Phosphoric acid, mono- and di-C12-14-alkyl esters

Endpoint summary

Description of key information

 In a reliable in vitro skin corrosion study human skin tissue (eipdermis keratinocytes) was exposed to undiluted substance for 3 and 60 minutes. There was 88.2% and 52.7 % tissue viability following the 3 and 60-minute exposure, respectively. Resultantly, the substance is considered to be a non-corrosive to human skin. In a reliable in vitro skin irritation study conducted on the substance the mean value of relative tissue viability was 104.3% following a 15-minutes exposure to undiluted substance. This value is above the threshold for skin irritation (50%). Therefore, the substance is considered to be a non-irritant to human skin.

In a reliable in vitro eye irritation study employing bovine cornea were exposed to the 20% w/v substance (750 µL) for 4 hours. Opacity and permeability values were measured. There was a mean IVIS of 87.4 following the 4-hour exposure point. Resultantly, the test material is considered to cause serious damage to the eye.

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:

2nd July 2019 - 21st August 2019

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:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

Commission Regulation (EC) No 440/2008, of 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

CAS Number: 68412-59-9
Batch: 1023S17201
Purity: 100%
Physical state/Appearance: White solid
Expiry Date: 09 July 2020
Storage Conditions: Room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: not applicable

Details on animal used as source of test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit.
Supplier: MatTek In Vitro Life Sciences Laboratories
Date received: 02 July 2019
EpiDermTM Tissues (0.63cm2) lot number: 30804
Assay Medium lot number: 062719MSC
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

Justification for test system used:

Recommended test by OECD TG.

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

0.1777 g.

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

Additionally for the 3 minute treatment time point, the media was aspirated and replaced with fresh medium and samples were incubated further to bring the total time to 60 min of incubation. At the end the samples were rinsed and incubated for further 3 h for the MTT-loading stage (for each treatment time point).

Number of replicates:

Duplicate

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 minute exposure time point

Run / experiment:

1

Value:

ca. 1.94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Remarks:

60 minute exposure time point

Run / experiment:

1

Value:

0.965

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 minute exposure time point

Run / experiment:

2

Value:

ca. 1.72

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Remarks:

60 minute exposure time point

Run / experiment:

2

Value:

ca. 1.435

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Mean OD570 Values and Viabilities for the Negative Control, Positive Control and Substance.

	3 -minute application viability (%)				1 -hour application viability (%)
	A (OD570)	B (OD570)	Mean (OD570)	SD (+/-)	A (OD570)	B (OD570)	Mean (OD570)	SD (+/-)
Negative control	2.073	2.066	2.070	0.005	2.234	2.320	2.277	0.061
Substance	1.940	1.720	1.830	0.156	0.965	1.435	1.200	0.332
Positive control	0.104	0.069	0.087	0.025	0.094	0.069	0.082	0.018

SD = Standard deviation

Duplicate exposures are indicated by A and B.

The relative mean viabilities for each treatment group were as follows:

Exposure period (min)	Viability (%)
	Negative control	Substance	Positive control
3	100*	88.4	4.2
60	100*	52.7	3.6

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

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is considered to be non-corrosive to skin based on a reliable in vitro skin corrosion study.

Executive summary:

In a reliable in vitro skin corrosion study, conducted according to the OECD Guideline 431, 'In Vitro Skin Corrosion: reconstructed human epidermis (RHE) test method', the undiluted substance (0.1777g) was applied onto reconstructed human skin tissue (epidermal model, EpiDerm^TM tissue (0.63cm2)) in duplicate for a period of 3 or 60 minutes.

 

Skin corrosion is expressed as the remaining cell viability after exposure to the substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the substance compared to the negative control tissues was 88.2% and 52.7%, respectively. Because the mean relative tissue viability for the substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the substance is considered to be not corrosive.

 

In conclusion, the substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this study.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1st October 2019 - 7th October 2019

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 18 June 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

CAS Number: 68412-59-9
Batch: 1023S17201
Purity: 100%
Physical state: White solid
Expiry Date: 09 July 2020
Storage Conditions: Room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

other: not applicable

Details on animal used as source of test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: EpiSkin Laboratories, Lyon, France
Date received: 01 October 2019
EpiSkinTM Tissues (0.38cm2) lot number: 19-EKIN-040
Maintenance Medium lot number: 19-MAIN3-043
Assay Medium lot number: 19-ESSC-041

Justification for test system used:

Recommended test system according to the OECD 439 TG.

