Tall oil, sodium salt

Endpoint summary

Description of key information

The following information is taken into account for any hazard / risk assessment:

There is some variability in the pH of manufactured samples of TOS. A suite of in vitro skin irritation/corrosion and eye irritation tests were therefore performed in accordance with OECD TG 431, 439 and 437 for three representative samples with measured pH 12.5 (TOS30), pH 12.4 (TOS31) and pH 12.4 (TOS32).

The results of the studies concluded the following:

TOS30: Skin corrosivity: not corrosive; Skin irritation: irritating (Cat.2); Eye irritation: not determinable

TOS31: Skin corrosivity: not corrosive; Skin irritation: irritating (Cat.2); Eye irritation: not determinable

TOS 32: Skin corrosivity: not corrosive; Skin irritation: irritating (Cat.2); Eye irritation: not determinable

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:

08 February 2017 until 17 March 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UN GHS (2003, last rev. 2009)

Principles of method if other than guideline:

• Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test system:

human skin model

Details on test system:

Pre-warming of EpiSkin™ Tissues
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for about 24 hours.

Treatment
The negative control, positive control and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material, but complete removal of test item was not possible. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

IL-1 α Immunoassay
Samples of all treatment groups were taken from the wells. The plates were shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well were taken and were stored in the freezer at –20 °C. Since the results derived from the MTT assay (see chapter 4.4 and 8.1) were not unclear or borderline, the IL-1 α concentration in the medium was not determined and the taken samples will be discarded after reporting finalisation.

MTT Assay
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for nearly 3.5 hours at room temperature with gentle agitation.
Per tissue sample 2  200 µL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

Data Recording
The data generated were recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups with the test item, negative and positive controls. 

Amount/concentration applied:

10 µL (26.3 µL/cm2) of the undiluted test item were applied to each of the triplicate tissues.

Species:

other: reconstituted human epidermis model

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test substance

Value:

10.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results after treatment with Tall Oil Soap, TOS30 and controls

Test Group	Absor-bance 570 nm Tissue 1*	Absor-bance 570 nm Tissue 2*	Absor-bance 570 nm Tissue 3*	Mean Absor-bance of 3 Tissues	Relative Absor-bance [%] Tissue 1, 2 + 3**	Standard Deviation	Relative Standard Deviation [%]	Rel. Absor-bance [% of Negative Control]***
Negative Control	0.792	0.760	0.888	0.813	97.4 93.4 109.2	8.2	8.2	100.0
Positive Control	0.032	0.034	0.029	0.032	3.9 4.2 3.6	0.3	7.6	3.9
Test Item	0.081	0.080	0.089	0.084	10.0 9.8 11.0	0.6	6.2	10.3
Negative Controladd. viable tissue	0.032	0.042		0.037****	3.9 5.1	0.9	19.3	4.5
Test Itemadd. viable tissue	0.015	0.020		0.017****	1.2 1.3	0.4	20.8	2.1
Negative Controladd. freeze-killed tissue	0.027	0.033		0.030****	3.3 4.0	0.5	13.9	3.7
Test Itemadd. freeze-killed tissue	0.082	0.057		0.070****	10.1 7.0	2.2	25.8	8.6

*         Mean of two replicate wells after blank correction

**       relative absorbance per tissue [rounded values]:

***     relative absorbance per treatment group [rounded values]:

****  Mean absorbance of 2 tissues

 

Optical pre-experiment (colour interference pre-experiment) to investigate the test item’s water dying potential led to a change in colour and optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent showed blue colour. Therefore, additional tests with viable tissues (without MTT addition) and with freeze-killed tissues were performed to determine correction factors for calculating the true viability in the main experiment.

Even without taking the determined correction factors into consideration the mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 10.3% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin. Therefore, the calculation of the true viability was not necessary, since the outcome of the study is not affected.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In conclusion, in this study and under the experimental conditions reported, Tall Oil Soap, TOS30 is irritating to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of Tall Oil Soap, TOS30 by means of the Human Skin Model Test.

The test item dyed water in the colour interference pre-test slightly brown, and it reduced MTT (pre-test for direct MTT reduction). Consequently, additional tests with freeze-killed and viable tissues (without MTT addition) had to be performed to determine correction factors for calculating the true viability in the main experiment. Since the test item proved to possess a clear skin irritating potential in the main experiment, consideration of correction factors was not necessary, since the outcome of the study would not be influenced.

Each three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes.

The test item and the positive and negative controls were washed off the skin tissues treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following nearly 3.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.6 till ≤ 1.5 for the 15 minutes treatment interval (values between 0.760 and 0.888) thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.9% thus ensuring the validity of the test system.

The rel. standard deviations between tissues of the same treatment group (main experiment) was below 9% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

After treatment with the test item the mean relative absorbance value was reduced to 10.3%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 December 2017 until 21 January 2018

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)

Version / remarks:

Updated Guideline adopted July 29, 2016

Deviations:

no

Principles of method if other than guideline:

EC Directive 2000/33/EC, OJ L136200, dated June 08, 2000, adopting the 27th time to technical progress the Dangerous Substances Directive 67/548/EEC, Annex V, part B40 and EC regulation No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation No 1907/2006 of the European Parliament and of the Council on REACH, 1st ATP, section B40.

MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”, 07 November 2014.

There was the following alteration from the MatTek test protocol:
Concerning formazan salt extraction (chapter 3.6.3): The formazan salt extraction period was extended up to 72 hours (in the refrigerator without shaking). According to an expert statement (Dr. Helena Kandarova from MatTek Corporation) this procedure does not have an impact on the outcome of the study, if sufficient covering of the plates to prevent isopropanol evaporation is ensured.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Details on test system:

Epi-200 SCT Kit Components Needed for the Assay

EpiDerm™ Kit Lot No.: 25874
1 Sealed 24-well plate Contains 24 inserts with EpiDerm™ tissues on agarose
2 24-well plates For MTT viability assay
4 6-well plates For storing inserts, or for topically applying test agents
1 bottle Serum-Free Assay Medium DMEM-based medium
1 bottle DPBS Rinse Solution For rinsing the inserts in MTT assay

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant Solution (Isopropanol) For extraction of formazan crystals

MTT-Solution
The MTT-solution was prepared freshly on day of use (resulting: 1 mg/mL).

