Fatty acids, lanolin, lithium salts

Endpoint summary

Description of key information

Skin

The test item was not irritant to skin based on the study according to OECD 439 guideline. The mean relative absorbance value of the test item, corresponding to the cell viability did not significantly decrease (103.2 %; threshold for irritancy: ≤ 50%). The test item was non-corrosive in a study following OECD 431 guideline. The mean viability of cells exposed to the test item was 98.1% after a 3-minute exposure period and 95.7% after a 1-hour exposure (corrected viability calculated for MTT reducing test items using freeze-killed control tissues).

Eye

In the BCOP test, the calculated In Vitro Irritation Score (IVIS) of the test substance was below the cut-off value of 3 (UN GHS no category) for identifying test substances as inducing serious eye damage (UN GHS Category 1) therefore the test item should not be classified as a severe irritant and is not corrosive according to UN GHS classification. In the EpiOcular™ Eye Irritation Test (EIT) the mean percentage viability of the EpiOcular™ tissues treated with test item was 3.6%, relative to negative controls. Under the present test conditions the OD540 values obtained from Fatty acids, lanolin, lithium salts were below the cut-off percentage of cell viability value that distinguishes irritant from non-irritant test items of ≤ 60%, therefore it was concluded that the test item is predicted to be eye irritant.

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

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC method B.40 tris: In vitro Skin Corrosion: Human Skin Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SOAP 012-2
- Expiration date of the lot/batch: November 01, 2022
- Purity test date: 13 December 2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10 to +25°C, kept containers tightly closed in a cool, well-ventilated place, kept away from heat, sparks and open flame.


FORM AS APPLIED IN THE TEST (if different from that of starting material) 25 mg of test item were applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface. The test item is a fine powder.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The test item was applied topically to a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum. Corrosive materials are identified by their ability to produce a decrease in cell viability (as determined, for example, by using the MTT reduction assay) below defined threshold levels at specified exposure periods. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SCT)
- Tissue batch number(s): Lot no. 25878

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg MMT /mL
- Incubation time: 3 h
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.968 ± 0.147 (acceptance criteria: 1.0-3.0)
- Barrier function: 6.8 h (acceptance criteria:4.77-8.72)
- Contamination: No contamination

NUMBER OF REPLICATE TISSUES: Two replicate tissues for each treatment (exposure periods) were employed

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The frozen tissues were stored in the freezer (-20 ± 5°C).
The test item was applied to two freeze-killed tissues. In addition, two freeze-killed tissues were left untreated. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDerm tissues.

- Fresh tissues / killed tissues: Freeze-killed tissues. Due to interacting of the test item with the MTT measurement (reduction of MTT) an additional test with freeze-killed tissues had to be performed.
- N. of replicates : 2
- Method of calculation used:

For the MTT reducing test item, calculations were corrected by values of correspondent additional controls.
True viability = Viability of treated tissue – Interference from test chemical = OD tvt – OD kt
where OD kt = (mean OD tkt – mean OD ukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)
If the interference by the test substance is greater than 30% of the negative control value, additional steps must be taken into account or the test substance may be considered incompatible with this test.

PREDICTION MODEL / DECISION CRITERIA
-The criteria of corrosivity associated with the EpiDermTM model are as follows:
- the test item is considered to be corrosive to skin and classified as category 1 (or optional category 1A ), if the viability after 3 minutes exposure is less than 50%;
- the test item is considered to be corrosive to skin and classified as sub-category 1B-and-1C, if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%;
- the test item is considered to be non-corrosive to skin, if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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

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


POSITIVE CONTROL : 8 N KOH
- Amount(s) applied (volume or weight): 50 µL

Duration of treatment / exposure:

1st period: 3 min; 2nd period: 60 min

Number of replicates:

Two replicate tissues for each treatment (exposure periods) were employed.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute ecposure

Value:

98.1

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

Run / experiment:

1 hour exposure

Value:

