Phosphoric acid, hexadecyl phosphate esters, potassium salts with cetostearyl alcohol

Endpoint summary

Description of key information

Based on the in vitro and ex vivo study results, the test substance is considered to be non-irritating to both skin and eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 16, 2013 to July 22, 2013

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)

Test system:

human skin model

Remarks:

human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM))

Cell type:

non-transformed keratinocytes

Justification for test system used:

The test is based on the experience that irritant chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test was designed to predict and classify the skin irritation potential of a test substance by assessment of its effect on a three dimensional human epidermis model. In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

- Triplicates skin tissues were moistened with sterile distilled water and then treated with 10 mg test substance for 15 ± 0.5 minutes. Negative (PBS) and positive (SDS 5%) controls were included as well in the experiment. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment (optical density measurement). Skin irritation was expressed as the remaining cell viability after exposure to the test substance.
- Possible inflammatory mediator (IL-1a) was also determined.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Expressed as the reduction of mitochondrial dehydrogenase activity

Value:

ca. 108

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% relative mean viability

Positive controls validity:

valid

Remarks:

19.5% relative mean viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- Skin irritation was expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance compared to the negative control tissues was 108.0%. Since the mean relative tissue viability for the test substance was above 50%, it was considered to be non-irritant. It was unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal.
- The positive control had a mean cell viability of 19.5% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range.
- The standard deviation value of the percentage viability of three tissues treated identically was 2.0% or less, indicating that the test system functioned properly.

Interpretation of results:

other: not classified based on EU CLP criteria

Conclusions:

Under the study conditions, the test substance was determined to be non-irritating to the skin.

Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and C16-18-OH', using Reconstructed Human Epidermis Test Method – EpiskinTM, according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Triplicates skin tissues were moistened with sterile distilled water and then treated with 10 mg test substance for 15 ± 0.5 minutes. Positive (5% Sodium Dodecyl Sulphate) and negative (Phosphate Buffered Saline Dulbecco's) controls were included in the experiment as well. After a 42 h post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment (optical density measurement). Skin irritation was expressed as the remaining cell viability after exposure to the test substance. Possible inflammatory mediator (IL-1a) was also determined. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 108.0%. Since the mean relative tissue viability for the test substance was above 50%, it was considered to be non-irritant. It was unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The positive control had a mean cell viability of 19.5% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was 2.0% or less, indicating that the test system functioned properly. The study was considered to have met all the validity criteria. Under the study conditions, the test substance was determined to be non-irritating to the skin (Harlan, 2014).

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Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

Bovine Corneal Opacity and Permeability (BCOP) test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 09, 2013 to August 09, 2013

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes from young adult cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20% w/v in 0.9% w/v sodium chloride

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

2 h (followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein)

Number of animals or in vitro replicates:

3

Details on study design:

The Bovine Corneal Opacity and Permeability (BCOP) test is an organic model that provides short-term maintenance of normal physiological and biological function of the bovine cornea in an isolated system. In this test method, damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitymeter and an ultraviolet/visible spectrophotometer, respectively.

1. Preparation of corneas
The isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.

2. Opacity reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded.

3. Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 µl of the negative control and 20% (w/v) imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 +/- 10 minutes at 32 +/- 1°C. After the incubation the solutions and the test substance were removed and the epithelium was washed at least three times with MEM with phenol red. Possible pH effects of the test substance on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

4. Opacity measurement
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated.
Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1°C. After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 492 nm (OD492) of each sampling tube was measured in triplicate using a microplate reader.

Irritation parameter:

in vitro irritation score

Remarks:

Based on Opacity and Permeability measurements

Run / experiment:

Test substance

Value:

ca. 0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

3.6 IVIS

Positive controls validity:

valid

Remarks:

96.9 IVIS

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- The corneas treaed with the test substance and the negative control were clear post treatment where as the one treated with the positive control were cloudy. The In Vitro irritancy scores were determined to be 0.7, 3.6 and 96.9 following application of the test substance, the negative control and the positive control, respectively.

Interpretation of the results:

- Determination of the In vitro irritancy score (IVIS):

The mean opacity and mean permeability values (OD492) were used for each treatment group to calculate an in vitro score.

- The IVIS cut-off values for identifying the test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:

< or = 3: no category,

>3 and < or = to 55: no prediction can be made,

> 55: category 1.

Based on the study results, the test substance was determined to be non-irritant to the eye.

Interpretation of results:

other: not classified based on EU CLP criteria

Conclusions:

Under the study conditions, the test substance was determined to be non-irritating or non-corrosive to the eye.

Executive summary:

An in vitro study was conducted to determine the eye corrosion potential of the test substance, 'mono- and di- C16 PSE, K+ and C16-18-OH', using the Bovine Corneal Opacity and Permeability (BCOP) method, according to OECD Guideline 437, in compliance with GLP. Preparation, selection and opacity reading of the corneas were performed as per guideline. Prepared corneas in triplicates were treated with each, test substance (20% w/v in sodium chloride 0.9% w/v), negative control (Sodium chloride 0.9% w/v) and positive control (20% w/v Imidazole solution in sodium chloride 0.9% w/v) substances at 32 ± 1ºC for 240 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC for 90 minutes. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The corneas treated with the test substance and the negative controls were clear post treatment where as the one treated with the positive control was cloudy. The positive control group had an overall IVIS of 96.9. The negative control gave opacity of ≤2.4 and permeability ≤0.072, the negative control acceptance criteria were therefore satisfied. The test substance IVIS score obtained was 0.7, which was well below the threshold for non-classification. The study has met the validity criteria. Under the study conditions, the test substance was determined to be non-irritating or non-corrosive to the eye (Harlan, 2014).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

SkinEthic Reconstructed Human Corneal Epithelial Model

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From October 23, 2013 to October 24, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg

Duration of treatment / exposure:

10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

Triplicate SkinEthic tissues were treated with 30 mg test substance for 10 minutes. At the end of the exposure period, each tissue was rinsed and taken for MTT-loading. After MTT-loading, the tissues were removed and immersed in isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. Following extraction, the absorbency of the extracted MTT solution for each tissue was measured. The optical density was recorded at 562 nm. Data were presented as the percentage viability (MTT conversion relative to negative controls).