Pre-warming of EpiSkin™ Tissues:
18 hours and 25 minutes before dosing, EpiDerm™ tissues were removed from the refrigerator, and the inserts were transferred into 6-well plates containing the pre-warmed assay medium under sterile conditions using sterile forceps. A 24-well plate was prepared as holding plate containing 300 µL assay medium. The holding plate was pre-warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.

Treatment:
Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control for the following exposure times:
• Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
• Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
• Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes

After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

MTT Assay:
Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 µL) was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required.
Following rinsing, the tissues were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the tissues were rinsed three times with DPBS and carefully dried with blotting paper. The inserts were transferred into new 24-well plates. The tissues were each immersed in 2 mL of extractant solution (isopropanol) pipetted in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for about 70 hours without shaking in the refrigerator.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour.
3  200 µL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

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 (79.4 µL/cm2 according to guideline) of the test item were dispensed directly onto duplicate EpiDermTM tissue surface


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


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

Duration of treatment / exposure:

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

Duration of post-treatment incubation (if applicable):

After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

Number of replicates:

two replicates

Type of coverage:

other: topical

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Results after 3 minutes treatment

Value:

101.66

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

results after 60 minutes treatment

Value:

87.21

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: results within historical range

Results after treatment with Tall Oil Soap, TOS30 and controls

Dose Group	Mean Absorbance (OD) of 3 Wells Blank corrected	Mean Absorbance (OD) of 2 Tissues	Mean Rel. Viability [%]
Negative Control Tissue 1	1.584
Negative Control Tissue 2	1.544	1.564	100.00
Positive Control Tissue 1	0.438
Positive Control Tissue 2	0.419	0.429	27.4
Test Item Tissue 1	1.656
Test Item Tissue 2	1.645	1.650	101.66*
Negative ControlViable Tissue  Tissue 1	0.006
Negative ControlViable Tissue  Tissue 2	0.007	0.006	0.4
Test ItemViable Tissue Tissue 1	0.015
Test ItemViable Tissue Tissue 2	0.004	0.010	0.6
Negative ControlFreeze Killed Tissue  Tissue 1	0.099
Negative ControlFreeze Killed Tissue  Tissue 2	0.100	0.099	6.3
Test ItemFreeze Killed Tissue  Tissue 1	0.143
Test ItemFreeze Killed Tissue  Tissue 2	0.156	0.150	9.6

* Corrected Relative Viability [%]

Results after treatment with Tall Oil Soap, TOS30 and the controls / exposure interval 60 minutes

Dose Group	Mean Absorbance (OD) of 3 Wells Blank corrected	Mean Absorbance (OD) of 2 Tissues	Mean Rel. Viability [%]
Negative Control Tissue 1	1.356
Negative Control Tissue 2	1.328	1.342	100.0
Positive Control Tissue 1	0.082
Positive Control Tissue 2	0.058	0.070	5.2
Test Item Tissue 1	1.270
Test Item Tissue 2	1.249	1.260	87.21*
Negative ControlViable Tissue  Tissue 1	0.007
Negative ControlViable Tissue  Tissue 2	0.014	0.010	0.8
Test ItemViable Tissue  Tissue 1	0.004
Test ItemViable Tissue Tissue 2	0.009	0.007	0.5
Negative ControlFreeze Killed Tissue  Tissue 1	0.095
Negative ControlFreeze Killed Tissue  Tissue 2	0.093	0.094	7.0
Test ItemFreeze Killed Tissue  Tissue 1	0.176
Test ItemFreeze Killed Tissue  Tissue 2	0.176	0.176	13.1

 * Corrected Relative Viability [%]

Optical pre-experiment (colour interference pre-experiment) to investigate the test item’s water dying potential led to a change in colour and optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent showed blue colour. Therefore, additional tests with viable tissues (without MTT addition) and with freeze-killed tissues were performed to determine correction factors for calculating the true viability in the main experiment.

Even without taking the determined correction factors into consideration the mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 10.3% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin. Therefore, the calculation of the true viability was not necessary, since the outcome of the study is not affected.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, Tall Oil Soap, TOS30 is non corrosive to skin according to EU CLP and UN GHS.

Executive summary:

This in vitro study was performed to assess the corrosive potential of Tall Oil Soap, TOS30 by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item reduced MTT (pre-test for direct MTT reduction), and it changed colour when mixed with deionised water (pre-test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were necessary.

Independent duplicate tissues of EpiDerm^TM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO₂) with MTT solution followed. MTT solution was then rinsed from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for about 70 hours in the refrigerator.

The required acceptability criteria were met.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (27.4%) and for the 1 hour exposure period (5.2%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item Tall Oil Soap, TOS30 the relative absorbance value increased slightly to 101.66% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 87.21%. Both values did not fall below the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 February 2017 until 17 March 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UN GHS (2003, last rev. 2009)

Principles of method if other than guideline:

• Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test system:

human skin model

Details on test system:

Pre-warming of EpiSkin™ Tissues
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for about 24 hours.

Treatment
The negative control, positive control and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material, but complete removal of test item was not possible. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

IL-1 α Immunoassay
Samples of all treatment groups were taken from the wells. The plates were shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well were taken and were stored in the freezer at –20 °C. Since the results derived from the MTT assay (see chapter 4.4 and 8.1) were not unclear or borderline, the IL-1 α concentration in the medium was not determined and the taken samples will be discarded after reporting finalisation.

