Potassium Lauroyl Wheat Amino Acids

Endpoint summary

Description of key information

Based on the results of the in vitro skin irritation/corrosion studies, the test substance is considered to be irritating to the skin and eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 28, 2017 to September 01, 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:

yes

Remarks:

However, Guideline or SOP deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

The reconstructed human epidermal model EpiDermTM (EPI-200-MatTek Corporation) consists of normal human-derived epidermal keratinocytes which have been cultured to form a multi-layered highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Justification for test system used:

Initially the predictive capacity of the modified EpiDerm™ Skin Irritation Test (SIT) test method, using MatTek EpiDermTM tissue model EPI-200, underwent full prospective validation from 2003-2007. The test method components of this method were used to define the essential test methods components of the original and updated ECVAM Performance Standards (PS). A modification of the original EpiDerm™ SIT was validated using the original ECVAM PS in 2008. In 2008, ESAC concluded that the Modified EpiDerm™ SIT has sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non-skin irritating) test substances.

Vehicle:

water

Details on test system:

Characterisation of the test system:

MatTek’s EpiDermTM model has been extensively characterised for multiple parameters including morphology, tissue viability, skin barrier function and sterility. QC results for the specific lot of models received (Lot# 25839) were checked in-house for MatTek acceptance ranges with following outcome:
Morphology - PASS
Tissue viability - PASS
Skin barrier function (ET50 value for 1% Triton X-100) where ET50 is the time taken for 1% Triton X-100 to reduce the viability of the skin model to 50% relative to the negative control)- PASS
Sterility testing showed no contamination during long term antibiotic and antimycotic free culture- PASS

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µL of neat test substance (60.7% active)

Duration of treatment / exposure:

60 ± 1 minute (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, ≥95% RH)

Duration of post-treatment incubation (if applicable):

42 ± 4h

Number of replicates:

Three tissues replicates per condition

Irritation / corrosion parameter:

% tissue viability

Remarks:

and c(Compared to the negative control)

Run / experiment:

Mean of 3 replicates

Value:

ca. 4.021

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results

Prior to the study, the required compatibility checks confirmed that the test substance did not interfere with MTT and no water colouration was observed.

 

Table 1: Results summary

	Percentage of viability (relative to negative control)	Classification Irritant (I)/non irritant (NI)
Test substance	4.021%	Irritant (I)

 The test substance reduced the viability to below 50% and should be considered as Irritant to the skin.

Data Analysis:

Table 2: Viability measurements after 60 min (± 1min) of application and 46h (+9 mins) post-incubation of test and reference substance and controls

Condition	Tissue #	Raw data		Blank corrected data		Mean OD	% of Viability
		Aliquot 1	Aliquot 2	Aliquot 1	Aliquot 2
NC	Tissue 1	1.911	1.865	1.737	1.691	1.714	105.0
	Tissue 2	1.265	1.371	1.091	1.197	1.144	77.8
	Tissue 3	1.856	1.595	1.682	1.421	1.552	95.0
PC	Tissue 1	0.265	0.282	0.091	0.108	0.100	6.1
	Tissue 2	0.232	0.232	0.058	0.058	0.058	3.6
	Tissue 3	0.231	0.291	0.057	0.117	0.087	5.3
TA1	Tissue 1	0.284	0.294	0.110	0.120	0.115	7.1
	Tissue 2	0.217	0.215	0.043	0.041	0.042	2.6
	Tissue 3	0.213	0.213	0.039	0.039	0.039	2.4

NC: negative control (DPBS), PC: Positive control (SDS 5%), TA1: Test substance. 

Note: Rounded figures used. NC tissue 2 was removed from further analysis as it was an outlier value as can be seen from the OD values and % viability.

  

Table 3: Mean and SD of cell viability measurements and of viability percentages after 60 min (± 1min) of application and 46h (+9 mins) post-incubation

Name	Code	Mean of OD	SD of OD	Mean of viability (%)	SD of viability (%)	CV %	Classification
DPBS	NC	1.633	0.115	100	7.036	7.036	Non-Irritant
SDS 5%	PC	0.082	0.021	5.011	1.304	26.016	Irritant
Test substance	TA1	0.066	0.043	4.021	2.635	65.541	Irritant

NC: Negative control (DPBS), PC: Positive control (SDS 5%), TA1: Test substance.

