Fatty acids, C18-unsatd., phosphates

Endpoint summary

Description of key information

Skin irritation/corrosion

In in vitro study according to OECD 439 fatty acids, C18-unsatd. is irritant in the in vitro skin irritation test under the experimental conditions described and should be classified category 1 or category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

In in vitro skin irritation study (OECD 431) the mean relative tissue viability for C18-unsatd., phosphates. was below 15% after the 1-hour treatment thus indicating it is considered to be corrosive. Subsequently, after 3 -min exposure the mean tissue viability was above 25%. In conclusion, C18-unsatd., phosphates is corrosive, category 1B.

Eye irritation

In vitro Bovine Corneal Opacity (BCOP) test conducted according to OECD 437 indicated an inconclusive result. An in vitro test using a Cornea Epithelial Model,

OECD 492) indicated either irritant or corrosive potential. In a follow-up in vivo eye irritation study (OECD 405) in a single rabbit sample of 0.1 mL of Fatty acids, C18-unsatd., phosphates. was instilled into one eye of one rabbit. Observations were made after 1, 24, 48 and 72 hours and 7, 14 and 21 days. Instillation of the test item resulted in effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity and epithelial damage. As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent 7, 14 and 21 days after instillation. The corneal injury did not completely resolve within 21 days. Based on the results of this study test substabce is corrosive, Category 1.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12.07.2017-06.09.2017

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)

GLP compliance:

yes

Specific details on test material used for the study:

IDENTIFICATION OF THE TEST MATERIAL
- Identification: Fatty acids, C18-unsatd., phosphates.
- CAS number: 68604-99-9
- EC number: 271-708-7
- Appearance: Yellow liquid

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0101891886
- Expiration date of the lot/batch: 02 November 2017
- Purity test date: 100% UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No specific handling conditions required
- Solubility and stability of the test substance in the solvent/vehicle: no
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not expected

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 25 μl of the undiluted test item was added into 12-well plates on top of the skin tissues.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm Skin Model (EPI-200, Lot no.: 26708 Kit L and M). The model consists of normal, human-derived epidermal keratinocytes which have been
cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum
containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of
10 mm cell culture inserts.
The skin tissues were kept in the refrigerator the day they were received. The next day, at
least 1 hour before the assay was started the tissues were transferred to 6-well plates
containing 0.9 ml DMEM per well. The level of the DMEM was just beneath the tissue (see
figure 1). The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0ºC. The
medium was replaced with fresh DMEM just before C18-unsatd., phosphates. was applied.
The test was performed on a total of 4 tissues per test item together with a negative control
and positive control. Two tissues were used for a 3-minute exposure to C18-unsatd.,
phosphates. and two for a 1-hour exposure. Fifty μl of the undiluted test item was added into
the 6-well plates on top of the skin tissues.
For the negative and positive controls, 2 tissues were treated with 50 μl Milli-Q water
(negative control) and 2 tissues were treated with 50 μl 8N KOH (positive control) for both
the 3-minute and 1-hour time point.
After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen
Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were
carefully dried. Rinsed tissues were kept in 24 well plates on 300 μl DMEM until 6 tissues
(= one application time) were dosed and rinsed.
4.7.5. Cell Viability Measurement
The DMEM was replaced by 300 μl MTT-medium and tissues were incubated for 3 hours at
37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and
formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room
temperature. The amount of extracted formazan was determined spectrophotometrically at
570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5%

Duration of treatment / exposure:

3-minute and 1-hour time points.

Number of replicates:

2

Amount / concentration applied:

- Amount(s) applied (volume or weight): 25 μl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5%

Irritation / corrosion parameter:

% tissue viability

Value:

6.8

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 μl of the test item was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed. No color change were observed.
- Colour interference with MTT: The test item was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the color interference, 10 μl of the test item was added to 90 μl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μl Milli-Q water was tested concurrently. At the end of the shaking period a color check was performed. No color changes were observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 6.8%. The absolute mean OD570(optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range.
- Acceptance criteria met for positive control: the positive control had a mean cell viability of 16% after 15 ± 0.5 minutes exposure. The mean relative tissue viability of the positive control should be <=50% relative to the negative control.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was less than 12%. The SD calculated from individual % tissue viabilities of the three identically treated replicates should be <=18.