MTT Assay
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for nearly 3.5 hours at room temperature with gentle agitation.
Per tissue sample 2  200 µL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

Data Recording
The data generated were recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups with the test item, negative and positive controls. 

Amount/concentration applied:

10 µL (26.3 µL/cm2) of the undiluted test item were applied to each of the triplicate tissues.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test material

Value:

34.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results after treatment with Tall Oil Soap, TOS31 and controls

Test Group	Absor-bance 570 nm Tissue 1*	Absor-bance 570 nm Tissue 2*	Absor-bance 570 nm Tissue 3*	Mean Absor-bance of 3 Tissues	Relative Absor-bance [%] Tissue 1, 2 + 3**	Standard Deviation	Relative Standard Deviation [%]	Rel. Absor-bance [% of Negative Control]***
Negative Control	0.792	0.760	0.888	0.813	97.4 93.4 109.2	8.2	8.2	100.0
Positive Control	0.032	0.034	0.029	0.032	3.9 4.2 3.6	0.3	7.6	3.9
Test Item	0.271	0.247	0.318	0.279	33.3 30.4 39.0	4.4	12.9	34.2
Negative Controladd. viable tissue	0.032	0.042		0.037****	3.9 5.1	0.9	19.3	4.5
Test Itemadd. viable tissue	0.010	0.011		0.010****	1.2 1.3	0.1	6.4	1.3
Negative Controladd. freeze-killed tissue	0.027	0.033		0.030****	3.3 4.0	0.5	13.9	3.7
Test Itemadd. freeze-killed tissue	0.118	0.093		0.070****	10.1 7.0	2.2	25.8	8.6

*         Mean of two replicate wells after blank correction

**       relative absorbance per tissue [rounded values]:

***     relative absorbance per treatment group [rounded values]:

****  Mean absorbance of 2 tissues

 

Optical pre-experiment (colour interference pre-experiment) to investigate the test item’s water dying potential led to a change in colour and optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent showed blue colour. Therefore, additional tests with viable tissues (without MTT addition) and with freeze-killed tissues were performed to determine correction factors for calculating the true viability in the main experiment.

Even without taking the determined correction factors into consideration the mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 10.3% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin. Therefore, the calculation of the true viability was not necessary, since the outcome of the study is not affected.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Tall Oil Soap, TOS31 is irritating to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of Tall Oil Soap, TOS31 by means of the Human Skin Model Test.

The test item dyed water in the colour interference pre-test slightly brown, and it reduced MTT (pre-test for direct MTT reduction). Consequently, additional tests with freeze-killed and viable tissues (without MTT addition) had to be performed to determine correction factors for calculating the true viability in the main experiment. Since the test item proved to possess a clear skin irritating potential in the main experiment, consideration of correction factors was not necessary, since the outcome of the study would not be influenced.

Each three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes.

The test item and the positive and negative controls were washed off the skin tissues treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following nearly 3.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.6 till ≤ 1.5 for the 15 minutes treatment interval (values between 0.760 and 0.888) thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.9% thus ensuring the validity of the test system.

The rel. standard deviations between tissues of the same treatment group (main experiment) was below 9% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

After treatment with the test item the mean relative absorbance value was reduced to 34.2%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Reference 4

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 December 2017 until 21 January 2018

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)

Version / remarks:

Updated Guideline adopted July 29, 2016

Deviations:

no

Principles of method if other than guideline:

EC Directive 2000/33/EC, OJ L136200, dated June 08, 2000, adopting the 27th time to technical progress the Dangerous Substances Directive 67/548/EEC, Annex V, part B40 and EC regulation No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation No 1907/2006 of the European Parliament and of the Council on REACH, 1st ATP, section B40.

MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”, 07 November 2014.

There was the following alteration from the MatTek test protocol:
Concerning formazan salt extraction (chapter 3.6.3): The formazan salt extraction period was extended up to 72 hours (in the refrigerator without shaking). According to an expert statement (Dr. Helena Kandarova from MatTek Corporation) this procedure does not have an impact on the outcome of the study, if sufficient covering of the plates to prevent isopropanol evaporation is ensured.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Details on test system:

Epi-200 SCT Kit Components Needed for the Assay

EpiDerm™ Kit Lot No.: 25874
1 Sealed 24-well plate Contains 24 inserts with EpiDerm™ tissues on agarose
2 24-well plates For MTT viability assay
4 6-well plates For storing inserts, or for topically applying test agents
1 bottle Serum-Free Assay Medium DMEM-based medium
1 bottle DPBS Rinse Solution For rinsing the inserts in MTT assay

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant Solution (Isopropanol) For extraction of formazan crystals

MTT-Solution
The MTT-solution was prepared freshly on day of use (resulting: 1 mg/mL).

Pre-warming of EpiSkin™ Tissues:
18 hours and 25 minutes before dosing, EpiDerm™ tissues were removed from the refrigerator, and the inserts were transferred into 6-well plates containing the pre-warmed assay medium under sterile conditions using sterile forceps. A 24-well plate was prepared as holding plate containing 300 µL assay medium. The holding plate was pre-warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.

Treatment:
Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control for the following exposure times:
• Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
• Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
• Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes

After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

MTT Assay:
Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 µL) was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required.
Following rinsing, the tissues were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the tissues were rinsed three times with DPBS and carefully dried with blotting paper. The inserts were transferred into new 24-well plates. The tissues were each immersed in 2 mL of extractant solution (isopropanol) pipetted in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for about 70 hours without shaking in the refrigerator.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour.
3  200 µL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

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 (79.4 µL/cm2 according to guideline) of the test item were dispensed directly onto duplicate EpiDermTM tissue surface


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


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

Duration of treatment / exposure:

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

Duration of post-treatment incubation (if applicable):

After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

Number of replicates:

two replicates

Type of coverage:

other: topical

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Results after 3 minutes treatment

Value:

79.76

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

results after 60 minutes treatment

Value:

48.56

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: results within historical range

