Phosphoric acid, C12-18-alkyl esters, potassium salts

Endpoint summary

Description of key information

Phosphoric acid, C12-18 alkyl esters, potassium salts was considered to be irritating to skin and eye based on in-vitro data of the substance and read-across information from a structural similar substance.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-06 to 2017-12-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

recommended by the OECD testing guideline 431

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissues
- Tissue batch number(s): Lot: 25867
-Certificate of analysis dated: 2017-12-20
- Date of initiation of testing: 2017-12-22

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min exposure at room temperature; 60 min exposure at 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 times with exess of DPBS
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL (1 mg/mL)
- Incubation time: 3 hours
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570)
- Wavelength: at 570 nm (OD570)
- Filter: no
- Linear OD range of spectrophotometer: no information

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: valid
- Barrier function: valid
- Morphology: valid
- Contamination: valid
- Reproducibility: valid

NUMBER OF REPLICATE TISSUES: two tissues

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Test item did not prove to be a MTT reducer

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. 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 [complete, e.g. 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%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 ± 2mg /tissue

VEHICLE
No vihicle

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL deionised water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL Potassium Hydroxide
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes

Duration of post-treatment incubation (if applicable):

no

Number of replicates:

Duplicate EpiDermTM tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

71.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 houre

Value:

46.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Results after treatment with Phosphoric acid, C12-18 alkyl esters, potassium salts and the controls

 

Dose Group	Ex-posure Inter-val	OD570 Well 1 (Tissue 1/2)	OD570 Well 2 (Tissue 1/2)	OD570 Well 3 (Tissue 1/2)	Mean OD570(Tissue 1/2)	OD570of 3 Wells blank corrected	OD570of 2 Tissues	Rel. Viability of Tissue 1/2 [%]*	CV [%]	Mean Rel. Viability [%]**
Blank		0.039	0.038	0.038	0.039
Negative Control	3 minutes	1.576	1.503	1.526	1.535	1.496	1.535	97.5	3.6	100.0
		1.633	1.596	1.609	1.613	1.574		102.5
Positive Control		0.265	0.258	0.258	0.260	0.222	0.282	14.5	30.1	18.4
		0.384	0.377	0.380	0.380	0.342		22.3
Test Item		1.218	1.175	0.187	1.193	1.155	1.095	75.2	7.7	71.3
		1.066	1.056	1.101	1.074	1.036		67.5
Blank		0.039	0.038	0.038	0.039
Negative Control	1 hour	1.714	1.682	1.705	1.700	1.662	1.654	100.4	0.6	100.0
		1.676	1.692	1.689	1.686	1.647		99.6
Positive Control		0.082	0.080	0.081	0.081	0.042	0.039	2.6	11.7	2.4
		0.075	0.074	0.074	0.074	0.036		2.2
Test Item		0.815	0.797	0.819	0.810	0.772	0.773	46.7	0.3	46.8
		0.839	0.797	0.805	0.814	0.775		46.8

 

Conclusion

The test item is considered to be non-corrosive to skin:

·        since the viability after 3 minutes exposure is greater than 50% and

·        the viability after 1 hour exposure is greater than 15%.

Interpretation of results:

GHS criteria not met

Remarks:

non-corrosive to skin

Conclusions:

Phosphoric acid, C12-18-alkyl esters, potassium salts is considered to be not corrosive, based on GHS criteria.

Executive summary:

In an in-vitro skin irritation study performed in accordance with OECD Guideline 431 (In Vitro Skin Corrosion, RHE) (adopted July 29, 2016), Phosphoric acid, C12-18-alkyl esters, potassium salts (99 % a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 3 minutes and 1 hour in duplicate.

Approximately 25 mg of the test item were applied to the surface of tissues, wetted with 25 µL of deionised water prior to application in order to improve contact between the solid and the tissue.

The test item was spread to match the surface of the tissue. Each 50 µL of negative and positive control were applied to sets of duplicate tissues, respectively.

After exposure period of 3 minutes (room temperature) and 1 hour (37 °C) the tissues were rinsed off, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. After rinsing, the formazan salt was extracted for about 20 hours at room temperature.

Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (18.4%) and for the 1 hour exposure period (2.4%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item Phosphoric acid, C12-18 alkyl esters, potassium salts the relative absorbance value decreased to 71.3% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 46.8%. Both values did not exceed the threshold for corrosivity which is defined to be < 50% after the 3 minutes exposure and < 15% after the 1 hour exposure. Therefore, the test item is considered to be not corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-03 to 2017-10-20

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)

Version / remarks:

adopted July 28, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended by the OECD testing guideline 439

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Tissue batch number(s): Lot: 25849
- Delivery date: 2017-10-17
- Date of initiation of testing: 2017-10-17

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: total 60 min (35 min at 37 °C and rest of time at room temperature)
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C (41 hours)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: at least 15 times in order to remove any residual test material.
- Observable damage in the tissue due to washing: no information


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL/ tissue (1mg/mL MTT)
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1
- Wavelength: 570 nm


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: valid
- Barrier function: valid
- Morphology: valid
- Contamination: valid
- Reproducibility: valid

NUMBER OF REPLICATE TISSUES: triplicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.


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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
Cut-off point(s) as recommended in TG 439

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test item: approx. 25 mg/ tissue (wetted with 25 µL DPBS) spread to match the surface of the tissue
Negative Control: 30 µL/tissue
Positive Control: 30 µL/tissue

Duration of treatment / exposure:

60 minutes (35 min at 37 °C and rest of time at room temperature)

Duration of post-treatment incubation (if applicable):

41 hours (37 ± 1.5 °C, 5 ± 0.5 % CO2)

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1

Value:

17.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.

Results after treatment with Phosphoric acid, C12-18 alkyl esters, potassium salts and the controls

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	OD 570 nm Well 3	Mean OD of 3 Wells	Mean OD of 3 Wells blank corrected	Mean OD of 3 tissues blank corrected	Rel. Viability [%] Tissue 1, 2 + 3*	Relative Standard Deviation [%]	Mean Rel. Viability [%]**
Blank		0.039	0.038	0.044	0.040	0.000
Negative Control	1	1.396	1.399	1.398	1.398	1.357	1.495	90.8	8.1	100.0
	2	1.580	1.593	1.581	1.584	1.544		103.3
	3	1.631	1.626	1.612	1.623	1.583		105.9
Positive Control	1	0.103	0.105	0.104	0.104	0.064	0.063	4.3	3.6	4.2
	2	0.104	0.105	0.105	0.104	0.064		4.3
	3	0.100	0.101	0.100	0.100	0.060		4.0
Test Item	1	0.283	0.279	0.285	0.282	0.242	0.264	16.2	7.3	17.7
	2	0.316	0.313	0.321	0.317	0.276		18.5
	3	0.316	0.315	0.313	0.315	0.274		18.3

 

Conclusion

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 17.7% (threshold for irritancy:≤50%), consequently the test item was irritant to skin. 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Phosphoric acid, C12-18-alkyl esters, potassium salts is considered to be irritating (Cat.2), based on GHS criteria.

Executive summary:

In an in-vitro skin irritation study performed in accordance with OECD Guideline 437 (In Vitro Skin Irritation, RHE) (adopted July 29, 2016), Phosphoric acid, C12-18-alkyl esters, potassium salts (99 % a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 60 minutes in triplicate.

Approximately 25 mg of the test item were applied to the surface of tissues, wetted with 25 µL of DPBS prior to application in order to improve contact between the solid and the tissue.

The test item was spread to match the surface of the tissue. Each 30 µL of negative and positive control were applied to sets of triplicate tissues, respectively.

After exposure period of 60 minutes (35 min at 37 °C and rest of time at room temperature) the tissues were rinsed off. Following a further incubation for 41 hours the tissues were rinsed and treated with the MTT solution for 3 hours.

After rinsing, the formazan salt was extracted for about 3 hours at room temperature while shaking.

Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 4.2% thus ensuring the validity of the test system.

After exposure to the test item Phosphoric acid, C12-18 alkyl esters, potassium salts the relative absorbance value decreased to 17.7 % after 60 minutes exposure.

This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye 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:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to attached document

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

iris score

Basis:

animal: 1,2,3

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.67

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal: 2,3

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal: 2,3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Classification is based on individual mean scores of 24, 48 and 72 hour scorings, considering reversibility.

Executive summary:

Available data from 1-Octadecanol, phosphate, potassium salt were used to evaluate eye irritation for Phosphoric acid, C12-18-alkyl esters, potassium salts.

In a primary eye irritation study according to OECD guideline 405, 0.1 mL of 1-Octadecanol, phosphate, potassium salt was evaluated to be irritating.