Prediction model of irritancy: test substance that reduce the viability to 50% or below are irritant (I), test substance with a percentage viability above 50% are considered to be non-irritant (NI).Note: Rounded figures used.

Evaluation of the results

 

Results were checked against the following acceptance criteria:

	Description	Actual values	PASS/FAIL
Acceptance criterion 1	The mean OD570of the negative control (treated with DPBS) tissues is≥ 0.8 and ≤ 2.8	1.633	PASS
Acceptance criterion 2	The mean of the positive control relative percentage viability must be ≤ 20% of the mean of the negative controls.	5.011	PASS
Acceptance criterion 3	The standard deviation of OD values for triplicate skin models in each experimental condition must be < 18%	NC: 7.036 PC: 1.304 TA1: 2.635	PASS
Acceptance criterion 4	The mean OD of the 6 wells containing extraction solvent alone (blanks) should be ≤ 0.1.	0.174	FAIL

All acceptance criteria were met with the exception of criterion 4:

Optical Density (OD) values obtained with blanks were higher than 0.1 (0.174) causing a deviation from Acceptance Criteria 4. However, the spectrophotometer was fully validated and had passed all required tests. The OD values for blanks observed in this study are consistent with historical data using this spectrophotometer in the XCellR8 laboratory and meet laboratory internal acceptance criteria of blank OD values <0.194 (mean of XCellR8 historical data, based on blanks obtained during 66 historical runs), therefore this is not considered to be an issue in the interpretation of this study data. The study author concluded that this SOP and guideline deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained.

 

Interpretation of results following Prediction Model

1) A test substance is considered to be an irritant (I) to skin in accordance with UN GHS Category 2 or EU R38 if the skin model viability after exposure and post-treatment incubation is ≤50%.

2) A test substance may be considered as a non-irritant (NI) if the skin model viability after exposure and post-treatment incubation is >50%.

The percentage of viability obtained with the test substance test substance was 4.021%, therefore it was considered as Irritant to the skin.

Interpretation of results:

other: Category 2 (irritant) based on EU CLP criteria

Conclusions:

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

Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'potassium lauroyl wheat amino acids' (active: 69.2%), using Reconstructed Human Epidermis Test method, according to OECD Guideline 439, in compliance with GLP. EpiDermTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% relative humidity (RH), after which 30 µL test substance and reference substances (negative control: sterile Dulbecco’s phosphate buffered saline and positive control: sodium dodecyl sulphate (5% in water)) were applied topically for 60 ± 1 minute in triplicate, followed by rinsing steps and a 46 h + 9 mins post-dose incubation at 37°C, 5% CO2, ≥95% RH. Medium was changed on Day 2. MTT viability test was conducted and readings at 570 nm without reference filter were taken on Day 3. As per the guideline criteria, a test substance is considered to be an irritating to skin if the skin model viability after exposure and post-treatment incubation is ≤50%. Based on the results, the final percentage of viability obtained with the test substance was determined to be 4.021%, which is well below the non-irritant limit of 50%; therefore the test substance was considered as irritating to the skin. The Optical Density (OD) values obtained with blanks were higher than 0.1 (0.174) causing a deviation from Acceptance Criteria 4. The study author concluded that this deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained. Hence, the study results qualified the acceptance criteria. Under the study conditions, the test substance was determined to be irritating to the skin (XCellR8, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 20, 2017 to September 22, 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:

yes

Remarks:

Deviations were considered to have not affected the integrity or validity of the study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviations were considered to have not affected the integrity or validity of the study

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source of Bovine Eyes
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 h of receipt.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL test substance or reference substances (Positive control: neat Ethanol, Negative control: Sodium chloride 0.9% w/v)

Duration of treatment / exposure:

At 32 ± 1 ºC for 10 minutes

Duration of post- treatment incubation (in vitro):

At 32 ± 1 ºC for 90 minutes for permeablity assessment

Number of animals or in vitro replicates:

Three corneas to each test substance and reference substances

Details on study design:

Preparation of corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
 
Selection of corneas and opacity reading
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substance.
 
Treatment of corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test substance or control substances were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 10 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number PQ52DX and HN60FW.
 