Results after treatment with Tall Oil Soap, TOS31 and controls

Dose Group	Mean Absorbance (OD) of 3 Wells Blank corrected	Mean Absorbance (OD) of 2 Tissues	Mean Rel. Viability [%]
Negative Control Tissue 1	1.584
Negative Control Tissue 2	1.544	1.564	100.00
Positive Control Tissue 1	0.438
Positive Control Tissue 2	0.419	0.429	27.4
Test Item Tissue 1	1.461
Test Item Tissue 2	1.365	1.413	79.76*
Negative ControlViable Tissue  Tissue 1	0.006
Negative ControlViable Tissue  Tissue 2	0.007	0.006	0.4
Test ItemViable Tissue Tissue 1	0.008
Test ItemViable Tissue Tissue 2	0.010	0.009	0.5
Negative ControlFreeze Killed Tissue  Tissue 1	0.099
Negative ControlFreeze Killed Tissue  Tissue 2	0.100	0.099	6.3
Test ItemFreeze Killed Tissue  Tissue 1	0.240
Test ItemFreeze Killed Tissue  Tissue 2	0.273	0.257	16.4

* Corrected Relative Viability [%]

Results after treatment with Tall Oil Soap, TOS31 and the controls / exposure interval 60 minutes

Dose Group	Mean Absorbance (OD) of 3 Wells Blank corrected	Mean Absorbance (OD) of 2 Tissues	Mean Rel. Viability [%]
Negative Control Tissue 1	1.356
Negative Control Tissue 2	1.328	1.342	100.0
Positive Control Tissue 1	0.082
Positive Control Tissue 2	0.058	0.070	5.2
Test Item Tissue 1	1.773
Test Item Tissue 2	0.730	0.752	48.56*
Negative ControlViable Tissue  Tissue 1	0.007
Negative ControlViable Tissue  Tissue 2	0.014	0.010	0.8
Test ItemViable Tissue  Tissue 1	0.025
Test ItemViable Tissue Tissue 2	0.018	0.021	1.6
Negative ControlFreeze Killed Tissue  Tissue 1	0.095
Negative ControlFreeze Killed Tissue  Tissue 2	0.093	0.094	7.0
Test ItemFreeze Killed Tissue  Tissue 1	0.172
Test ItemFreeze Killed Tissue  Tissue 2	0.173	0.172	12.8

 * Corrected Relative Viability [%]

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water led to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent showed purple colour.

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

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

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

The acceptance criteria are met:

·        the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (1.328 to 1.584)

·        the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (5.2%)

·        the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range:1.5% to 4.0%)

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, Tall Oil Soap, TOS31 is non corrosive to skin according to EU CLP and UN GHS.

Executive summary:

This in vitro study was performed to assess the corrosive potential of Tall Oil Soap, TOS31 by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item reduced MTT (pre-test for direct MTT reduction), and it changed colour when mixed with deionised water (pre-test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were necessary.

Independent duplicate tissues of EpiDerm^TM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO₂) with MTT solution followed. MTT solution was then rinsed from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for about 70 hours at room temperature.

The required acceptability criteria were met.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (27.4%) and for the 1 hour exposure period (5.2%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item Tall Oil Soap, TOS31 the relative absorbance value decreased to 79.76% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 48.56%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

Reference 5

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 March 2017 until 17 March 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UN GHS (2003, last rev. 2009)

Principles of method if other than guideline:

• Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Details on test system:

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative and the positive control for 60 minutes.
Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of triplicate tissue.
30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.
The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 43 hours the tissues were treated with the MTT solution for 3 hours following 69.75 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

Amount/concentration applied:

10 µL (26.3 µL/cm2) of the undiluted test item were applied to each of the triplicate tissues.

Duration of treatment / exposure:

15 minutes

Species:

other: reconstituted human epidermis model

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test material

Value:

17.7

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results after treatment with Tall Oil Soap, TOS32 and controls

Test Group	Absor-bance 570 nm Tissue 1*	Absor-bance 570 nm Tissue 2*	Absor-bance 570 nm Tissue 3*	Mean Absor-bance of 3 Tissues	Relative Absor-bance [%] Tissue 1, 2 + 3**	Standard Deviation	Relative Standard Deviation [%]	Rel. Absor-bance [% of Negative Control]***
Negative Control	0.943	0.817	0.864	0.875	107.8 93.4 98.8	7.3	7.3	100.0
Positive Control	0.037	0.042	0.042	0.040	4.2 4.9 4.7	0.3	7.6	4.6
Test Item	0.156	0.167	0.141	0.155	17.8 19.1 16.1	1.5	8.4	17.7
Negative Controladd. viable tissue	0.018	0.027		0.022****	2.0 3.0	0.7	28.0	2.5
Test Itemadd. viable tissue	0.060	0.084		0.072****	6.8 9.6	2.0	24.2	8.2
Negative Controladd. freeze-killed tissue	0.026	0.031		0.028****	2.9 3.6	0.5	14.0	3.3
Test Itemadd. freeze-killed tissue	0.126	0.101		0.114****	14.4 11.6	2.0	15.2	13.0

*         Mean of two replicate wells after blank correction

**       relative absorbance per tissue [rounded values]:

***     relative absorbance per treatment group [rounded values]:

****  Mean absorbance of 2 tissues

 

Optical pre-experiment (colour interference pre-experiment) to investigate the test item’s water dying potential led to a change in colour and optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent showed blue colour. Therefore, additional tests with viable tissues (without MTT addition) and with freeze-killed tissues were performed to determine correction factors for calculating the true viability in the main experiment.

Even without taking the determined correction factors into consideration the mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 17.7% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin. Therefore, the calculation of the true viability was not necessary, since the outcome of the study is not affected.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Tall Oil Soap, TOS32 is irritating to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of Tall Oil Soap, TOS32 by means of the Human Skin Model Test.

