Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)

Endpoint summary

Description of key information

The in vitro skin corrosion potential of Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) was assessed in vitro according to OECD Test Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis Test Method). This study was conducted in accordance with GLP and assigned a Klimisch score of 1 using the criteria for assessing data quality set forth by Klimisch et al. (1997)

This in vitro assay predicts the skin corrosion potential of a chemical by measurement of its cytotoxic effect on the EpiDerm™ tissue model. The test item is applied directly to the skin surface, cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from the skin tissues. The test item is classified "corrosive", if the relative mean of tissue viability after 3 minute treatment with a test material is decreased below 50%. In addition, those materials classified "non-corrosive" after 3 minute treatment (viability ≥ 50%) are classified "corrosive" if the relative mean of tissue viability after 1 hour treatment with a test material is decreased below 15%.

 In the OECD TG 431 study, the percentage viabilities obtained after 3 minutes and 1hr exposure were 84.23% and 5.37% respectively, therefore Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) is classified as Corrosive to skin and eyes. The corrosive properties of Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) are likely to be a result of the dissociation of the main constituents of this salt, which yeilds free octanoic acid (CAS 124 -07 -2), a substance which is classified as Skin Corrosive Category 1C.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The is a OECD 431 guideline study conducted in accordance with GLP on the substance Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1), which has been a Klimisch rating of 1.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch 1115-80.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiDermTM skin model and assay for skin corrosion testing is endorsed by OECD TG 431.

Details on 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 differential 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Single topical application of 50µl of test item, negative control (water) or positive control (potassium hydroxide) was applied to the surface of the EpiDerm(TM) skin models.

Duration of treatment / exposure:

3 and 60 minutes at 37°C, 5% CO2, ≥95% RH (Relative Humidity).

Number of replicates:

Three tissues per condition (n=3).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Viability after 3 minutes

Value:

ca. 84.23

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Viability after 1 hour

Value:

ca. 5.37

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Cell viability measurements after 3 minutes of application.

3 Minute Endpoint		NC	PC	TA1
Tissue 1	Aliquot 1 OD	2.38	0.14	2.23
	Aliquot 2 OD	2.38	0.13	2.18
Tissue 2	Aliquot 1 OD	2.50	0.22	1.69
	Aliquot 2 OD	2.57	0.22	1.64
Tissue 3	Aliquot 1 OD	2.31	0.06	2.21
	Aliquot 2 OD	2.30	0.07	2.22
All tissues	Average OD	2.41	0.14	2.03
	Average % Viability	100.00	5.82	84.23
	Average % SD	4.01	2.63	10.71
	Average % CV	4.01	45.10	12.71

NC: negative control (H₂O), PC: Positive control (KOH 8N), TA1: Octanoic Acid Triethanolamine Salt. *CV value of the PC is above 30% (45.10%) however, as the percentage of viability is below 20% (5.82%) acceptance criterion 3 is met.

Cell viability measurements after 1 hour of application.

1 Hour Endpoint		NC	PC	TA1
Tissue 1	Aliquot 1 OD	2.18	0.12	0.15
	Aliquot 2 OD	2.20	0.11	0.14
Tissue 2	Aliquot 1 OD	2.08	0.21	0.09
	Aliquot 2 OD	2.03	0.23	0.08
Tissue 3	Aliquot 1 OD	1.95	0.02	0.10
	Aliquot 2 OD	2.04	0.03	0.10
All tissues	Average OD	2.08	0.12	0.11
	Average % Viability	100.00	5.75	5.37
	Average % SD	4.01	3.76	1.18
	Average % CV	4.01	65.52*	22.06

NC: negative control (H₂O), PC: Positive control (KOH 8N), TA1: Octanoic Acid Triethanolamine Salt. *CV value of the PC is above 30% (65.52%) however, as the percentage of viability is below 20% (5.75%) acceptance criterion 3 is met (as per OECD TG431).