95.7

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:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean optical density (OD) of the negative control of 2 tissues was 1.456 (3 minute exposure) or 1.552 (1-hour exposure) and therefore well within the acceptable range of ≥ 0.8 to ≤ 2.8
- Acceptance criteria met for positive control: The viability of cells treated with the positive reference item 8 N KOH was 11.2% or 4.4% (3-minute or 1-hour exposure, respectively) of the negative control and, hence well below the 15% cut-off value at the 1-hour exposure
- Acceptance criteria met for variability between replicate measurements: The difference of viability between the two tissue replicates (at 20 - 100% viability) was below the limit of acceptance of 30%.
All acceptance criteria were fulfilled.

Historical data of negative and positive controls
(Most recent background data from GLP studies of the years 2015 - 2017 (n = 14).

Material	Average OD (mean% difference ±SD)	Average viability [%] (mean% difference ±SD)	Range		No. of unqualified experi-ments
			Viability [%]	% difference
Short time incubation – 3‑min
Negative control (Non-Corrosive)	1.624 (2.5±2.4)	100 (2.5±2.4)	93.7–106.3	0.14–8.6	0^#1
8 N KOH (Corrosive)	0.126 (8.8±7.4)	7.6 (8.8±7.4)	2.0–15.6	<0.01–23.7	0
Long time incubation – 60‑min
Negative control (Non-Corrosive)	1.650 (4.0±5.0)	100 (4.0±5.0)	85.6–114.4	0.13–18.3	0^#1
8 N KOH (Corrosive)	0.090 (5.7±9.8)	5.9 (5.7±9.8)	2.0–12.6	0.30–38.4	0^#2

OD: Optical density. Viability for negative control is set = 100%

SD: Standard deviation

CV: Coefficient of variation

^#1     Unqualified results =       if the mean OD of the NC tissues is < 0.8 or > 2.8

                                            if difference in viability for duplicate tissues > 30%

^#2     Unqualified results =       8 N KOH: viability > 15% (1-hour exposure)

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions Fatty acids, lanolin, lithium salts tested at two exposure periods of 3 minutes or 1 hour was non-cytotoxic and, hence, predicted to be non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to assess the corrosive properties of Fatty acids, lanolin, lithium salts to human skin,in an experiment with an artificial three-dimensional model of human skin. TheEpiDerm™model was employed.

The test item Fatty acids, lanolin, lithium salts, the negative control sterile deionised water or the positive reference item 8 N KOH were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.

Two tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined byusing the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assayand expressed as relative percentage of viability of the negative control-treated tissues.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 98.1% after a 3-minute exposure period and 95.7% after a 1-hour exposure (corrected viability calculated for MTT reducing test items using freeze-killed control tissues) and hence ,the 3-minute and the1-hour exposure values were above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of ≥50% and ≥15%, respectively.Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

Conducted according to OECD Test Guideline 431 and GLP, the study is considered to be reliable without restriction (Klimisch 1).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 February 2018 - 23 February 2018

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 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No. 640/2012, Method B.46: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, adopted July 06, 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: SOAP 012-2
- Expiration date of the lot/batch: November 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Physical characteristics: Solid
- Storage condition of test material: At ambient temperature (10 - 25°), container kept tightly closed and stored in a dry and well-ventilated place.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The optical properties of the test item were evaluated under aqueous conditions (25 mg of test item was mixed with 300 μL deionized water and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes). No discolorations were noted. The test item was also evaluated for its potential to interfere with the MTT assay reagent, via reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt. Therefore, 25 mg of test item was added to 1 mL MTT medium and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. Untreated MTT solution was used as control. A discoloration of the test item to greenish-black was noted. Hence, due to interacting of the test item with the MTT measurement (reduction of MTT) an additional test with freeze-killed tissues was performed.
The frozen tissues were stored in the freezer (-20 ± 5°C).
The test item was applied to two freeze-killed tissues. In addition, two freeze-killed tissues were left untreated. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDerm tissues. The correction factor in this test resulted in a negative value, therefore no influence of the test item on the optical density is assessed and, hence, no correction for test item reduction was calculated.