Table 1. Mean absorption in the in vitro skin corrosion test with C18 -unsatd. phosphates. 3 -minute application

	A(OD570)	B(OD570)	Mean (OD570)	SD
Negative control	1.692	1.836	1.764	+-0.102
Test item	1.464	1.647	1.555	+-0.129
Positive control	0.115	0.135	0.125	+-0.014

SD= standard deviation, Duplicate exposures are indicated by A and B

Table 2. Mean tissue viability in the in vitro skin corrosion test with C18 -usatd. phosphates, 1 -hour application

	A(OD570)	B(OD570)	Mean (OD570)	SD
Negative control	1.702	1.566	1.634	+-0.096
Test item	0.090	0.106	0.098	+-0.011
Positive control	0.188	0.153	0.171	+-0.025

Table 3. Mean tissue vability in the in vitro skin corrosion test with C18 -usatd. phosphates.

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	88	6.0
Positive control	7.1	10

 

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

In conclusion, C18-unsatd., phosphates. is corrosive in the in vitro skin corrosion test under the experimental conditions described.

Executive summary:

The objective of this study was to evaluate C18-unsatd., phosphates. for its ability to induce skin corrosion on on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of C18-unsatd., phosphates. was tested through topical application for 3 minutes and 1 hour. The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 0101891886 of C18-unsatd., phosphates. was a yellow liquid. C18-unsatd., phosphates. was applied undiluted (50 μl) was applied directly on top of the skin tissue. The positive control had a mean relative tissue viability of 10% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit 2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was  11%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with C18-unsatd., phosphates. compared to the negative control tissues was 88% and 6.0%, respectively. Because the mean relative tissue viability for C18-unsatd., phosphates. was below 15% after the 1-hour treatment it is considered to be corrosive. Subsequently, after 3 -min exposure the mean tissue viability was above 25%. In conclusion, C18-unsatd., phosphates. is corrosive, category 1B in the in vitro skin corrosion test under the experimental conditions described.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27.03.2017-01.05.2017

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)

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Fatty acids, C18-unsatd., phosphates.
CAS number: 68604-99-9
EC number: 271-708-7
Appearance: Yellow liquid
Batch: 0101891886
Purity/Composition: 100% UVCB
Test item storage: At room temperature
Stable under storage conditions until: 02 November 2017 (retest date)
Test Facility test item number: 208073/A
Chemical name (IUPAC, synonym or trade name: Phosphorylated fatty acid
pH (1% in water, indicative range): 2.28 – 2.21 (determined by Charles RiverDen Bosch)
Specific gravity / density: 1.04
Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not expected

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 25 μl of the undiluted test item was added into 12-well plates on top of the skin tissues.

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: EPISKIN Small Model TM
- Tissue batch number(s): 17-EKIN-013 and 17-EKIN-017
- Production date:
- Shipping date:
- Delivery date:
- Date of initiation of testing:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: incubator 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 ml MTT solution (0.3 mg/ml in PBS)
- Incubation time: 3h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 ± 0.5 minutes

Observation period:

42 hours

Number of animals:

not used, in vitro study

Details on study design:

TEST SITE
- Area of exposure:
- % coverage:
- Type of wrap if used:

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
- Time after start of exposure:

OBSERVATION TIME POINTS
(indicate if minutes, hours or days)

SCORING SYSTEM:
- Method of calculation:

Irritation / corrosion parameter:

% tissue viability

Value:

6.8

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 μl of the test item was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed. No color change were observed.
- Colour interference with MTT: The test item was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the color interference, 10 μl of the test item was added to 90 μl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μl Milli-Q water was tested concurrently. At the end of the shaking period a color check was performed. No color changes were observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 6.8%. The absolute mean OD570(optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range.
- Acceptance criteria met for positive control: the positive control had a mean cell viability of 16% after 15 ± 0.5 minutes exposure. The mean relative tissue viability of the positive control should be <=50% relative to the negative control.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was less than 12%. The SD calculated from individual % tissue viabilities of the three identically treated replicates should be <=18.