Evalutation of the results - Results were checked against the following acceptance criteria:

		Actual values	Pass/Fail
Acceptance criterion 1	The mean OD570of the negative control tissues must be ≥0.8.	3 min = 2.41 1 hr = 2.08	Pass
Acceptance criterion 2	The mean relative percentage viability of the positive control, after 1hour exposure must be < 15% of the mean of the negative control.	5.75	Pass
Acceptance criterion 3	In the range between 20% and 100% viability, the coefficient of variation (CV) is an additional acceptance criterion.It should not exceed 0.3(30%).	CV 3 minutes 1 hour NC 4.01 4.01 PC 45.10* 65.52* TA1 12.71 22.06	Pass

*Note that the CV values of the PC after 3 min and 1hr were above 30% (45.10% and 65.52% respectively). However, as the percentage of cell viability was below 20% (5.82% and 5.75% respectively), acceptance criterion 3 was met (as per OECD TG431).

 Results Summary

Test item	Test Item ID	Viability after 3 minutes application (% to negative control)	Viability ≥ 50% after 3 minutes (Yes/No)	Viability after 1h application (% to negative control)	Viability ≥ 15% after 1 hour (Yes/No)	Corrosive (C) / Non corrosive (NC)
Octanoic Acid Triethanolamine Salt	TA1	84.23	Yes	5.37	No	Corrosive

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

After a 3 minute and 1 hour exposure on the surface of EpiDermTM reconstructed human epidermis, the viability of the tissues was assessed and compared to a negative control. The percentage viabilities obtained after 3 minutes and 1hr were 84.23% and 5.37% respectively and therefore Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) is classified as Corrosive to human skin, and can be sub-categorised as 1B/1C.

Executive summary:

The skin corrosion potential of Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) was assessed in vitro according to OECD Test Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis Test Method). This study was conducted in accordance with GLP and assigned a Klimisch score of 1 using the criteria for assessing data quality set forth by Klimisch et al. (1997)

 

This in vitro assay predicts the skin corrosion potential of a chemical by measurement of its cytotoxic effect on the EpiDerm™ tissue model. The test item is applied directly to the skin surface, cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from the skin tissues.

 

Prior to the execution of the study, the required compatibility checks confirmed that the test item did not interfere with MTT, and following the study results were checked and validated against acceptance criteria.

 

A test item is classified "corrosive", if the relative mean of tissue viability after 3 minute treatment with a test material is decreased below 50%. In addition, those materials classified "non-corrosive" after 3 minute treatment (viability ≥ 50%) are classified "corrosive" if the relative mean of tissue viability after 1 hour treatment with a test material is decreased below 15%.

 

The classification can be then further sub-categorized to 1 A or B/ C. A test item is classified "corrosive, optional Sub-Category 1A", if the relative tissue viability after 3 minute treatment with a test material is decreased below 25%. A test item is classified "corrosive, optional Sub-Category 1B/1C", if the relative tissue viability after 3 minute treatment with a test material is ≥25%.

 

In this study, the percentage viabilities obtained after 3 minutes and 1hr were 84.23% and 5.37% respectively therefore Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) is classified as Corrosive to human skin, and can be sub-categorised as 1B/1C.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Dec 2018 - Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This is a OECD 439 guideline study conducted in accordance with GLP using the test substance octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1), which has been assigned a Klimish Rating of 1.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch 1115-80

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

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.

Details on 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

A single topical application of 30 µl of neat test item, negative control (DPBS) or positive control (SDS 5%) to the surface of EpiDermTM model.

Duration of treatment / exposure:

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

Duration of post-treatment incubation (if applicable):

42±4 hours post-treatment incubation.

Number of replicates:

Three tissues per condition (Negative Control, Positive Control and Test Item).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

ca. 2.469

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Viability measurements after 60±1 minutes of application and 42±4 hours post-incubation of test and reference items and controls.