OTHER SPECIFICS:
Administration of the test, negative and positive reference item: As a fine powder, 25 mg of test item was applied to the skin model with a surface area of 0.63 cm2 moistened with 25 mL of Dulbecco’s phosphate buffered saline to uniformly cover the skin surface.

Test system:

human skin model

Source species:

human

Cell type:

other: Differentiated epidermal keratinocytes

Cell source:

other: Keratinocyte strain 00267

Source strain:

other: All cells used to produce EpiDerm tissue are purchased or derived from tissue obtained from accredited institutions, from the donor or the donor's legal next of kin for use of the tissues or derivatives of the tissue for research purposes

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Human
- Tissue: normal epidermal keratinocytes

Justification for test system used:

The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis
- Source: MatTek in vitro Life Sceince Laboratories (Bratislava, Slovakia)
- Tissue batch number(s): 23349
- Date of analysis of tissue functionality and quality: 21 February 2018
- Date of initiation of testing: February 2018

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm
- Replicates: The spectrophotometer measurements were made for each of the three tissues in two replicates.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Manufacturer MTT assay, 1.52 ± 0.086 OD (acceptable range: 1.0 to 3.0)
- Barrier function: Manufacturer ET-50 assay, 7.27 hrs (acceptable range: 4.77 to 8.72 hrs)
- Contamination: Manufacturer long term antibiotic and antimycotic free culture, sterile (acceptable criteria: no contamination)

NUMBER OF REPLICATE TISSUES: 3

ASSAY ACCEPTABILITY CRITERIA
- Negative control: The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 1.0 and ≤ 2.5.
- Positive control: A 5% SDS (in H2O) solution was used as a positive control (PC) and tested concurrently with the test chemical. Concurrent means here that the PC has to be tested in each assay, but only one PC is required per testing day. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%.
- Standard deviation: Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%.

PREDICTION MODEL / DECISION CRITERIA
- Justification for the selection of the cut-off point(s): According to the EU and GHS classification (H314 or H315 / Category 1/2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.
mean tissue viability ≤ 50% Irritant (I), (H314 or H315 or GHS Category 1 or 2)
mean tissue viability > 50% non-irritant (NI).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of the test item was applied to 0.63 cm2 skin model

NEGATIVE CONTROL
- Negative control: Dulbecco’s Phosphate Buffered Saline (D-PBS)
- Source and lot/batch number: GIBCO Invitrogen GmbH (76131, Germany) batch no# 1908933
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Positive control: Sodium dodecyl sulphate
- Source and lot/batch number: MatTek Corporation batch no# 031617 MGKA
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% aqueous solution

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hour

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

results presented as decimal figure (i.e. 100% = 1)

Run / experiment:

Mean % optical density (OD540) relative to negative controls (n= 3)

Value:

103.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: absolute optical density (OD 540)

Run / experiment:

Tissue 1

Value:

1.537

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: absolute optical density (OD 540)

Run / experiment:

Tissue 2

Value:

1.691

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: absolute optical density (OD 540)

Run / experiment:

Tissue 3

Value:

1.675

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: absolute optical density (OD540)

Run / experiment:

Mean of tissues (n=3)

Value:

1.634

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:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean optical density (OD) of 3 negative control tissues was 1.584 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.
- Acceptance criteria met for positive control: The viability of cells treated with the positive reference item, 5% SDS, was 7.7 % of the negative control and fulfilled the acceptance criterion of ≤ 20%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation determined for all triplicates was below the limit of acceptance of 18%.
- Range of historical values if different from the ones specified in the test guideline: Results for positive and negative control were in line with historical data. All acceptance criteria required were fulfilled.

Historical data of negative and positive controls

Summary of EpiDerm Skin Irritation tested in independent lots of EpiDerm. If tissue viability is≤50% the test material is labelled as an irritant; if viable > 50%, the test material is labelled as a non-irritant.