The test item dyed water in the colour interference pre-test slightly brown, and it reduced MTT (pre-test for direct MTT reduction). Consequently, additional tests with freeze-killed and viable tissues (without MTT addition) had to be performed to determine correction factors for calculating the true viability in the main experiment. Since the test item proved to possess a clear skin irritating potential in the main experiment, consideration of correction factors was not necessary, since the outcome of the study would not be influenced.

Each three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes.

The test item and the positive and negative controls were washed off the skin tissues treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following nearly 3.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.6 till ≤ 1.5 for the 15 minutes treatment interval (values between 0.852 and 0.978) thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.6% thus ensuring the validity of the test system.

The rel. standard deviations between tissues of the same treatment group (main experiment) was below 9% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

After treatment with the test item the mean relative absorbance value was reduced to 17.7%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Reference 6

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 December 2017 until 21 January 2018

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)

Version / remarks:

Updated Guideline adopted July 29, 2016

Deviations:

no

Principles of method if other than guideline:

EC Directive 2000/33/EC, OJ L136200, dated June 08, 2000, adopting the 27th time to technical progress the Dangerous Substances Directive 67/548/EEC, Annex V, part B40 and EC regulation No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation No 1907/2006 of the European Parliament and of the Council on REACH, 1st ATP, section B40.

MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”, 07 November 2014.

There was the following alteration from the MatTek test protocol:
Concerning formazan salt extraction (chapter 3.6.3): The formazan salt extraction period was extended up to 72 hours (in the refrigerator without shaking). According to an expert statement (Dr. Helena Kandarova from MatTek Corporation) this procedure does not have an impact on the outcome of the study, if sufficient covering of the plates to prevent isopropanol evaporation is ensured.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Details on test system:

Epi-200 SCT Kit Components Needed for the Assay

EpiDerm™ Kit Lot No.: 25874
1 Sealed 24-well plate Contains 24 inserts with EpiDerm™ tissues on agarose
2 24-well plates For MTT viability assay
4 6-well plates For storing inserts, or for topically applying test agents
1 bottle Serum-Free Assay Medium DMEM-based medium
1 bottle DPBS Rinse Solution For rinsing the inserts in MTT assay

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant Solution (Isopropanol) For extraction of formazan crystals

MTT-Solution
The MTT-solution was prepared freshly on day of use (resulting: 1 mg/mL).

Pre-warming of EpiSkin™ Tissues:
18 hours and 25 minutes before dosing, EpiDerm™ tissues were removed from the refrigerator, and the inserts were transferred into 6-well plates containing the pre-warmed assay medium under sterile conditions using sterile forceps. A 24-well plate was prepared as holding plate containing 300 µL assay medium. The holding plate was pre-warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.

Treatment:
Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control for the following exposure times:
• Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
• Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
• Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes

After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

MTT Assay:
Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 µL) was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required.
Following rinsing, the tissues were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the tissues were rinsed three times with DPBS and carefully dried with blotting paper. The inserts were transferred into new 24-well plates. The tissues were each immersed in 2 mL of extractant solution (isopropanol) pipetted in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for about 70 hours without shaking in the refrigerator.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour.
3  200 µL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

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 (79.4 µL/cm2 according to guideline) of the test item were dispensed directly onto duplicate EpiDermTM tissue surface


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


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

Duration of treatment / exposure:

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

Duration of post-treatment incubation (if applicable):

After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

Number of replicates:

two replicates

Type of coverage:

other: topical

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Results after 3 minutes treatment

Value:

87.29

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

results after 60 minutes treatment

Value:

47.96

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: results within historical range

Results after treatment with Tall Oil Soap, TOS32 and controls

Dose Group	Mean Absorbance (OD) of 3 Wells Blank corrected	Mean Absorbance (OD) of 2 Tissues	Mean Rel. Viability [%]
Negative Control Tissue 1	1.584
Negative Control Tissue 2	1.544	1.564	100.00
Positive Control Tissue 1	0.438
Positive Control Tissue 2	0.419	0.429	27.4
Test Item Tissue 1	1.408
Test Item Tissue 2	1.388	1.398	87.29*
Negative ControlViable Tissue  Tissue 1	0.006
Negative ControlViable Tissue  Tissue 2	0.007	0.006	0.4
Test ItemViable Tissue Tissue 1	0.003
Test ItemViable Tissue Tissue 2	0.006	0.005	0.3
Negative ControlFreeze Killed Tissue  Tissue 1	0.098
Negative ControlFreeze Killed Tissue  Tissue 2	0.100	0.099	6.3
Test ItemFreeze Killed Tissue  Tissue 1	0.122
Test ItemFreeze Killed Tissue  Tissue 2	0.133	0.127	8.1

* Corrected Relative Viability [%]

Results after treatment with Tall Oil Soap, TOS32 and the controls / exposure interval 60 minutes

Dose Group	Mean Absorbance (OD) of 3 Wells Blank corrected	Mean Absorbance (OD) of 2 Tissues	Mean Rel. Viability [%]
Negative Control Tissue 1	1.356
Negative Control Tissue 2	1.328	1.342	100.0
Positive Control Tissue 1	0.082
Positive Control Tissue 2	0.058	0.070	5.2
Test Item Tissue 1	0.878
Test Item Tissue 2	0.980	0.929	47.96*
Negative ControlViable Tissue  Tissue 1	0.007
Negative ControlViable Tissue  Tissue 2	0.014	0.010	0.7
Test ItemViable Tissue  Tissue 1	0.018
Test ItemViable Tissue Tissue 2	0.013	0.016	1.2
Negative ControlFreeze Killed Tissue  Tissue 1	0.095
Negative ControlFreeze Killed Tissue  Tissue 2	0.093	0.094	7.0
Test ItemFreeze Killed Tissue  Tissue 1	0.354
Test ItemFreeze Killed Tissue  Tissue 2	0.372	0.363	27.1

 * Corrected Relative Viability [%]

Optical pre-experiment (colour interference pre-experiment) to investigate the test item’s water dying potential led to a change in colour and optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent showed blue colour. Therefore, additional tests with viable tissues (without MTT addition) and with freeze-killed tissues were performed to determine correction factors for calculating the true viability in the main experiment.