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

Approxi.10 mg (26.3 mg/cm2) of substance.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative viability for tissue 1

Run / experiment:

1

Value:

102.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative viability for tissue 2

Run / experiment:

2

Value:

102.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative viability for tissue 3

Run / experiment:

3

Value:

107.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Direct MTT Reduction
The MTT solution containing the substance did not turn blue or purple which indicated that the test item did not directly reduce MTT.
Assessment of Color Interference with the MTT endpoint.
The solution containing the substance was a white color. This color was attributed to the intrinsic color of the substance itself. It was therefore unnecessary to run color correction tissues.

It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

Mean OD570 Values and Viabilities

	Optical density (OD570) of tissues				Relative viability of tissues (%)
	Tissue 1	Tissue 2	Tissue 3	Mean (+/-SD)	Tissue 1	Tissue 2	Tissue 1	Mean (+/-SD)
Negative control	0.818	0.978	1.002	0.933 (0.100)	87.7	104.8	107.4	*100 (10.7)
Substance	0.957	0.955	1.006	0.973 (0.029)	102.6	102.4	107.8	104.3 (3.1)
Positive control	0.066	0.081	0.078	0.075 (0.008)	7.1	8.7	8.4	8.1 (0.9)

SD = Standard deviation

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

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is considered to be non-irritant to the skin based on a reliable in vitro skin irritation study.

Executive summary:

In an in vitro skin irritation study conducted according to OECD TG 439 ‘In vitro Skin Irritation: Reconstructed Human Epidermis Test Method’ human skin tissue (eipdermis keratinocytes) was exposed to the undiluted substance for 15-minutes. Following the exposure, the substance was rinsed and incubated for further 42 hours in fresh medium. There was 104.3 % relative tissue viability following the 15-minute exposure point. This value is above the threshold for skin irritation (50%). Resultantly, the substance is considered to be a non-irritant to human skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

19th September 2019 - 10th October 2019

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:

Updated 09 October 2017

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)

Version / remarks:

EC No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on ‘The Collection of Tissues for Historical Evaluation and Collection of Data’. Series on Testing and Assessment, No. 160.

Version / remarks:

Adopted July 6, 2018 Paris.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

CAS Number: 68412-59-9
Batch: 1023S17201
Purity: 100%
Physical state/Appearance: White solid
Expiry Date: 09 July 2020
Storage Conditions: Room temperature in the dark

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Vehicle:

physiological saline

Remarks:

sodium chloride 0.9% w/v

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of Substance 20% w/v solution in sodium chloride 0.9% w/v;
0.75 mL of negative control;
0.75 mL of positive control;

Duration of treatment / exposure:

240 min at 32 ± 1 ºC.

Duration of post- treatment incubation (in vitro):

Following opacity measurements the corneas were used further for pereability measurement. The corneas were rinsed to remove the test/control substances and further incubated for 90 min with sodum fluorescein at 32 ± 1 ºC.

Number of animals or in vitro replicates:

triplicate

Details on study design:

Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 75 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading.
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.
Three corneas were randomly allocated to the test substsance, negative and positive control.

Treatment of Corneas.
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the substance preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire
cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein.
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 ± 1 ºC for 90 minutes.

Permeability Determinations.
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology.
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Data Evaluation.
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity Measurement.
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability Measurement.
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score.
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

Visual Observation.
The condition of the cornea was visually assessed post treatment.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

ca. 87.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

IVIS = 2.1

Positive controls validity:

valid

Remarks:

IVIS=108.1

Remarks on result:

positive indication of irritation

Treatment	Mean Opacity^1	Mean Permeability^1	Mean In vitro Irritation Score^1, 2
Negative control	2.0	0.003	2.1
Positive control (20% Imidazole)	85	1.538	108.1
Substance	86	0.095	87.4

¹    Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and substance.

²    In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

Corneal epithelium post-treatment condition was visually examined. For positive control and substance treated corneas appeared cloudy; the negative control treated corneas appeared clear.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The mean in vitro irritancy score (IVIS) of the test substance is 87.4. Therefore, the substance is classified to cause serious eye damage (Category 1).

Executive summary:

In a reliable in vitro eye irritation study, conducted according to 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’, the ability of the substance to induce opacity and permeability in an isolated bovine cornea were determined. The substance was applied 20% w/v solution (in sodium chloride 0.9% w/v) at 750 µL onto corneas (n=3) for a period of 240 min at 32 ± 1 ºC, followed by rinsing of the substance. 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 (following further 90 min incubation) was evaluated. According to opacity and permeability measurements, the substance resulted in a mean in vitro irritancy score (IVIS) of 87.4. In conclusion, since the substance induced an IVIS > 55, the substance is classified to cause serious eye damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the findings of reliable in vitro studies for skin and eye irritation conducted on the substance, it is classified as Category 1 Eye Damage.