 

Material	Average OD (mean CV%)	Average Viability%(mean CV%)	Range		Irritant (I) / Non-irritant (NI)	No. of Unqualified Experiments
			Viability [%]	CV [%]
Negative control (DPBS)	1.776 (5.0±3.0)	100 (5.0±3.0)	87.4–110.8	1.1–11.8	NI	0^#1
Positive control (SDS5%)	0.085 (7.8±3.6)	5.1 (8.3±4.0)	1.3–10.2	1.8–15.1	I	0^#2

 ^#1Unqualified results = if the mean OD of the NC tissues is < 1.0 or > 2.5
if difference in viability for duplicate tissues > 18%

^#2Unqualified results = viability > 20%

CV coefficient of variation

SDS Sodium dodecyl sulphate

DPBS Dulbecco's Phosphate-Buffered Saline

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, test item was non-cytotoxic in an in vitro experiment employing an artificial three-dimensional model of human skin. The test item did not show irritant properties and should not be classified as irritant (UN GHS no category).

Executive summary:

Skin irritation of the test item was evaluated with the EpiDerm Reconstructed Human Epidermis Model. Cell viability of the multi-layered tissue culture of highly differentiated epidermal keratinocytes topically exposed to the test substance was evaluated using the MTT assay, which measures the conversion of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt.

Undiluted test item was applied to the EpiDerm tissue for 60 minutes, alongside a negative and positive control. The mean relative absorbance value of the test item, corresponding to the cell viability did not significantly decrease (103.2 %; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

Conducted according to OECD Test Guideline 439 and GLP, the study is considered to be reliable without restriction (Klimisch 1).

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 February - 14 February 2018

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:

Adopted July 26, 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No. 1152/2010 method B.47 (published in the Official Journal of the European Union L324, dated December 09, 2010)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: sponsor batch# SOAP 012-2
- Manufacture date of the lot/batch: Not reported
- Expiration date of the lot/batch: November 2022
- Purity test date: 13 December 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Physical characteristics: Beige to brown, solid
- Storage condition of test material: At ambient temperature (10 - 25°), container kept tightly closed and stored in a dry and well-ventilated place.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Vehicle: 0.9% sodium chloride solution
- Final dilution of a dissolved solid, stock liquid or gel: 10% (w/v)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspended

OTHER SPECIFICS: The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 10% test item (w/v) as recommended in the test guideline 437 for surfactant solids. 0.9% NaCl solution was used as the negative control and 1% NaOH solution in 0.9% NaCl solution as the positive control item.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST MODEL
- Test model: Bovine eyes from cattle obtained from slaughterhouse at age 6-12 months
- Source: Hubert Bahlmann GmbH & Co. Versandschlachterei Speziamichfutterwerk KG 49699, Lindern, Germany
- Storage: Bovine tissues were stored in Hanks’ Balanced Salt Solution (HBSS) (GIBCO) containing penicillin at 100 IU/mL (GIBCO) and streptomycin at 100 µg/mL (GIBCO).
- Quality criteria: Eyes were examined for opacity, scratches and neovascularisation, only corneas from eyes free of defects were used. Prior to the assay, corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders, were also discarded.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 1°C

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL of the 10% test item suspension in 0.9% sodium chloride solution (w/v)
- Concentration (if solution): 10%

NEGATIVE CONTROL
- Negative control: Physiological saline solution (0.9% sodium chloride solution)
- Amount(s) applied (volume or weight): 750 µL

POSITIVE CONTROL
- Positive control: NaOH
- Amount(s) applied (volume or weight): 750 µL of the 1% NaOH solution in highly purified water
- Concentration (if solution): 1%

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

90 ± 5 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL. Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.
The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded. The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment, solvent and positive control groups

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED
- Negative control: Physiological saline solution (0.9% sodium chloride solution)
- Amount(s) applied (volume or weight): 750 µL