Application of sodium fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.
 
Permeability determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.360 µL of media representing each cornea was dispensed into the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
 
Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin. In this study histopathology was not required.
 
Data evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
 
Opacity measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
 
Permeability measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
 
In Vitro irritancy score
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
 
Additionally, the opacity and permeability values were evaluated independently to determine whether the test substance induced a response through only one of the two endpoints.
 
Visual observation
The condition of the cornea was visually assessed post treatment.

The negative and positive control data was shared with Envigo study number PQ52DX and HN60FW.

Irritation parameter:

in vitro irritation score

Run / experiment:

Test substance

Value:

ca. 19.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: IVIS is within the range: ≥3-≤55; therefore no predication can be made

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive control

Value:

ca. 45.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative control

Value:

ca. 0.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied.

Results

Corneal Opacity and Permeability measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in following table 1:

Treatment	Cornea Number	Opacity					Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation - Pre‑Treatment	Corrected Value		Corrected Value
Negative Control*	1	3	3	3	0		0.000
	2	5	6	6	1		0.002
	3	3	3	4	1		0.001
	Mean				0.7		0.001		0.7
Positive Control*	4	4	36	34	30	29.3	0.659	0.658
	5	4	35	35	31	30.3	1.276	1.275
	6	2	35	35	33	32.3	1.061	1.060
	Mean					30.7		0.998	45.6
Test Substance	10	2	16	17	15	14.3	0.265	0.264
	11	6	22	20	14	13.3	0.402	0.401
	12	3	13	16	13	12.3	0.588	0.587
	Mean					13.3		0.417	19.6

OD= Optical density 

* = The negative and positive control data was shared with Envigo study number PQ52DX and HN60FW.

Corneal Epithelium Condition

The condition of each cornea is given in below table :

Treatment	Cornea Number	Observation
		Post Treatment	Post Incubation
Negative Control*	1	Clear	Clear
	2	Clear	Clear
	3	Clear	Clear
Positive Control*	4	Cloudy	Cloudy
	5	Cloudy	Cloudy
	6	Cloudy	Cloudy
Test Substance	10	Partly Cloudy	Cloudy
	11	Partly Cloudy	Cloudy
	12	Partly Cloudy	Cloudy

*= Control data shared with Envigo study number PQ52DX and HN60FW

The corneas treated with the test substance were partly cloudy post treatment and post incubation. The corneas treated with the negative control substance were clear post treatment and post incubation. The corneas treated with the positive control substance were cloudy post treatment and post incubation.

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Substance	19.6
Negative Control	0.7
Positive Control	45.6

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied.

Conclusion

No prediction of eye irritation can be made.

Interpretation of results:

other: no prediction could be made

Conclusions:

Under the study conditions, no prediction of eye irritation could be made for the test substance.

Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'potassium lauroyl wheat amino acids' (active: 69.2%), using the Bovine corneal Opacity Test (BCOP) method, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. Preparation, selection and opacity reading of the corneas were performed as per the guideline. Prepared corneas in triplicates were treated with each, test substance (undiluted), negative control (sodium chloride 0.9% w/v) and positive control (neat ethanol) substances at 32 ± 1ºC for 10 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete Eagle’sMinimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number PQ52DX and HN60FW. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC for 90 minutes. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The IVIS of the test substance was determined to be 19.6 which is well below the corrosive limit of 55 and above the non-corrosive limit of 3. The positive control IVIS was within the range of 31.6 to 58.7; therefore acceptance criterion was satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077; therefore the negative control acceptance criteria were satisfied. T The quality criteria required for acceptance of results in the test were satisfied. Under the study conditions, no prediction of eye irritation could be made for the test substance (Envigo, 2018).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