Mean Absorption in the In Vitro Skin Irritation Test with the test item

First experiment

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)	SD
Negative control	1.005	1.002	1.096	1.035	± 0.053
Test item	0.420	0.560	0.718	0.566	± 0.053
Positive control	0.031	0.037	0.048	0.039	± 0.053

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C. In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

Second experiment

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)	SD
Negative control	1.000	0.990	1.103	1.031	± 0.062
Test item	0.074	0.073	0.061	0.070	± 0.007
Positive control	0.093	0.102	0.303	0.166	± 0.119

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C. In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

Mean Tissue Viability in the In Vitro Skin Irritation Test with the test item

First experiment

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	5.2
Test item	55	14
Positive control	3.7	0.86

Second experiment

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	6.1
Test item	6.8	0.69
Positive control	16	12

Individual OD Measurements at 570nm

First experiment

		A (OD570)	B (OD570)	C (OD570)
Negative control	OD570 measurement 1	1.0115	1.0503	1.1360
	OD570 measurement 2	1.0819	1.0371	1.1392
Test item	OD570 measurement 1	0.4606	0.6141	0.7844
	OD570 measurement 2	0.4616	0.5890	0.7339
Positive control	OD570 measurement 1	0.0732	0.0797	0.0897
	OD570 measurement 2	0.0710	0.0767	0.0894

Second experiment

		A (OD570)	B (OD570)	C (OD570)
Negative control	OD570measurement 1	1.0618	1.0242	1.1788
	OD570measurement 2	1.0212	1.0376	1.1089
Test item	OD570measurement 1	0.1317	0.1230	0.1208
	OD570measurement 2	0.0989	0.1058	0.0842
Positive control	OD570measurement 1	0.1439	0.1496	0.4049
	OD570measurement 2	0.1248	0.1356	0.2839

Historical Control Data for In Vitro Skin Irritation Studies

	Negative control (absorption; OD570)	Postive control (absorption; OD570)	Positive control (viability; %)
Range	0.676 -1.336	0.036 -0.549	2.85 -45.43
Mean	1.01	0.16	15.74
SD	0.16	0.10	9.22
n	155	154	163

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

In conclusion, Fatty acids, C18-unsatd. is irritant in the in vitro skin irritation test under the experimental conditions described and should be classified category 1 or category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

The objective of this study was to evaluate Fatty acids, C18-unsatd. for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM^TM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 0101891886 of the test item was a yellow liquid. The test item was applied undiluted (25 µl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive control had a mean cell viability of 3.7% 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 less than 15%, indicating that the test system functioned properly.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 55%. Since the individual values were both above and below 50% (41, 54 and 69% respectively) the test was inconclusive and a repeat experiment was performed.

In the second test,the positive control had a mean cell viability of 16% after 15 ± 0.5 minutes exposure. The relative mean tissue viabilityobtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 6.8%. The standard deviation value of the percentage viability of three tissues treated identically was less than 12%, for the negative and positive control indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was below or equal to 50% after 15 ± 0.5 minutes treatment it is considered to be irritant. 

In conclusion, Fatty acids, C18-unsatd. is irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26-Jul-2017- 10-Oct-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Fatty acids, C18-unsatd., phosphates.
CAS number: 68604-99-9
EC number: 271-708-7
Appearance: Yellow liquid
Batch: 0101891886
Purity/Composition: 100% UVCB
Test item storage: At room temperature
Stable under storage conditions until: 02 November 2017 (retest date)
Test Facility test item number: 208073/A
Chemical name (IUPAC, synonym or trade name: Phosphorylated fatty acid
pH (1% in water, indicative range): 2.28 – 2.21 (determined by Charles RiverDen Bosch)
Specific gravity / density: 1.04

Species:

rabbit

Strain:

New Zealand White

Remarks:

1 male

Details on test animals or tissues and environmental conditions:

Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19 to 20°C with an actual daily mean relative humidity of 58 to 90%

12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Pelleted diet for rabbits (Global Diet 2030 Teklad®, Mucedola, Milanese, Italy) was provided once daily throughout the study. In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was available during the study period. The feed was analyzed by the supplier for nutritional components and environmental
contaminants. Results of the analysis were provided by the supplier and are on file at the TestFacility.It is considered that there were no known contaminantsin the feed that would interfere withthe objectives of the study.