POSITIVE CONTROL USED
- Positive control: NaOH
- Amount(s) applied (volume or weight): 750 µL of the 1% NaOH solution in highly purified water
- Concentration (if solution): 1%

APPLICATION DOSE AND EXPOSURE TIME
- Amount(s) applied (volume or weight with unit): 750 µL of the 10% test item suspension in 0.9% sodium chloride solution (w/v)
- Exposure time: 10 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the exposure period of 10 minutes (the recommended exposure time for surfactant solids) the test item, the negative and positive controls, were removed from each chamber. Subsequently, the epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

- POST-EXPOSURE INCUBATION:
- Post-exposure preparation: To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM.
- Incubation period: 90 ± 5 minutes
- Examination of corneas: Sodium fluorescein measurements (Tecan Sunrise Magellan Version 7.2)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of (Tecan Sunrise Magellan Version 7.2 Microtiter plate reader) (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
After correcting the opacity and mean permeability (OD490) values for background opacity and the negative control permeability OD490 values, the mean opacity, and permeability OD490 values for each treatment group were combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The opacity and permeability values were also evaluated independently to determine whether the test item induced corrosivity or severe irritation through only one of the two endpoints.

DECISION CRITERIA:
A test item that induces an IVIS > 55 is defined as a corrosive or severe irritant.
The BCOP test method can also be used to identify chemicals that do not require classification for eye irritation or serious eye damage under the UN GHS classification system. When used for this purpose, the BCOP test method has an overall accuracy of 69% (135/169), a false positive rate of 69% (61/89), and a false negative rate of 0% (0/107), when compared to in vivo rabbit eye test method data classified according to the UN GHS classification system.
The IVIS cut-off values for identifying test chemicals as inducing serious damage (UN GHS Category 1) and test chemicals not requiring classification for irritation or serious eye damage (UN GHS No Category) are >55 and ≤3, respectively.

Irritation parameter:

cornea opacity score

Run / experiment:

mean opacity (n=3), relative to negative control

Value:

-0.252

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Remarks:

(permeability)

Run / experiment:

Mean permeability score (n=3) relative to negative controls

Value:

0.002

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Calculated IVIS of 3 corneas exposed to strontium apatite copper-doped

Value:

-0.217

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:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Range of historical values if different from the ones specified in the test guideline: The most recent control data for GLP BCOP studies at LPT (2015-2017) are as follows:

Negative control (0.9% NaCl): presented as mean ± standard deviation (limits of acceptance)
IVIS: 0.579 ± 1.097 (-1.615 - 2.773)
Opacity: 0.062 ± 0.934 (-1.806 - 1.931)
Permeability: 0.043 ± 0.073 (-0.102 - 0.189)

Positive control (NaOH 1%): presented as mean ± standard deviation (limits of acceptance)
IVIS: 102.903 ± 21.283 (60.337 - 145.469)
Opacity: 72.668 ± 20.908 (30.852 - 114.484)
Permeability: 2.016 ± 0.415 (1.186 - 2.847)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: TThe corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of <0.001 and a mean permeability value of 0.005 ± 0.003. The calculated IVIS value of <0.001 was well below the cut-off value of 3 (UN GHS no category).
- Acceptance criteria met for positive control: The corneas treated with the positive control item 1% NaOH in highly purified water revealed a mean opacity value of 68.858 ± 14.992 and a mean permeability value of 2.547 ± 0.266 compared to the solvent control. The calculated IVIS value of 107.068 ± 11.486 was within two standard deviations of the current historical mean and well above the cut-off value of 55.
- The acceptance criteria for the test were fulfilled.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions Fatty acids, lanolin, lithium salts tested in the in vitro BCOP test method, had an IVIS value of <0.001, which is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.

Executive summary:

Eye irritation is primarily defined by the extent of corneal injury; substances which damage the superficial epithelium may cause slight irritation, whereas further penetration to the corneal stroma or endothelium may induce mild or severe irritation, respectively. Eye irritation of the test item was evaluated in the Bovine Corneal Opacity and Permeability Test (BCOP, OECD 437), which utilises measurements of corneal opacity as an indicator of protein denaturation, swelling, vacuolation and tissue damage, and corneal fluorescein retention/leakage provides a measure of permeability in vitro.