Even without taking the determined correction factors into consideration the mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 10.3% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin. Therefore, the calculation of the true viability was not necessary, since the outcome of the study is not affected.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, Tall Oil Soap, TOS32 is non corrosive to skin according to EU CLP and UN GHS.

Executive summary:

This in vitro study was performed to assess the corrosive potential of Tall Oil Soap, TOS32 by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item reduced MTT (pre-test for direct MTT reduction), and it changed colour when mixed with deionised water (pre-test for colour interference). Consequently, additional tests with freeze-killed and viable tissues to determine correction factors for calculating the true viability in the main experiment were necessary.

Independent duplicate tissues of EpiDerm^TM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO₂) with MTT solution followed. MTT solution was then rinsed from the wells and the wells were rinsed with DPBS. Inserts were transferred into new24 well plates. The formazan salt was extracted for about 70 hours at room temperature.

The required acceptability criteria were met.

Exposure to the positive control induced a decrease in the relative absorbance as compared tothe negative control, both for the 3 minutes exposure period (27.4%) and for the 1 hour exposure period (5.2%) thus confirming the validity of the test system and the specific batchof tissue models.

After exposure to the test item Tall Oil Soap, TOS32 the relative absorbance value only decreased to 87.29 after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 47.96%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

weight of evidence

Study period:

The test was performed on 10 February 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance was tested undiluted.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3 cornea per group (test item, negative control, positive control)

Details on study design:

Three corneas were exposed to each 0.75 mL of the undiluted item for 10 minutes.
Following treatment of the corneas, the test material was rinsed off and the opacity was determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.

Incubation Medium

The incubation medium consists of MEM, supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin per 500 mL medium (final concentration of 100 units penicillin per mL medium, and 100 µg streptomycin per mL medium). Immediately before starting the test, MEM was supplemented with 1% foetal calf serum (FCS).
The OECD guideline 437 recommends the use of EMEM which is in composition and osmolarity equivalent to the MEM, thus MEM can be used without restriction.

Opacity measurement

The opacitometer determines changes in the light transmission passing through the cornea, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the cornea was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the cornea to the test groups, after rinsing and further incubation of the cornea for two hours, the opacity value was determined again (t130).

Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Cornea were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and thereafter was the optical density at 490 nm (OD490) determined with a spectrophotometer.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
Opacity
The change of opacity value of each treated cornea or positive and negative control cornea is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea.
The average change in opacity of the negative control cornea is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.

Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1


Criteria for Determination of a Valid Test

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine cornea treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Run / experiment:

main experiment

Value:

13.65

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: fulfilled, no category (IVIS 1.18)
- Acceptance criteria met for positive control: fulfilled, category 1 (IVIS 86.01)

Results after 10 Minutes Treatment Time

Test Group	Opacity value #		Permeability ##		IVIS	Mean IVIS	Proposed IVIS
		Mean		Mean
Neg. Control	1		0.049		1.74
	0	0.33	0.071	0.057	1.07	1.18	Not categorized
	0		0.050		0.75
Pos. Control	48.67*		1.544*		71.83
	61.67*		1.984*		91.43	86.01	Category 1
	70.67*		1.607*		94.78
Leuco Sulphur Blue 15	5.67*		0.302*		10.20
	7.67*		0.415*		13.90	13.65	No prediction can be made
	7.67*		0.612*		16.85

# Opacity value = Difference (t₁₃₀ - t₀) of Opacity

## Permeability at 490 nm (OD₄₉₀)

*corrected values

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, a prediction for the damage hazard cannot be made (GHS) for Tall Oil Soap, TOS30.

Executive summary:

This in vitro study was performed to assess the corneal damage potential of Tall Oil Soap, TOS30 by means of the BCOP assay using fresh bovine cornea.

After a first opacity measurement of the fresh bovine cornea (t₀), the neat test item, the positive, and the negative controls were applied to cornea fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1°C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the cornea. Further, the corneas were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t₁₃₀).

After the opacity measurements permeability of the cornea was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horisontal position for 90 minutes at 32 ± 1°C.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the cornea corresponding to a classification as serious eye damaging (EU/EPA/GHS(Cat 1)).

 

Relative to the negative control, the test item Tall Oil Soap, TOS30 caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 13.65. According to OECD 437 the test item’s hazard for eye damage cannot be predicted.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The test was performed on 10 February 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance was tested undiluted.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3 cornea per group (test item, negative control, positive control)

Details on study design:

Three corneas were exposed to each 0.75 mL of the undiluted item for 10 minutes.
After treatment the test item was rinsed off the corneas and the corneas' opacity were determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.

Incubation Medium

The incubation medium consists of MEM, supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin per 500 mL medium (final concentration of 100 units penicillin per mL medium, and 100 µg streptomycin per mL medium). Immediately before starting the test, MEM was supplemented with 1% foetal calf serum (FCS).
The OECD guideline 437 recommends the use of EMEM which is in composition and osmolarity equivalent to the MEM, thus MEM can be used without restriction.

Opacity measurement

The opacitometer determines changes in the light transmission passing through the cornea, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the cornea was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Following exposure of the corneae to the test groups, rinsing and further incubation of the cornea for two hours, the opacity value was determined again (t130).

Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Cornea were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
Opacity
The change of opacity value of each treated cornea or positive and negative control cornea is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea.
The average change in opacity of the negative control cornea is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.

Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1


Criteria for Determination of a Valid Test

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine cornea treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Run / experiment:

main experiment

Value:

19.78

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: fulfilled, no category (IVIS 1.18)
- Acceptance criteria met for positive control: fulfilled, category 1 (IVIS 86.01)

Results after 10 Minutes Treatment Time

Test Group	Opacity value #		Permeability ##		IVIS	Mean IVIS	Proposed IVIS
		Mean		Mean
Neg. Control	1		0.049		1.74
	0	0.33	0.071	0.057	1.07	1.18	Not categorized
	0		0.050		0.75
Pos. Control	48.67*		1.544*		71.83
	61.67*		1.984*		91.43	86.01	Category 1
	70.67*		1.607*		94.78
Leuco Sulphur Blue 15	11.67*		0.791*		23.54
	10.67*		0.437*		17.23	19.78	No prediction can be made
	9.67*		0.593*		18.57

# Opacity value = Difference (t₁₃₀ - t₀) of Opacity

## Permeability at 490 nm (OD₄₉₀)

*corrected values

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, a prediction for the damage hazard cannot be made (GHS) for Tall Oil Soap, TOS31.

Executive summary:

This in vitro study was performed to assess the corneal damage potential of Tall Oil Soap, TOS31 by means of the BCOP assay using fresh bovine cornea.

After a first opacity measurement of the fresh bovine cornea (t0), the neat test item, the positive, and the negative controls were applied to cornea fixed in an incubation chamber in horisontal position for 10 minutes at 32 ± 1°C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the cornea. Furthermore, the cornea were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t130).

After the opacity measurements permeability of the cornea was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horisontal position for 90 minutes at 32 ± 1°C.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the cornea corresponding to a classification as serious eye damaging (EU/EPA/GHS(Cat 1)).

 

Relative to the negative control, the test item Tall Oil Soap, TOS31 caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 19.78. According to OECD 437 the test item’s hazard for eye damage cannot be predicted.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The test was performed on 10 March 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance was tested undiluted.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3 cornea per group (test item, negative control, positive control)

Details on study design:

Three corneas were exposed to each 0.75 mL of the undiluted item for 10 minutes.
After treatment the test item was rinsed off the corneas and the corneas' opacity were determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.

Incubation Medium

The incubation medium consists of MEM, supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin per 500 mL medium (final concentration of 100 units penicillin per mL medium, and 100 µg streptomycin per mL medium). Immediately before starting the test, MEM was supplemented with 1% foetal calf serum (FCS).
The OECD guideline 437 recommends the use of EMEM which is in composition and osmolarity equivalent to the MEM, thus MEM can be used without restriction.

Opacity measurement

The opacitometer determines changes in the light transmission passing through the cornea, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the cornea was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the cornea to the test groups, after rinsing and further incubation of the cornea for two hours, the opacity value was determined again (t130).

Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated again in a horisontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
Opacity
The change of opacity value of each treated cornea or positive and negative control cornea is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea.
The average change in opacity of the negative control cornea is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.

Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1


Criteria for Determination of a Valid Test

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine cornea treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Run / experiment:

main experiment

Value:

17.19

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: fulfilled, no category (IVIS 1.41)
- Acceptance criteria met for positive control: fulfilled, category 1 (IVIS 132.54)

Results after 10 Minutes Treatment Time

Test Group	Opacity value #		Permeability ##		IVIS	Mean IVIS	Proposed IVIS
		Mean		Mean
Neg. Control	0		0.339**		5.09**
	1	0.33	0.062	0.062	1.93	1.41	Not categorized
	0		0.059		0.89
Pos. Control	107.67*		1.398*		128.63
	132.67*		1.715*		158.39	132.54	Category 1
	94.67*		1.062*		110.59
Leuco Sulphur Blue 15	22.67**		0.708**		33.28**
	10.67*		0.420*		16.96	17.19	No prediction
	6.67*		0.717*		17.42

# Opacity value = Difference (t₁₃₀ - t₀) of Opacity

## Permeability at 490 nm (OD₄₉₀)

*corrected values

** outlier, not taken into account for IVIS calculation

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, a prediction for the damage hazard cannot be made (GHS) for Tall Oil Soap, TOS32.

Executive summary:

This in vitro study was performed to assess the corneal damage potential of Tall Oil Soap, TOS32 by means of the BCOP assay using fresh bovine cornea.

After a first opacity measurement of the fresh bovine cornea (t₀), the neat test item, the positive, and the negative controls were applied to the cornea fixed in an incubation chamber in horisontal position for 10 minutes at 32 ± 1°C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the cornea. Furthermore, the cornea were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t₁₃₀).

After the opacity measurements permeability of the cornea was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horisontal position for 90 minutes at 32 ± 1°C.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the cornea corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). 

Relative to the negative control, the test item Tall Oil Soap, TOS32 caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 17.19. According to OECD 437 the test item’s hazard for eye damage cannot be predicted.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion:

In vitro skin corrosion studies were performed to assess the corrosive potential of three representative manufactured samples of Tall Oil Soap, (TOS30, TOS31 and TOS32) by means of the Human Skin Model Test with EpiDerm™ tissue models, according to OECD TG 431 and in compliance with GLP. The measured pH of the three samples were 12.5, 12.4 and 12.4 respectively.

The test substances reduced MTT (pre-test for direct MTT reduction), and it changed colour when mixed with deionised water (pre-test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were necessary.

Independent duplicate tissues of EpiDerm^TM were exposed to the test substances, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour respectively.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1°C, 5 ± 0.5 % CO₂) with MTT solution followed. MTT solution was then rinsed from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for about 70 hours in the refrigerator.

The required acceptability criteria were met.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (27.4%) and the 1 hour exposure period (5.2%) in all three studies, thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item Tall Oil Soap, TOS30, the relative absorbance value increased slightly to 101.66% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 87.21%. Both values did not fall below the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test substance was not considered to be corrosive.

After exposure to the test item Tall Oil Soap, TOS31, the relative absorbance value decreased to 79.76% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 48.56%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test substance was not considered to be corrosive.