Municipal tap-water was freely available to each animal via water bottles. Periodic analysis of the water was performed.It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

For psychological/environmental enrichment, the animal was provided with shelter (Ebeco, Germany, dimensions 40 x 32 x 23 cm) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) except when interrupted by study procedures/activities.

Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel).

One hour prior to instillation of the test item, buprenorphine (Buprenodale®) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia. Five minutes prior to instillation of the test item, two drops of the topical anaesthetic 0.5% proparacaine hydrochloric ophthalmic solution (Alcaine eye drops®) were applied to botheyes.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

0.1 ml

Observation period (in vivo):

21 d

Details on study design:

The animal was treated by instillation of 0.1 mL of the test item, in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test item. The other eye remained untreated and served as the reference control. Additional injections of buprenorphine 0.01 mg/kg were administered immediately after the 1-hour observation and at the end of the first day to reduce pain and distress.Additional injections of buprenorphine 0.03 mg/kg were supplied approximately 4.5 hours after dosing to reduce pain and distress.Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt,Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. This procedure was repeated to assess recovery. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area. Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH, Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection. Additional injections of buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg were administered immediately after the 48-hour observation to reduce pain and distress.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24 h

Score:

1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

48 h

Score:

1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

72 h

Score:

1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

7 d

Score:

1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

14 d

Score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

21 d

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24 h

Score:

1

Irritation parameter:

iris score

Basis:

animal #1

Time point:

48 h

Score:

1

Irritation parameter:

iris score

Basis:

animal #1

Time point:

72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

7 d

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

14 d

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

21 d

Score:

0

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24 h

Score:

3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

48 h

Score:

3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

72 h

Score:

3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

7 d

Score:

3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

14 d

Score:

2

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

21 d

Score:

1

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24 h

Score:

3

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

48 h

Score:

2

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

72 h

Score:

2

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

7 d

Score:

1

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

14 d

Score:

1

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

21 d

Score:

1

Irritation parameter:

other: conjuctivae disharge score

Basis:

animal #1

Time point:

24 h

Score:

2

Irritation parameter:

other: conjuctivae disharge score

Basis:

animal #1

Time point:

48 h

Score:

2

Irritation parameter:

other: conjuctivae disharge score

Basis:

animal #1

Time point:

72 h

Score:

2

Irritation parameter:

other: conjuctivae disharge score

Basis:

animal #1

Time point:

7 d

Score:

1

Irritation parameter:

other: conjuctivae disharge score

Basis:

animal #1

Time point:

14 d

Score:

1

Irritation parameter:

other: conjuctivae disharge score

Basis:

animal #1

Time point:

21 d

Score:

1

Irritant / corrosive response data:

The corneal injury consisted of opacity and epithelial damage. As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent between 7 and 21 days after instillation. The corneal injury did not completely resolve within 21 days. Iridial irritation was observed and resolved within 72 hours. The irritation of the conjunctivae consisted of redness, chemosis and discharge and did not completely resolve within 21 days.

Mean value eye irritation scores (mean 24, 48, 72 hours): corneal opacity: 1.0, iris 0.7, conjuctivae redness 3.0, conjuctivae chemosis 2.3.

Interpretation of results:

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

Conclusions:

According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), Fatty acids, C18-unsatd., phosphates. should be classified as: having irreversible effects on the eyes (Category 1).

Executive summary:

The objective of this acute eye irritation study was to assess the possible irritation or corrosion potential when a single dose of Fatty acids, C18-unsatd., phosphates. was placed in the conjunctival sac of the rabbit eye. The study was carried out in compliance with the guidelines described in:

• OECD No.405 (2012) "Acute Eye Irritation / Corrosion"

• EC No 440/2008, part B: "Acute Toxicity: Eye Irritation/Corrosion"

• EPA, OPPTS 870.2400 (1998), "Acute Eye Irritation"

• JMAFF Guidelines (2000), including the most recent revisions.

A single sample of 0.1 mL of Fatty acids, C18-unsatd., phosphates. was instilled into one eye of one rabbit. Observations were made 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation.

Instillation of the test item resulted in effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity and epithelial damage. As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent 7, 14 and 21 days after instillation. The corneal injury did not completely resolve within 21 days.

Iridial irritation was observed and resolved within 72 hours. The irritation of the conjunctivae consisted of redness, chemosis and discharge and did not completely resolve within 21 days.