A 10% suspension of the test item was applied to bovine corneas in 750 µL of physiological saline solution (0.9% NaCl) for 10 minutes, alongside positive and negative controls. Following treatment with Fatty acids, lanolin, lithium salts a mean opacity of <0.001 and a mean permeability value of 0.002±0.002 compared to the negative control were determined. The calculated IVIS of<0.001 is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.

The BCOP (EU B.47; OECD 437) is an accepted in vitro test method to detect serious eye damage (Category 1 under CLP) and/or absence of effects requiring classification for serious eye damage/eye irritation (i.e. not classified under CLP), as described in the Annex to the EU Test Methods (TM) Regulation (Council Regulation (EC) No 440/2008). Conducted according to the aforementioned guidelines and GLP, the BCOP passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 February - 22 February 2018

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: sponsor batch# SOAP 012-2
- Manufacture date of the lot/batch: Not reported
- Expiration date of the lot/batch: November 2022
- Purity: Min. 95%
- Purity test date: 13 December 2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Physical characteristics: Beige to brown, solid
- Storage condition of test material: At ambient temperature (10 - 25°), container kept tightly closed and stored in a dry and well-ventilated place.

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST MODEL
- Test model: EpiOcular™-EIT Model (Lot. No. 27024; OCL-200-EIT)
- Source: MatTek In Vitro Life Science, Mlynské Nivy 73, Bratislava, Slovakia
- Analysis: The MatTek EpiOcular™ corneal model is derived from normal human keratinocytes, cultured to form a stratified, 3D structure similar to that found in the cornea. Each batch of EpiOcular™ tissues is safety tested for HIV, Hepatitis-B, Hepatitis-C and bacteria, yeast and fungi by the manufacturer.

ANALYSIS FOR TISSUE FUNCTIONALITY AND QUALITY
- Tissue viability (MTT QC assay): OD 1.944 ± 0.116 (1.1 - 3.0 acceptance range)
- Barrier function (ET50 assay): ET50 17.9 minutes (12.2 - 37.5 acceptance range)
- Sterility (Long term antibiotic and antimycotic free culture): sterile (no contamination)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C
- CO2 level: 5%

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 mg

POSITIVE CONTROL:
- Positive control(s): Methyl acetate
- Amount(s) applied (volume or weight with unit): 50 µL

NEGATIVE CONTROL:
- Negative control(s): Sterilie deionised water

Duration of treatment / exposure:

Exposure 6 hours ± 15 minutes

Duration of post- treatment incubation (in vitro):

- Following rinsing and drying, tissues were submerged in medium and incubated at room temperature for 25 ± 2 minute post-soak period, to ensure removal of the absorbed test item. Tissues were blotted dry, then returned to prewarmed assay medium and incubated for a further 18 hours prior to the MTT assay.

Number of animals or in vitro replicates:

3 Replicates

Details on study design:

TEST MODEL
- Test model: EpiOcular™-EIT Model (Lot. No. 27024; OCL-200-EIT)
- Source: MatTek In Vitro Life Science, Mlynské Nivy 73, Bratislava, Slovakia

ANALYSIS FOR TISSUE FUNCTIONALITY AND QUALITY
- Analysis: The MatTek EpiOcular™ corneal model is derived from normal human keratinocytes, cultured to form a stratified, 3D structure similar to that found in the cornea. Each batch of EpiOcular™ tissues is safety tested for HIV, Hepatitis-B, Hepatitis-C and bacteria, yeast and fungi by the manufacturer.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C
- CO2 level: 5%

PREDOSE TREATMENT
- Prior to application of the test items or controls, the surface of each EpiOcular™ tissue was moistened with DPBS (20 µL). The tissues were incubated for a further 30 ± 2 minutes prior to dosing.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg
- Application of test item to tissues: Solid test item was applied directly to the EpiOcular™ surface, in triplicate.

