Octadecanoic acid, reaction products with 2-amino-2-methyl-1-propanol

Endpoint summary

Description of key information

Based on the in vitro test results, the test substance, C16 -18 AMP is considered to be non-irriating to 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 May 16, 2017 to June 16, 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:

see "Principles of method if other than guideline

Principles of method if other than guideline:

Optical Density (OD) values obtained with blanks were higher than 0.1 (0.191) causing a deviation from Acceptance Criteria. However, this is not considered to be an issue in the interpretation of this study data.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other:

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed human epidermal model

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:

other: Sterile Dulbecco’s Phosphate Buffered Saline

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.

Lot No.: 25819
Keratinocyte Strain: 00267

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# 25819) were checked in-house for MatTek acceptance ranges with the 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:

Nominal 25 mg neat test substance

Duration of treatment / exposure:

60 minutes (25 minutes at room temperature and 35 minutes at 37 °C, 5 % CO2)

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Three tissues per condition (n=3).

Irritation / corrosion parameter:

% tissue viability

Value:

93.66

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All validity criteria for the test were met:
- Criteria: the mean OD570 of the negative control (treated with DPBS) tissues is ≥ 0.8 and ≤ 2.8
Result for the test: 1.777

- The mean of the positive control relative percentage viability must be ≤ 20 % of the mean of the negative controls.
Result for the test: 3.8 %

- The standard deviation of OD values for triplicate skin models in each experimental condition must be < 18 %
Results for the test:
NC: 5 %
PC: 0.72 %
TA2: 5.89 %

Optical Density (OD) values obtained with blanks were higher than 0.1 (0.191) 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 <0.194 (mean of XCellR8 historical data, based on blanks obtained during the last 66 studies), therefore this is not considered to be an issue in the interpretation of this study data.
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:

other: CLP criteria not met (not classified)

Conclusions:

Under the study conditions, based on the percentage of viability of 93.66 %, the test substance was concluded to be non-irritant to the skin.

Executive summary:

A study was conducted to determine the skin irritation potential of the test substance, C16-18 AMP, using Reconstructed Human Epidermis (RhE) Test Method, according to OECD 439 Guideline, in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated with the test substance, positive or negative control for 60 minutes (25 minutes at room temperature and 35 minutes at 37 °C, 5 % CO2) and 42 h post incubation period. Tissues were pre-wetted with 25 μl of DPBS (Sterile dulbecco’s phosphate buffered saline) prior to topical application of 25 mg of the neat test substance. 30 μl of DPBS was used as negative control and 5 % of sodium dodecyl sulphate solution in water was used as positive control. Subsequently, viability of the tissues was assessed and compared to the negative control. Under the study conditions, based on the percentage of viability of 93.66 %, the test substance was concluded to be non-irritant to the skin (XCellR8, 2017).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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 May 25, 2017 to June 22, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EpiOcularTM tissue model (OCL-200-MatTek Corporation)

Strain:

other: Keratinocyte 4F1188

Details on test animals or tissues and environmental conditions:

Test system:
The EpiOcularTM tissue model (OCL-200-MatTek Corporation) is composed of stratified human keratinocytes in a three-dimensional structure, reflecting the morphology and function of the human corneal epithelium found in vivo.

Lot No.: 23787
Keratinocyte Strain: 4F1188

MatTek’s EpiOcularTM system consists of normal, human-derived keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. Cultured on specially prepared cell culture inserts using serum-free culture medium, the cells differentiate to form a multi-layered structure with progressively stratified, but not cornified cells which closely parallel the corneal epithelium (for information see www.mattek.com). QC results for the specific lot of models received (Lot# 23787) were checked in-house for MatTek acceptance ranges with the following outcome:

- Morphology - TBD
- Tissue viability - TBD
- Skin barrier function (ET50 value for 0.3 % Triton X-100) where ET50 is the time taken for 0.3 % Triton X-100 to reduce the viability of the skin model to 50 % relative to the negative control) - TBD
- Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - TBD

Vehicle:

other: PBS (Sterile Dulbecco’s Phosphate Buffered Saline)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20 µl of PBS (Sterile Dulbecco’s Phosphate Buffered Saline) plus 50 mg of test substance.

Duration of treatment / exposure:

6h ± 15 minutes post-treatment immersion, and 18 hours ± 15 minutes post-treatment incubation.