Based on these results:

• according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), Fatty acids, C18-unsatd., phosphates. should be classified as: having irreversible effects on the eyes (Category 1).

• according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Fatty acids, C18-unsatd., phosphates. should be classified as Irreversible effects on the eye (Category 1) and labeled as H318: Causes serious eye damage.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03.03.2017-04.05.2017

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)

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Fatty acids, C18-unsatd., phosphates.
CAS number: 68604-99-9
EC number: 271-708-7
Appearance: Yellow liquid
Batch: 0101891886
Purity/Composition: 100% UVCB
Test item storage: At room temperature
Stable under storage conditions until: 02 November 2017 (retest date)
Test Facility test item number: 208073/A
Chemical name (IUPAC, synonym or trade name: Phosphorylated fatty acid
pH (1% in water, indicative range): 2.28 – 2.21 (determined by Charles RiverDen Bosch)
Specific gravity / density: 1.04

Species:

other: in vitro study

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Protocol: Bovine eyes were used as soon as possible after slaughter.
- Source: Bovine eyes from young cattle were obtained from the
slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands),
where the eyes were excised by a slaughterhouse employee as
soon as possible after slaughter.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32±1°C
- Cell Culture Conditions: The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential
Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine
(Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 ul
- The test item was tested neat.

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minute

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine(Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
yes

SOLVENT CONTROL USED (if applicable)
no

POSITIVE CONTROL USED
yes

APPLICATION DOSE AND EXPOSURE TIME
750ul 10 min +/- 1min

TREATMENT METHOD:
open chamber

POST-INCUBATION PERIOD:
yes 120 min no.

REMOVAL OF TEST SUBSTANCE
Number of washing steps after exposure period: 2

- POST-EXPOSURE INCUBATION:
the corneas were incubated for 120 +/- 10 minutes at 32 +/- 1 degC.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
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 according to:

opacity =I0/I − 0.9894 / 0.0251

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging corrected opacity values of the treated corneas for each treatment group.

- Corneal permeability:
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 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/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 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range(linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints. The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1

DECISION CRITERIA:
The assay is considered acceptable if:
 The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
 The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Irritation parameter:

in vitro irritation score

Value:

15.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0,2

Positive controls validity:

valid

Remarks:

50,6

Remarks on result:

not determinable

Remarks:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Irritation parameter:

cornea opacity score

Value:

8.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0,1

Positive controls validity:

valid

Remarks:

19,8

Irritation parameter:

fluorescein leakage

Value:

0.431

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0,007

Positive controls validity:

valid

Remarks:

2,056

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item showed opacity values ranging from 3.8 to 14.2 and permeability values ranging from 0.342 to 0.535 The corneas were translucent after the
10 minutes of treatment with the test item. In addition, some test item residue was visible on the corneas. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no color change of the medium was observed. Hence, the in vitro irritancy scores ranged from 10 to 22 after 10 minutes of treatment with the test item.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The negative control responses for opacity and permeability were less than the upper limits of
the laboratory historical range indicating that the negative control did not induce irritancy on
the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 51 and
within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test
system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The individual in vitro irritancy scores for the negative controls ranged from -0.3 to 0.5. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: Yes, The individual positive control in vitro irritancy scores ranged from 45.2 to 61.5. The mean in vitro irritancy score of the positive control (Ethanol) was 51 and within two standard deviations of the current historical positive control mean
- Range of historical values if different from the ones specified in the test guideline:

Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity^1	Mean Permeability^1	MeanIn vitroIrritation Score^1, 2
Negative control	0.1	0.007	0.2
Positive control (Ethanol)	19.8	2.056	50.6
The test item	8.9	0.431	15.4

¹  Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

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

Opacity Score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1		Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	2.1	2.5	0.4			0.1
	1.6	1.3	-0.3
	3.5	3.6	0.2
Positive control	2.9	22.8	20.0	19.9		19.8
	3.6	21.8	18.1	18.1
	4.7	26.3	21.6	21.5
Test item	2.1	6.0	3.9	3.8		8.9
	3.9	18.2	14.3	14.2
	2.3	11.1	8.8	8.7

1    Final Opacity = Opacity after treatment – Opacity before treatment.

²    Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control.