POSITIVE CONTROL:
- Positive control(s): Methyl acetate
- Amount(s) applied (volume or weight with unit): 50 µL

NEGATIVE CONTROL: Sterile deionised water


MTT DIRECT REDUCTION TEST
After the Post-treatment Incubation the MTT assay was performed. Tissues were transferred into wells of a 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) . The tissues were incubated at 37°C, 5% CO2 and 95% relative humidity for 180 ± 10 minutes.
After incubation each insert was removed from the 24-well plate. The bottom of the insert blotted on absorbent material, and then the insert was transferred to a pre-labeled 24-well plate containing 1.0 mL of isopropanol in each designated well so that isopropanol flowed into the insert on the tissue surface. The plates were sealed with parafilm and were immediately extracted on a plate shaker for 3 hours at room temperature.
At the end of the non-submerged extraction, inserts and tissues were discarded without piercing and 1 mL of isopropanol was added into each well. The extract solution was mixed and transferred to a 96-well plate.
The absorbance (Optical Density (OD) at 540 nm) was determined with a microplate reader.

IDENTIFICATION OF COLOUR INTERFERING TEST ITEMS
50 mg test item were mixed with 1000 µL sterile deionised water and incubated in the dark at 37°C, 5% CO2 and 95% relative humidity for 3 hours. At the end of exposure time, the mixture was evaluated of the presence and intensity of the staining. No discolouration of the test item was noted. In addition, 50 mg test item were mixed with 2 mL isopropanol and incubated at room temperature for three hours. No discolouration of the test item was noted. Hence, the criteria of the test on colour interference were met. No further additional test on colourant controls, no OD measurement was performed.
Furthermore the test item was evaluated for its potential to interfere with the MTT assay reagent (e.g. reduction). Therefore, 50 mg test item were dissolved in 1 mL MTT solution (1 mg/mL) and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for approximately 180 ± 10 minutes. A negative control (50 µL of deionised water) was run concurrently. As the MTT solution turned black, the test item reduced MTT and an additional functional check on freeze-killed tissues was performed.

EXPOSURE
- Exposure: 6 hours ± 15 minutes
- Rinsing procedure: The test item or control treatments were removed by extensively rinsing the tissues with DPBS (without Mg2+ and Ca2+) at room temperature. The rinsing was performed by dipping the tissues three times in containers prefilled with fresh 100 mL DPBS (without Mg2+ and Ca2+). For each treatment different fresh DPBS (without Mg2+ and Ca2+) solutions were used. The rinsing was repeated twice, each with fresh DPBS (without Mg2+ and Ca2+).
- Post-exposure incubation: After rinsing, the tissues were immediately transferred in 5 mL pre-warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for 25 ± 2 minutes.


ACCEPTANCE OF RESULTS:
The assay is acceptable if:
- The negative control OD is >0.8 and <2.5.
- The mean relative viability of the positive control is below 50% of control viability.
- The difference of viability between two replicate tissues is <20%

Irritation parameter:

other: Mean calculated viability

Run / experiment:

The Mean corrected viability (%)

Value:

3.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Other effects / acceptance of results:

TEST ITEM RESULTS
The mean corrected viability of the cells exposed to the test item was 3.6% of the mean negative control value (corrected viability calculated for MTT reducing test items using freeze-killed control tissues).
Hence, the OD540 values were below the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of ≤ 60%, so no predictions on classification could be made. The test item is predicted to be irritant.


ACCEPTANCE OF RESULTS:
- The mean optical density (OD) of the negative control of 2 tissues was 1.285 and was well within the acceptable range of > 0.8 to < 2.5.
- The viability of cells treated with the positive reference item, methyl acetate, was 32.1% of the negative controls and below the 50% cut-off value of the negative controls.
- The results were within the established historical data.

Interpretation of results:

other: Predicted to be irritant.