Condition	Tissue #	Raw data		Blank corrected data		Mean OD	% of Viability
		Aliquot 1	Aliquot 2	Aliquot 1	Aliquot 2
NC	Tissue 1	2.25	2.185	2.095	2.030	2.063	98.567
	Tissue 2	2.502	2.461	2.347	2.306	2.327	111.181
	Tissue 3	2.038	2.049	1.883	1.894	1.889	90.252
PC	Tissue 1	0.234	0.22	0.079	0.065	0.072	3.456
	Tissue 2	0.214	0.214	0.059	0.059	0.059	2.835
	Tissue 3	0.199	0.206	0.044	0.051	0.048	2.286
TA1	Tissue 1	0.221	0.212	0.066	0.057	0.062	2.955
	Tissue 2	0.205	0.205	0.050	0.050	0.050	2.405
	Tissue 3	0.196	0.199	0.041	0.044	0.043	2.047

NC: negative control (DPBS), PC: Positive control (SDS 5%), TA1: Octanoic Acid Triethanolamine Salt.

Mean and SD of cell viability measurements and of viability percentages after 60±1 minutes of application and 42±4 hours post-incubation.

Name	Code	Mean of OD	SD of OD	Mean of viability (%)	SD of viability (%)	CV %	Classification
DPBS	NC	2.093	0.221	100.000	10.538	10.538	Non-Irritant
SDS 5%	PC	0.060	0.012	2.859	0.586	20.486	Irritant
Test Item	TA1	0.052	0.010	2.469	0.457	18.522	Irritant

NC: Negative control (DPBS), PC: Positive control (SDS 5%), TA1: Octanoic Acid Triethanolamine Salt.

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

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 must be≥ 0.8 and ≤ 2.8	2.093	PASS
Acceptance criterion 2	The mean of the positive control relative percentage viability must be ≤ 20% of the mean of the negative controls.	2.859	PASS
Acceptance criterion 3	Thes tandard deviation of viability percentages for triplicate skin models in each experimental condition must be < 18%	NC: 10.538 PC: 0.586 TA1: 0.457	PASS
Acceptance criterion 4	The mean OD of the 6 wells containing extraction solvent alone (blanks) must be ≤ 0.1.	0.155	ACCEPT*

*Optical Density (OD) values obtained with blanks were higher than 0.1 (0.155) 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 our current internal acceptance criteria of blank OD values <0.244 (mean + 2SD of XCellR8 historical data, based on blanks obtained during 88 historic runs), therefore this is not considered to be an issue in the interpretation of this study data.

Result Summary

Test Item	Percentage of viability (relative to negative control)	Classification Irritant / Non irritant
Octanoic Acid Triethanolamine Salt	2.469	Irritant

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

Conclusions:

The percentage of viability obtained with the test item Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) was 2.469%, therefore it was considered as Irritant to the skin.

Executive summary:

The skin irritation potential of Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) was assessed using an in vitro study conducted according to OECD guideline TG439: In vitro Skin Irritation: Reconstructed Human Epidermis (RHE) Test Method and GLP. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

 

This in vitro assay predicts the skin irritation potential of a chemical by measurement of its cytotoxic effect on the EpiDerm™tissue model. The test item is applied directly to the skin surface, providing a good model of “real life”exposure. Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from the skin tissues. Irritant test items are identified by their ability to decrease cell viability below defined threshold levels (below or equal to 50% for UN GHS Category 2). If the viability is greater than 50%, the test item is classified as Non-Irritant (no-label).

 

Prior to the execution of the study, the required compatibility checks confirmed that the test item, Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) did not interfere with MTT and following the study results were checked and validated against acceptance criteria.

 

The percentage of viability obtained with the test item Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) was 2.469%, therefore it was considered as Irritant to the skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Justification for classification or non-classification

Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) is classified as corrosive to skin and eyes, as in the in vitro OECD TG 431 study, the percentage viabilities obtained after 3 minutes and 1hr exposure were 84.23% and 5.37% respectively.