Mean grades of ocular reactions at 24, 48 and 72 h after instillation were evaluated for classification purpose. Based on mean conjunctivae scores of 2.67/ 2/ 2, classification in category 2 is required according to CLP; EU GHS (Regulation (EC) No 1272/2008).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-09-15

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

July, 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS Test System:
Freshly isolated bovine cornea (at least 9 month old donor cattle)
Rationale: OECD 437
Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany

Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.

All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.

Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration:
Since the test item is a surfactant substance, it was tested at a final concentration of 10% (a.i) as a solution (w/v) in saline as stated in the OECD guideline 437.

Duration of treatment / exposure:

The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted ten minutes.

Duration of post- treatment incubation (in vitro):

After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added into the anterior compartment. Then the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position.

Number of animals or in vitro replicates:

Sets of three corneae were used for treatment with the test item and for the negative and positive controls.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): rinsed off from the application side with saline, fresh cMEM
- Time after start of exposure: 10 min.

SCORING SYSTEM:
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 – corrected opacity value mean negative control) + (15 x corrected OD490 value)

TOOL USED TO ASSESS SCORE:
Opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France)
Permeability was assessed using fluorescein

Irritation parameter:

in vitro irritation score

Value:

8.24

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.53).

- Acceptance criteria met for positive control:
The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 70.24) corresponding to a classification as serious eye damaging (Cat 1 according to CLP).

Results after 10 Minutes Treatment Time

Test Group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	-1	-0.33	0.050	0.058	-0.25	0.53	Not categorized
	0		0.070		1.05
	0		0.053		0.80
Positive Control	49.33*		1.323*		69.18	70.24	Category 1
	53.33*		1.296*		72.78
	53.33*		1.028*		68.76
Test Item	5.33*		0.278*		9.51	8.24	No prediction can be made

*corrected values

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 (t₁₃₀– t₀).

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.

Interpretation of results:

other: not severely irritating/ corrosive

Conclusions:

The calculated mean in vitro irritation score for Phosphoric acid, C12-18-alkyl esters, potassium salts was 8.24.
Since the mean in vitro irritancy score of the test substance was <55, the substance is considered to not be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Phosphoric acid, C12-18-alkyl esters, potassium salts by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted July, 2013. Since the test item is a surfactant substance, it was tested as 10% solution (w/v) in saline as recommended by OECD guideline 437.

 

The corneae were incubated with the test substance and controls for 10 min. After rinsing with saline, the corneae were incubated for another 2 h at 32±1°C.

The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value). Substance that induces an IVIS > 55 Cat. 1 for serious eye damage has to be assigned. For substances that induces an IVIS < 3, no classification is required.

 

The 10% dilution of the test substance in saline caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 8.24.

The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 70.24) corresponding to a classification “serious eye damaging Cat 1” according to GHS/CLP criteria.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.53).

Relative to the negative control, the test item Phosphoric acid, C12-18 alkyl esters, potassium salts caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 8.24. Since the mean in vitro irritancy score of the test substance was <55, the substance is considered to be not severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

In an in-vitro skin irritation study performed in accordance with OECD Guideline 431, Phosphoric acid, C12-18-alkyl esters, potassium salts (99 % a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 3 minutes and 1 hour in duplicate.

Approximately 25 mg of the test item were applied to the surface of tissues, wetted with 25 µL of deionised water prior to application in order to improve contact between the solid and the tissue.

The test item was spread to match the surface of the tissue. Each 50 µL of negative and positive control were applied to each set of duplicate tissues.

After exposure period of 3 minutes (room temperature) and 1 hour (37 °C) the tissues were rinsed off, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. After rinsing, the formazan salt was extracted for about 20 hours at room temperature. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (18.4%) and for the 1 hour exposure period (2.4%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item Phosphoric acid, C12-18 alkyl esters, potassium salts the relative absorbance value decreased to 71.3% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 46.8%. Both values did not exceed the threshold for corrosivity which is defined to be < 50% after the 3 minutes exposure and < 15% after the 1 hour exposure. Therefore, the test item is considered to be not corrosive.

In an in-vitro skin irritation study performed in accordance with OECD Guideline 437, Phosphoric acid, C12-18-alkyl esters, potassium salts (99 % a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 60 minutes in triplicate. Approximately 25 mg of the test item were applied to the surface of tissues, wetted with 25 µL of DPBS prior to application in order to improve contact between the solid and the tissue.

