threshold for serious eye damage: IVIS > 55). According to OECD 437, no prediction for the damage hazard of the test item to the eye can be made.

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the results a prediction for the damage hazard cannot be made (GHS) for the test item.

Executive summary:

An in vitro study was performed to assess the corneal irritation and damage potential of the test item by means of the BCOP assay using fresh bovine corneae. After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item as well as the positive and the negative controls were each applied to different corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240). After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (physiological saline), neither an increase of opacity nor permeability of the corneae was observed. The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (EU CLP/UN GHS Category 1). Relative to the negative control, the test item did cause a slight increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 28.35. According to OECD 437 a prediction for corneal irritation and damage potential cannot be made.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

In vitro study according to OECD 431 (Epiderm):

The objective of study was to evaluate the in vitro skin corrosion potential the test item by measurement of tissue viability on the Epidermal Model - Epiderm™ (EPI-200-SCT) as per the OECD Guideline for the testing of chemicals No. 431, “In vitro skin corrosion: reconstructed human epidermis (RHE) test method”, adopted on 29th July 2016. The test item did not develop any colour when dissolved in distilled water or isopropanol and was considered not to reduce MTT as no purple colour was developed when mixed and incubated with MTT solution. Tissue inserts were transferred to upper row of 6 well plates prefilled with 0.9 mL of assay medium and incubated in CO2 incubator for 60 minutes. Test items were exposed for 1 hour and 3 minutes separately. All the treatments were maintained in duplicates.For the 3 minutes treatment, a quantity of 50 µL of sterile distilled water (NC) was dispensed into the first insert atop the tissue. After 60 seconds the procedure was repeated with the second tissue and continued for the other tissues. A similar procedure was followed in the same manner until all the tissues were treated. The tissues were treated with 25 mg test item + 25µL distilled waterand 50 µL of positive control (glacial acetic acid). For the 1 hour treatment, a quantity 25 mg test item + 25 µL distilled water, 50 µL of negative control and 50 µL of positive control were dispensed directly atop Epiderm™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 1 hour. At the end of treatment time tissue inserts were rinsed with sterile PBS (fill and empty insert 20 times in a constant soft stream of 1xPBS) to remove any residual test item. The tissues were placed into 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and incubated for 3 hours at 37±1°C and 5±1% CO2. Post incubation, the tissue inserts were removed and blotted onto the tissue paper and transferred to a prelabeled 24-well plate containing 2.0 mL of isopropanol in each designated. The plates were placed on an orbital plate shaker and shaken (̴ 120 rpm/minute) for 5 hours and 19 minutes (for 1 hour exposure) and 4 hours and 44 minutes (for 3 minutes exposure) at room temperature. At the end of the extraction period, the tissue was pierced with an injection needle and allowed the extract to run into the well from which the insert was taken. The punctured inserts were discarded and solution was placed on mixer for 15 minutes until it becomes homogenous. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated. For 3 minutes exposure, percentage viability of negative control, positive control and test item was 100±3.1, 4.2±0.2 and 94.4±2.0 respectively. As the percentage viability of test item was greater than 50% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control, clearly representing the irritation potential of positive control. For the 1 hour exposure, the percentage viability of negative control, positive control and test item was 100±3.3, 4.3±0.0 and 90.2±6.6 respectively. As the percentage viability of test item was greater than 15% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control clearly represents the irritation potential of positive control.

Read across, in vivo study according to OECD 404:

The read across-item Phosphoric acid, dodecyl ester, potassium salt (CAS 39322-78-6) was tested for its skin irritant properties in 3 New Zealand White rabbits. The study was performed according to OECD Guideline 404. The only deviation was that the test item was applied as 77% paste in water, but since according to the guideline dry powders have to be moistened, the testing conditions and results are considered to be the same. Effects on the skin (erythema grades up to 4 and edema scores up to 3) were observed in all animals 24 hours after application. These signs were reversible within the 21 days observation period. With reference the reported scores and the reversibility of the observed effects the test item has to be classified as irritant to the skin (H315 - causes skin irritation, skin irritant category 2) according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (EC) No 1272/2008.

In the ECHA disseminated dossier of 1-Octadecanol, phosphate, potassium salt (CAS 68987-29-1) no signs of a skin irritating property are revealed for the C18 salt. The substance to be registered has been proven to not have a corrosive property on skin by the in vitro study. But, taken into account the above mentioned results for the C12 salt (read across), the substance to be registered (comprising of C12, C18 and C12/C18 alkyl phosphates) is considered as skin irritating (cat. 2) and labeled with H315: causes skin irritation. The approach taken therefore also reflects a worst case-approach.

 

Eyes:

Other phosphoric acid esters have been identified as being corrosive to the eyes (Phosphoric Acid, Butyl Ester, Na Salt, CAS 53126-67-3; Phosphoric acid, 2-ethylhexyl ester, Na salt, CAS 68186-64-1). As the test substance was shown to be not corrosive to skin, a BCOP-study has been conducted to decide if the test substance is to be classified as corrosive to the eyes. Relative to the negative control, the test item Phosphoric acid, C12/C18-alkylester, K salt did cause a slight increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 28.35. According to OECD 437 a prediction for corneal irritation and damage potential cannot be made.

For comparison, the read across –substance Phosphoric acid, dodecyl ester, potassium salt (CAS 39322-78-6) is classified as cat 1 (corrosive to the eyes), whereas the second read-across substance 1-Octanol, phosphate, potassium salt (CAS 68987-29-1) is classified as irritating to the eyes (cat. 2).

As a worst-case approach, the test item Phosphoric acid, C12/C18-alkylester, K salt was therefore considered to be classified as corrosive to the eyes (cat. 1).

Justification for classification or non-classification

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

The available data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is considered to be classified for skin irritation into cat. 2 and labeled with H315 (causes skin irritation) and is also classified as corrosive to the eyes (cat. 1) and labeled with H318 ( causes serious eye damage) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.