Irritation parameter:

other: relative mean tissue viability (%)

Run / experiment:

Test substance

Value:

ca. 89.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% relative mean tissue viability

Positive controls validity:

valid

Remarks:

12.8% relative mean tissue viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean tissue viability for the positive control was 12.8%.

Interpretation of results:

other: not classified based on EU CLP criteria

Conclusions:

Under the study conditions, the test substance was determined to be non-irritating to the eye.

Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, ‘mono- and di- C16 PSE, K+ and C16-18-OH’, using the SkinEthic Reconstructed Human Corneal model, according to OECD Guideline 429, in compliance with GLP. For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test substance for 10 minutes. Triplicate tissues treated with 30 µL of "Solution A" (containing Na2HPO4, glucose, HEPES, KCl and NaCl) served as the negative control and triplicate tissues treated with 30 µL of 2% w/v Sodium Dodecyl Sulphate served as the positive control. The plates were incubated at 37°C, 5% CO2 in air during the exposure time. At the end of the exposure period, each tissue was rinsed and taken for MTT-loading. After MTT-loading, the tissues were removed and immersed in isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. Following extraction, the absorbency of the extracted MTT solution for each tissue was measured. The optical density was recorded at 562 nm. Data were presented as the percentage viability (MTT conversion relative to negative controls). The relative mean tissue viability for the negative control and positive control were found to be 100 and 12.8%, respectively. Therefore, the study was considered to have met the validity criteria. The relative mean viability for the test substance treated tissues after 10 min exposure period was 89.2%, which was well below the threshold for non-classification. Under the study conditions, the test substance was concluded to be non-irritating to the eye (Harlan, 2014).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and C16-18-OH', using Reconstructed Human Epidermis Test Method – EpiskinTM, according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Triplicates skin tissues were moistened with sterile distilled water and then treated with 10 mg test substance for 15 ± 0.5 minutes. Positive (5% Sodium Dodecyl Sulphate) and negative (Phosphate Buffered Saline Dulbecco's) controls were included in the experiment as well. After a 42 h post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment (optical density measurement). Skin irritation was expressed as the remaining cell viability after exposure to the test substance. Possible inflammatory mediator (IL-1a) was also determined. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 108.0%. Since the mean relative tissue viability for the test substance was above 50%, it was considered to be non-irritant. It was unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The positive control had a mean cell viability of 19.5% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was 2.0% or less, indicating that the test system functioned properly. The study was considered to have met all the validity criteria. Under the study conditions, the test substance was determined to be non-irritating to the skin (Harlan, 2014).

Eye:

Study 1: An in vitro study was conducted to determine the in vitro eye irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and C16-18-OH', using the Bovine Corneal Opacity and Permeability (BCOP) method, according to OECD Guideline 437, in compliance with GLP. Preparation, selection and opacity reading of the corneas were performed as per guideline. Prepared corneas in triplicates were treated with each, test substance (20% w/v in sodium chloride 0.9% w/v), negative control (Sodium chloride 0.9% w/v) and positive control (20% w/v Imidazole solution in sodium chloride 0.9% w/v) substances at 32 ± 1ºC for 240 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC for 90 minutes. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The corneas treated with the test substance and the negative controls were clear post treatment where as the one treated with the positive control was cloudy. The positive control group had an overall IVIS of 96.9. The negative control gave opacity of ≤2.4 and permeability ≤0.072, the negative control acceptance criteria were therefore satisfied. The test substance IVIS score obtained was 0.7, which was well below the threshold for non-classification. The study has met the validity criteria. Under the study conditions, the test substance was determined to be non-irritating or non-corrosive to the eye (Harlan, 2014).

Study 2: An in vitro study was conducted to determine the eye irritation potential of the test substance, ‘mono- and di- C16 PSE, K+ and C16-18-OH’, using the SkinEthic Reconstructed Human Corneal model, according to OECD Guideline 429, in compliance with GLP. For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test substance for 10 minutes. Triplicate tissues treated with 30 µL of "Solution A" (containing Na2HPO4, glucose, HEPES, KCl and NaCl) served as the negative control and triplicate tissues treated with 30 µL of 2% w/v Sodium Dodecyl Sulphate served as the positive control. The plates were incubated at 37°C, 5% CO2 in air during the exposure time. At the end of the exposure period, each tissue was rinsed and taken for MTT-loading. After MTT-loading, the tissues were removed and immersed in isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. Following extraction, the absorbency of the extracted MTT solution for each tissue was measured. The optical density was recorded at 562 nm. Data were presented as the percentage viability (MTT conversion relative to negative controls). The relative mean tissue viability for the negative control and positive control were found to be 100 and 12.8%, respectively. Therefore, the study was considered to have met the validity criteria. The relative mean viability for the test substance treated tissues after 10 min exposure period was 89.2%, which was well below the threshold for non-classification. Under the study conditions, the test substance was concluded to be non-irritating to the eye (Harlan, 2014).

Justification for classification or non-classification

Based on the in vitro and ex vivo study results, the test substance, 'mono- and di- C16 PSE, K+ and C16-18 -OH' does not warrant classification for skin and eye irritation according to EU CLP criteria (Regulation 1272/2008/EC).