After exposure to the test item Tall Oil Soap, TOS32, the relative absorbance value only decreased to 87.29 after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 47.96%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test substance was not considered to be corrosive.

Additional in vitro skin corrosion studies are available for another three representative manufactured samples of Tall Oil Soap (TOS E, TOS, 23 and TOS 24). The studies were conducted in Epiderm Human Skin Model, according to OECD Test Guideline 431 and incompliance with GLP. The results of the three studies demonstrated that the Tall Oil Soap samples tested were not corrosive to skin, supporting the findings of the key studies (Harlan, 2012a,b,c).

Skin irritation:

In vitro skin irritation studies were performed to assess the irritation potential of three representative manufactured samples of Tall Oil Soap, (TOS30, TOS31 and TOS32) by means of the Human Skin Model Test, according to OECD 439 and in compliance with GLP. The measured pH of the three samples was 12.5, 12.4 and 12.4 respectively.

The test substances dyed water in the colour interference pre-test slightly brown, and it reduced MTT (pre-test for direct MTT reduction). Consequently, additional tests with freeze-killed and viable tissues (without MTT addition) had to be performed to determine correction factors for calculating the true viability in the main experiment. Since the test item proved to possess a clear skin irritating potential in the main experiment, consideration of correction factors was not necessary, since the outcome of the study would not be influenced.

Each of three tissues of the human skin model EpiSkin™ were treated with the test substances, the negative or the positive control for 15 minutes.

The test item and the positive and negative controls were washed off the skin tissues treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following nearly 3.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.6 till ≤ 1.5 for the 15 minutes treatment interval (values between 0.760 and 0.888) thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.9% thus ensuring the validity of the test system.

For the study with TOS30, the relative standard deviations between tissues of the same treatment group (main experiment) were below 9% (threshold OECD TG: 18%), thus ensuring the validity of the studies. After treatment with TOS30, the mean relative absorbance value was reduced to 10.3%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

For the study with TOS31, the relative standard deviations between tissues of the same treatment group (main experiment) was below 9% (threshold of OECD 439: 18%), thus ensuring the validity of the study. After treatment with the test substance the mean relative absorbance value was reduced to 34.2%. This value is below the threshold for irritancy of ≤50%. Therefore, TOS31 is considered to possess an irritant potential.

For the study with TOS32, the relative standard deviations between tissues of the same treatment group (main experiment) was below 9% (threshold of OECD TG 439: 18%), thus ensuring the validity of the study. After treatment with the test substance the mean relative absorbance value was reduced to 17.7%. This value is below the threshold for irritancy of ≤50%. Therefore, the TOS32 is considered to possess an irritant potential.

Eye irritation:

In vitro eye irritation studies were performed to assess the irritation potential of three representative manufactured samples of Tall Oil Soap, (TOS30, TOS31 and TOS32) by means of the Bovine Corneal Opacity (BCOP) Assay, according to OECD 437 and in compliance with GLP. The measured pH of the three samples was 12.5, 12.4 and 12.4 respectively.

After a first opacity measurement of the fresh bovine cornea (t0), the neat test substances, the positive, and the negative controls were applied to cornea fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1°C. The posterior chamber contained incubation medium. After the incubation phase the test substances, the positive, and the negative controls were each rinsed from the cornea. Further, the cornea were incubated for another 120 minutes at 32 ± 1°C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t130).

After the opacity measurements permeability of the cornea was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1°C.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the cornea could be observed.

The positive control (2-ethoxyethanol) showed clear opacity and distinctive permeability of the cornea corresponding to a classification as serious eye damaging (EU/EPA/GHS (Cat 1)).

Relative to the negative control, Tall Oil Soap, TOS30 caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 13.65.

Relative to the negative control, TOS31 caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 19.78.

Relative to the negative control, the test item Tall Oil Soap, TOS32 caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 17.19.

According to OECD TG 437, the potential for eye damage cannot be predicted for substances with irritancy scores in the range >3 - ≤55. Based on the available studies it is therefore not possible to reach a conclusion concerning the eye irritation classification for TOS.

Justification for classification or non-classification

An assessment of corrosivity/irritation potential can, however, be made based on the ‘free alkali’ approach. Measured data on pH and alkaline reserve are available from 34 samples of TOS collected during February and March 2019 from 22 pulp mills. Values of pH were in the range ca. 10.2 to 13.7 and measured free alkali was in the range 0.3 to 7.5% as NaOH. There was no correlation between the measured pH and free alkali, as shown in Figure 5.3.1 (attached in Section 13). This may be due to the complexity of constituents present in this UVCB substance.

Classification of individual products should therefore be made by comparison of the free alkali (as %NaOH) with the harmonised classification of NaOH in Annex VI of Regulation (EC) No 1272/2008:

≥5% NaOH - Skin Corr. 1A H314: Causes severe skin burns and eye damage

2-5% NaOH  - Skin Corr. 1B H314: Causes severe skin burns and eye damage

0.5-2% NaOH - Skin Irrit. 2 H315: Causes skin irritation

                      Eye Irrit. 2 H319: Causes serious eye irritation

 

 

Based on the measured in vitro data it can be concluded that any samples meeting the criteria for Skin Irrit. 2 should also be classified as Eye Irrit. 2. Samples meeting the criteria for Skin Corr. 1B are given the H314 hazard statement covering both severe skin burns and severe eye damage.

 

The free alkali content of samples TOS30, TOS31 and TOS32 was not measured. An earlier programme of testing skin corrosion testing according to OECD 431 gave results of “not corrosive” for three samples of TOS, the results are are reported in the table below. At the time, no measurements of pH or free alkali content were made. Measurements from the same mills for the 2019 programme were:

 

 

pH	Free alkali, %NaOH
13.1	0.2
12.2	0.34
12.8	0.5

 

 

These data indicate that the approach may be considered precautionary since a sample above the 2% threshold was not corrosive. However, this cannot be definitively concluded due to the variability of the measured data.