³  Calculations are made without rounding off.

     
Permeability ScoreIndividual Values (Uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2		OD490 3		Average OD		Final OD		Mean final negative control
Negative control	1	0.008		0.009		0.008		0.008		0.008		0.007
	1	0.001		0.001		0.002		0.001		0.001
	1	0.010		0.011		0.011		0.011		0.011
Positive control	6	0.473		0.466		0.468		0.469		2.814
	6	0.313		0.308		0.307		0.309		1.856
	6	0.269		0.270		0.271		0.270		1.620
Test item	1	0.428		0.418		0.420		0.422		0.422
	1	0.539		0.545		0.541		0.542		0.542
	1	0.352		0.340		0.354		0.349		0.349

¹    Calculations are made without rounding off.

    
Permeability Score Individual Values (Corrected)

Treatment	Dilution factor	Negative control corrected OD4901^1	Negative control corrected OD4902^1	Negative control corrected OD4903^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Positive control	6	0.466	0.459	0.461	0.462	2.773	2.056
	6	0.306	0.301	0.300	0.303	1.815
	6	0.262	0.263	0.264	0.263	1.579
Test item	1	0.421	0.411	0.413	0.415	0.415	0.431
	1	0.532	0.538	0.534	0.535	0.535
	1	0.345	0.333	0.347	0.342	0.342

¹    OD₄₉₀values corrected for the mean final negative control permeability (0.007).

²    Calculations are made without rounding off.

 

      
In Vitro Irritancy Score

Treatment	Final Opacity^2	Final OD490^2	In vitroIrritancy Score^1
Negative control	0.4	0.008	0.5
	-0.3	0.001	-0.3
	0.2	0.011	0.3
Positive control	19.9	2.773	61.5
	18.1	1.815	45.3
	21.5	1.579	45.2
Test item	3.8	0.415	10.0
	14.2	0.535	22.2
	8.7	0.342	13.9

¹  In vitro irritancy score (IVIS) = opacity value + (15 x OD₄₉₀value).

²  Positive control and test item are corrected for the negative control.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, Fatty acids, C18-unsatd., phosphates.induced an IVIS > 3 ≤ 55 in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the experimental conditions described in this report. No prediction on the classification can be made.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Fatty acids, C18-unsatd., phosphates. as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 51 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 

The test item was tested neat. The test item induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 15 after 10 minutes of treatment. In conclusion, since Fatty acids, C18-unsatd., phosphates. induced an IVIS > 3 ≤ 55, no prediction on the classification can be made. 

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.05.2017-05.05.2017

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)

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Fatty acids, C18-unsatd., phosphates.
CAS number: 68604-99-9
EC number: 271-708-7
Appearance: Yellow liquid
Batch: 0101891886
Purity/Composition: 100% UVCB
Test item storage: At room temperature
Stable under storage conditions until: 02 November 2017 (retest date)
Test Facility test item number: 208073/A
Chemical name (IUPAC, synonym or trade name: Phosphorylated fatty acid
pH (1% in water, indicative range): 2.28 – 2.21 (determined by Charles RiverDen Bosch)
Specific gravity / density: 1.04

Species:

other: in vitro study

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Protocol: In vitro EpiOcularTM eye irritation test (OCL-200-EIT)
- Source: MatTek Corporation
- Cell line: The EpiOcular™ human cell construct
- Lot: 23479
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37±1°C
- Cell Culture Conditions: a humidified atmosphere of 5±0.5% CO2 in air

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl

Duration of treatment / exposure:

30 ± 2 minutes

Observation period (in vivo):

120 ± 15 minutes

Duration of post- treatment incubation (in vitro):