Conclusions:

Under the present test conditions the OD540 values obtained from Fatty acids, lanolin, lithium salts were below the cut-off percentage of cell viability value that distinguishes irritant from non-irritant test items of ≤ 60%, so is it concluded that the test item is predicted to be an eye irritant.

Executive summary:

Eye irritation is primarily defined by the extent of corneal injury; substances which damage the superficial epithelium may cause slight irritation, whereas further penetration to the corneal stroma or endothelium may induce mild or severe irritation, respectively. A reconstructed human cornea-like epithelium (RhCE) test method, using the EpiOcular™ 3D model of the human corneal epithelium, was conducted for the test item.

50 mg of the test item, 50 µL of the concurrent negative control (sterile deionised water)or 50 µL of the positive control (methyl acetate)were administered by topical application onto the construct as cultures for 6 hours, followed by a 25-minute post-treatment immersion, and an 18-hours post-treatment incubation, prior to the MTT endpoint.

The mean corrected viability of the cells exposed to the test item was 3.6% of the mean negative control value (corrected viability calculated for MTT reducing test items using freeze-killed control tissues). The OD₅₄₀values were below the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of ≤ 60%, so it is predicted that the test item is an eye irritant.

Conducted according to the aforementioned guidelines and GLP, the study passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation of the test item was evaluated with the EpiDerm Reconstructed Human Epidermis Model according to OECD 439 guideline. The mean relative absorbance value of the test item, corresponding to the cell viability did not significantly decrease (103.2 %; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

The test item was evaluated for its corrosive properties using the EpiDerm Reconstructed Human Epidermis Model in a study following OECD 431 guideline. In comparison to the negative controls, the mean viability of cells exposed to the test item was 98.1% after a 3-minute exposure period and 95.7% after a 1-hour exposure (corrected viability calculated for MTT reducing test items using freeze-killed control tissues) and hence, the 3-minute and the 1-hour exposure values were above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of ≥50% and ≥15%, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

Eye irritation of the test item has been evaluated in two in vitro tests, The Bovine Corneal Opacity and Permeability Test (BCOP, OECD 437) and the Reconstructed human Cornea-like Epithelium Test (RhCE, OECD 492). The BCOP utilises measurements of corneal opacity as an indicator of protein denaturation, swelling, vacuolation and tissue damage, and corneal fluorescein retention/leakage provides a measure of permeability in vitro. Whereas the EpiOcular™ 3D model of the human corneal epithelium measures cell viability as a measure of ocular irritation in the RhCE test method.

 

In the BCOP test, the calculated In Vitro Irritation Score (IVIS) of the test substance was below the cut-off value of 3 (UN GHS no category) for identifying test substances as inducing serious eye damage (UN GHS Category 1). Consequently the test item should not be classified as a severe irritant and is not corrosive according to UN GHS classification. In the EpiOcular™ Eye Irritation Test (EIT) the mean percentage viability of the EpiOcular™ tissues treated with test item was 3.6%, relative to negative controls. Under the present test conditions the OD540 values obtained from Fatty acids, lanolin, lithium salts were below the cut-off percentage of cell viability value that distinguishes irritant from non-irritant test items of ≤ 60%, therefore it is potentially eye irritant. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is ≤60%. When the final mean percent tissue viability is ≤ 60% further testing with other test methods would be required because EpiOcular™ EIT shows a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2. In order to avoid further testing and in line with the principles of the Replacement, Refinement and Reduction (3Rs) of the use of animals for scientific purposes, no further testing has been initiated and a Category 2 classification has been assumed. 

Conducted according to the aforementioned guidelines and GLP, the studies passed all validity criteria and were considered to be reliable without restriction (Klimisch 1).

Justification for classification or non-classification

Skin : According to the available data, no classification is required according to Regulation (EC) No 1272/2008 (2017).

Eye: Based on the available data, the substance is classified as eye irritant Category 2 according to Regulation (EC) No 1272/2008 (2017).