Study 1:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'potassium lauroyl wheat amino acids' (active: 69.2%), using Reconstructed Human Epidermis (RHE) Test method, according to OECD Guideline 439, in compliance with GLP. EpiDermTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% relative humidity (RH), after which 30 µL test substance and reference substances (negative control: sterile Dulbecco’s phosphate buffered saline and positive control: sodium dodecyl sulphate (5% in water)) were applied topically for 60 ± 1 minute in triplicate, followed by rinsing steps and a 46 h + 9 mins post-dose incubation at 37°C, 5% CO2, ≥95% RH. Medium was changed on Day 2. MTT viability test was conducted and readings at 570 nm without reference filter were taken on Day 3. As per the guideline criteria, a test substance is considered to be an irritating to skin if the skin model viability after exposure and post-treatment incubation is ≤50%. Based on the results, the final percentage of viability obtained with the test substance was determined to be 4.021%, which is well below the non-irritant limit of 50%; therefore the test substance was considered as irritating to the skin. The Optical Density (OD) values obtained with blanks were higher than 0.1 (0.174) causing a deviation from Acceptance Criteria 4. The study author concluded that this deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained. Hence, the study results qualified the acceptance criteria. Under the study conditions, the test substance was determined to be irritating to the skin (XCellR8, 2017).

Study 2:

An in vitro study was conducted to determine the skin corrosion potential of the test substance, 'pPotassium lauroyl wheat amino acids' (69.2% active), using Reconstructed Human Epidermis (RHE) cells, according to OECD Guideline 431, in compliance with GLP. EpiDermTM tissues were kept overnight at 4°C. On Day 1, the tissues were pre-incubated for 1 h at 37°C, 5% CO2, 95% RH. After incubation, tissues were exposure to test (50 μL neat test substance) and reference substances (50 μL sterile water as negative control and 50 μL Potassium hydroxide as positive control) in triplicates for 3 and 60 minutes. After 3 minutes and 1 h treatment, the test substance and the reference substances were rinsed off from the tissues. Cell viability of the tissues was evaluated by addition of MTT on Day 2 and final MTT assay testing and measurements were performed. Results were compared to negative control. All validity criteria for the performed test were met. After 3 minutes and 1 h treatment, the mean viability values obtained with the test substance were determined to be 69% and 99.4%, respectively, which is well above the corrosive limits of 50 and 15% respectively. Under the study conditions, the test substance was determined to be non-corrosive to the skin (XCellR8, 2018).

Based on the available results from in vitro irritation/corrosion studies, the test substance, 'potassium lauroyl whear amino acids' is considered to be irritating to the skin.

Eye:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'potassium lauroyl wheat amino acids' (active: 69.2%), using the Bovine corneal Opacity Test (BCOP) method, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. Preparation, selection and opacity reading of the corneas were performed as per the guideline. Prepared corneas in triplicates were treated with each, test substance (undiluted), negative control (sodium chloride 0.9% w/v) and positive control (neat ethanol) substances at 32 ± 1ºC for 10 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete Eagle’sMinimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number PQ52DX and HN60FW. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC for 90 minutes. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The IVIS of the test substance was determined to be 19.6 which is well below the corrosive limit of 55 and above the non-corrosive limit of 3. The positive control IVIS was within the range of 31.6 to 58.7; therefore acceptance criterion was satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077; therefore the negative control acceptance criteria were satisfied. T The quality criteria required for acceptance of results in the test were satisfied. Under the study conditions, no prediction of eye irritation could be made for the test substance (Envigo, 2018).

Based on the in vitro BCOP study, no clear conclusion could be drawn as per the Guideline. However, given the IVIS score (i.e., 19.6), which is in between the threshold for corrosive (i.e., >55) and non-corrosive limits (i.e., <=3), together with positive skin irritation potential (which was assessed based on the in vitro skin irritation (RHE) study with the test substance), indicate that a corrosive potential can be ruled out and the test substance, 'potassium lauroyl wheat amino acids’ can be considered to be more likely to be irritating to the eyes (in a worst case).

Justification for classification or non-classification

Skin:

Based on the results of in vitro skin irritation/corrosion studies, the test substance, ‘potassium lauroyl wheat amino acids’, is concluded to warrant ‘Skin Irrit. 2; H315 - Causes skin irritation’ classification according to the EU CLP criteria (Regulation 1272/2008/EC).

 

Eye:

Based on the available weight of evidence from in vitro eye corrosion and in vitro skin irritation/corrosion studies, the test substance, ‘potassium lauroyl wheat amino acids’, is concluded to warrant ‘Eye Irrit. 2: H319 causes serious eye irritation’ classification according to the EU CLP criteria (Regulation 1272/2008/EC).