Duration of post- treatment incubation (in vitro):

25 ± 2 minutes

Number of animals or in vitro replicates:

Three tissues per condition (n=3).

Details on study design:

Preliminary test:
The test substance was first checked for its potential for MTT interference and solvent interference (water and isopropanol).

Main test overview:
Day 0: On the day of receipt, EpiOcularTM tissues were pre-incubated overnight at 37 °C, 5 % CO2.
Day 1: Exposure to and removal of test and reference substances (50 mg of test substance or 50 µl of reference substances for 6h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 hours ± 15 minutes post-treatment incubation). 
Day 2: End of MTT viability test, readings at 570 nm without reference filter.

Irritation parameter:

other: % viability

Value:

112.829

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All validity criteria for the test were met:
- Criteria: the mean OD570 of the negative control (treated with sterile water) tissues is ≥ 0.8 and ≤ 2.5.
Result for the test: 1.071
- The mean of the positive control relative percentage viability is below 50% of negative control viability after 6 hours exposure.
Result for the test: 36.696
- The SD between three tissues replicates should not exceed 18 % in the same run (for negative and positive control tissues and tissues of test substances).
Results for the test:
NC: 11.368
PC: 7.373
TA2: 7.528

Interpretation of results:

other: CLP criteria not met (not classified)

Conclusions:

Under the study conditions, based on the percentage viability of 112.829%, the test substance was concluded to be non-irritant to the human eye

Executive summary:

A study was conducted to determine the eye irritation potential of the test substance, C16-18 AMP using Reconstructed human Cornea-like Epithelium (RhCE) test method, according to OECD 492 Guideline, in compliance with GLP. Three tissues of the EpiOcularTM tissue model were treated with the test substance, positive or negative control. Tissues were pre-wetted with 20 μL of PBS (Sterile Phosphate Buffered Saline) prior to topical application of approximately 50 mg of neat test substance. Sterile water was used as negative control and methyl acetate as positive control. After 6 h exposure on the surface of EpiOcularTM reconstructed ocular epithelium and 18 h post-incubation time, the viability of the tissues was assessed and compared to a negative control. The MTT viability test readings were conducted at 570 nm without reference filter. Under the study conditions, based on the percentage viability of 112.829%, the test substance was concluded to be non-irritant to the human eye (XCellR8, 2017).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

A study was conducted to determine the skin irritation potential of the test substance, C16-18 AMP, using Reconstructed Human Epidermis (RhE) Test Method, according to OECD 439 Guideline, in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated with the test substance, positive or negative control for 60 minutes (25 minutes at room temperature and 35 minutes at 37 °C, 5 % CO2) and 42 h post incubation period. Tissues were pre-wetted with 25 μl of DPBS (Sterile dulbecco’s phosphate buffered saline) prior to topical application of 25 mg of the neat test substance. 30 μl of DPBS was used as negative control and 5 % of sodium dodecyl sulphate solution in water was used as positive control. Subsequently, viability of the tissues was assessed and compared to the negative control. Under the study conditions, based on the percentage of viability of 93.66 %, the test substance was concluded to be non-irritant to the skin (XCellR8, 2017).

Eye:

A study was conducted to determine the eye irritation potential of the test substance, C16-18 AMP using Reconstructed human Cornea-like Epithelium (RhCE) test method, according to OECD 492 Guideline, in compliance with GLP. Three tissues of the EpiOcularTM tissue model were treated with the test substance, positive or negative control. Tissues were pre-wetted with 20 μL of PBS (Sterile Phosphate Buffered Saline) prior to topical application of approximately 50 mg of neat test substance. Sterile water was used as negative control and methyl acetate as positive control. After 6 h exposure on the surface of EpiOcularTM reconstructed ocular epithelium and 18 h post-incubation time, the viability of the tissues was assessed and compared to a negative control. The MTT viability test readings were conducted at 570 nm without reference filter. Under the study conditions, based on the percentage viability of 112.829%, the test substance was concluded to be non-irritant to the human eye (XCellR8, 2017).

Justification for classification or non-classification

Based on the results of in vitro skin and eye irritation studies, the test substance, C16-18 AMP does not warrant classification for skin and eye irritation according to the EU CLP criteria (Regulation 1272/2008/EC).