The test item was spread to match the surface of the tissue. Each 30 µL of negative and positive control were applied to sets of triplicate tissues, respectivly.

After exposure period of 60 minutes (35 min at 37 °C and rest of time at room temperature) the tissues were rinsed off. Following a further incubation for 41 hours the tissues were rinsed and treated with the MTT solution for 3 hours.

After rinsing, the formazan salt was extracted for about 3 hours at room temperature while shaking.

Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 4.2% thus ensuring the validity of the test system.

After exposure to the test item Phosphoric acid, C12-18 alkyl esters, potassium salts the relative absorbance value decreased to 17.7 % after 60 minutes exposure. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

 

In conclusion, both in-vitro studies clearly indicate the irritating potential of Phosphoric acid, C12-18 alkyl esters, potassium salts.

 

Eye:

This in vitro study was performed to assess the corneal irritation and damage potential of Phosphoric acid, C12-18-alkyl esters, potassium salts by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted July, 2013. Since the test item is a surfactant substance, it was tested as 10% solution (w/v) in saline as recommended by OECD guideline 437.

The corneae were incubated with the test substance and controls for 10 min. After rinsing with saline, the corneae were incubated for another 2 h at 32±1°C.

The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value). Substance that induces an IVIS > 55 Cat. 1 for serious eye damage has to be assigned. For substances that induces an IVIS < 3, no classification is required.

The 10% dilution of the test substance in saline caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 8.24.

The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 70.24) corresponding to a classification “serious eye damaging Cat 1” according to GHS/CLP criteria.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.53).

Relative to the negative control, the test item Phosphoric acid, C12-18 alkyl esters, potassium salts caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 8.24. 

Since the mean in vitro irritancy score of the test substance was <55, the substance is considered to be not severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

 

In-vivo eye irritation

1-Octadecanol, phosphate, potassium salt

In a primary eye irritation study according to OECD guideline 405, 0.1 mL of 1-Octadecanol, phosphate, potassium salt was instilled into the conjunctival sac of 3 adult New Zealand white rabbits. Animals then were observed for 7 days. 

Irritation was scored according to OECD guideline 405. Very well-defined signs of irritation were observed on the treated eyes of all animals including effects on cornea, iris and conjunctivae. All effects were fully reversible within 7 days.

Mean grades of ocular reactions at 24, 48 and 72 h after instillation were evaluated for classification purpose. Based on mean conjunctivae scores of 2.67/ 2/ 2, classification in category 2 is required according to CLP; EU GHS (Regulation (EC) No 1272/2008).

 

Weight of evidence

In a weight of evidence approach it can be deduced that Phosphoric acid, C12-18-alkyl esters, potassium salts has a clear eye irritating potential. Taking into account the IVIS score of 8.24 from in-vitro BCOP study, which is far below the threshold of > 55 requiring classification as severely irritating/ corrosive, but also above the threshold of ≤ 3 requiring no classification,
irritating properties can be assumed. From a formal aspect from this in-vitro study no prediction can be made. However, in-vivo data from the structural similar substance 1-Octadecanol, phosphate, potassium salt clearly underpin the assumption of irritating properties.

 

In conclusion, Phosphoric acid, C12-18-alkyl esters, potassium salts has to be regarded as eye irritating and has to be classified accordingly.

 

Justification for read-across

Evaluation of structure-activity relationship is based on data from structural similar substances with the same basic structure, potassium salts of phosphoric acid alkyl ester, varying mainly in the chain length of fatty acid moiety. A detailed justification document for the read-across is attached in the respective target records of IUCID.

Justification for classification or non-classification

Based on data from in-vitro studies according to OECD Guideline 431 and 439, Phosphoric acid, C12-18-alkyl esters, potassium salts has to be classified as irritating to skin Cat. 2 according the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008) and labelled with H315 “Causes skin irritation” respectively.

Phosphoric acid, C12-18-alkyl esters, potassium salts has to be regarded as eye irritating based on a weight of evidence approach including data from an in-vitro study on the substance and an in vivo study with a structural similar substance.

Taken together, Phosphoric acid, C12-18-alkyl esters, potassium salts has to be classified for eye irritation Cat. 2, based on CLP criteria, EU GHS (Regulation (EC) No 1272/2008) and labelled with H319 “Causes serious eye irritation” accordingly.