12 ± 2 minute

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
The EpiOcularTM cultures were pre-incubated at 37±1°C in a humidified atmosphere (80-100%) containing 5±0.5% CO2 for 60 min. At the end of the first pre-incubation period, the medium was replaced by 1 mL of fresh assay medium. Further, the tissues were pre-incubated 20 ± 4 hours at 37°C. No correction was made for the purity/composition of the test item. After pre-incubation tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS, then 50 μL of control and test items were applied topically onto the tissue surface and incubated at standard culture conditions for 30 ± 2 minutes. Two tissues were used per treatment, negative and positive controls. After exposure period, each tissue was rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove any residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) for a 12 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to 1.0 ml of warm Assay Medium and were incubated for 120±15 minutes at 37°C.
- RhCE tissue construct used, including batch number
EpiOcular™ human cell construct Lot: 23479
- Doses of test chemical and control substances used
50 μL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Exposure: 30 ± 2 minutes at 37.0 ± 1.0°C
Post-exposure immersion: 12 ± 2 minute at room temperature
Post-exposure incubation: 120±15 minutes at 37°C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 μl of the test item was added to 1 ml MTT solution (1 mg/ml MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark. A negative control, 50 μl sterile Milli-Q water was tested concurrently. If the MTT solution color turned blue / purple or if a blue/purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
Spectrophotometer TECAN Infinite® M200 Pro Plate Reader 570nm
- Positive and negative control means and acceptance ranges based on historical data
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be >0.8 and <2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.
- Acceptable variability between tissue replicates for positive and negative controls
- Acceptable variability between tissue replicates for the test chemical
The SD calculated from individual % tissue viabilities of the two identically treated replicates should be <20%. All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Irritation parameter:

in vitro irritation score

Run / experiment:

The mean tissue viability (%)

Value:

11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not observed

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
The in vitro eye irritation test is considered acceptable if it meets the following criteria:
a)The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and <2.5.
b)The mean relative tissue viability of the positive control should be <50% relative to the negative control.
c)The SD calculated from individual % tissue viabilities of the two identically treated replicates should be <20%.All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Mean Absorption in the EpiOcularTM Test with the test item

	A (OD570)	B (OD570)	Mean (OD570)	SD
Negative control	1.077	1.563	1.320	±0.344
Test item	0.120	0.175	0.148	±0.039
Positive control	0.453	0.037	0.245	±0.295

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B. In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

Mean Tissue Viability in the EpiOcularTM Test with the test item

 

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	26
Test item	11	3.0
Positive control	19	22

Individual OD Measurements at 570Nm

		A (OD570)	B (OD570)
Negative control	OD570 measurement 1	1.1226	1.6181
	OD570 measurement 2	1.1140	1.5909
Test item	OD570 measurement 1	0.1607	0.2187
	OD570 measurement 2	0.1624	0.2148
Positive control	OD570 measurement 1	0.5052	0.0781
	OD570 measurement 2	0.4845	0.0781

Historical Control Data for EpiOcularTM Studies

	Negative control (absorption; OD570)	Positive control (absorption; OD570)	Positive control (viability; %)
Range	1.077 -1.805	0.029 -0.793	2.11 -48.25
Mean	1.52	0.42	26.86
SD	0.21	0.23	14.11
n	16	16	16

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, Fatty acids, C18-unsatd., phosphates. is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Fatty acids, C18-unsatd., phosphates. For this purpose Fatty acids, C18-unsatd., phosphates. was topically applied on the Reconstructed Human EpiOcular™ Model. The possible eye hazard potential of Fatty acids, C18-unsatd., phosphates. was tested through topical application for 30 minutes. The study procedures described in this report were based on the most recent OECD guideline. Batch 0101891886 of the test item was a yellow liquid. The test item was applied undiluted (50 μl) directly on top of the tissue for 30+-2 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control had a mean cell viability of 19% after 30+-2 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 two tissues treated with the test item was less than 3%, indicating that the test system functioned properly. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30+-2 minutes treatment with the test item compared to the negative control tissues was 11%. Since the mean relative tissue viability for the test item was below or equal to 60% after 30 +- 2 minutes treatment it is considered to be potentially irritant or corrosive to the eye. In conclusion, Fatty acids, C18-unsatd., phosphates. is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Additional information

Skin irritation

Table 1 . Mean tissue vability in the in vitro skin corrosion test (OECD 431) with C18 -usatd. phosphates.

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	88	6.0
Positive control	7.1	10

Eye irritation

Table 1. Mean Tissue Viability in the EpiOcularTM Test (OECD 492) with the test item

 

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	26
Test item	11	3.0
Positive control	19	22

Justification for classification or non-classification

Based on the in vitro study results, C18-unsatd., phosphates has to be classified as a hazard class Skin Corr. 1B H314 with the risk of serious damage to the eyes according to CLP Regulation 1272/2008.