Salts of 2-ethylhexyl phosphate esters with (Z)- octadec-9-enylamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 - 24 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Jul 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

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.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MatTek test protocol “In vitro EpiDerm Skin Corrosion Test (EPI-200-SCT)”

Version / remarks:

07 Nov 2014

Deviations:

yes

Remarks:

The formazan salt extraction period was extended up to 72 hours. According to an expert statement (from MatTek Corporation) this procedure does not have an impact on the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Salts of 2-ethylhexyl phosphate esters with (Z)- octadec-9-enylamine

EC Number:

949-054-5

Molecular formula:

not applicable, UVCB substance

IUPAC Name:

Salts of 2-ethylhexyl phosphate esters with (Z)- octadec-9-enylamine

Test material form:

liquid: viscous

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm tissues supplied by MatTek Corporation, Slovakia.

Justification for test system used:

Guideline specific test system.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm
- Tissue batch number(s): 288647
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: 21 Aug 2018
- Date of initiation of testing: 21 Aug 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature ( 3 min exposure group) and 37 ± 1.5 ºC (60 min exposure group)
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 times
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h incubation with MTT solution followd by 19 h extraction.
- Spectrophotometer: Microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570)
- Wavelength: 570 nm
- Filter: No
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Acceptable (2.097 ± 0.214). Acceptance criteria = 1.0 - 3.0.
- Barrier function: Acceptable (6.33 h). Acceptance criteria 4.77 - 8.72 h.
- Morphology: Acceptable.
- Contamination: Acceptable (sterile).
- Reproducibility: COV of mean replicates = < 30 %.

NUMBER OF REPLICATE TISSUES: 1

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : N/A
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : N/A
- Method of calculation used: N/A

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: N/A

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
- Concentration (if solution): Unchanged - applied as supplied

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): N/A

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

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

Incubated in MTT medium for 3 hours and followed by an 19 h extraction in isopropanol.

Number of replicates:

2 for each timepoint

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

97.62

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

100.04

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes inc. certificate.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time.
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 hour, is <15 % compared to the negative control.
- Acceptance criteria met for variability between replicate measurements: Coefficient of Variation (CV) in the range 20 – 100 % viability between tissue replicates is ≤ 30 %.
- Range of historical values if different from the ones specified in the test guideline:
Positive control: 3 min = 4.6 - 39.83 % viability, 60 min = 1.07 - 14.77 % viability (n=33).
Negative control: 3 min = 1.25-1.93 OD range, 60 min = 1.23-2.00 OD range (n=33)

Any other information on results incl. tables

Table.2       Viabilities for the negative control, positive control and test item

 

Treatment group	Tissue no.	Exposure interval (mins)	OD 570 nm			Mean OD of 3 wells (blank corrected)	Mean OD of 2 tissues (blank corrected)	SD (well 1-3)	Mean relative viability (%)	CV (%)
			Well 1	Well 2	Well 3
Blank	-	3	0.038	0.038	0.038
Negative control	1		2.034	1.980	2.078	1.992	1.970	0.049	100.00	1.6
	2		2.035	1.967	1.954	1.947		0.043
Positive control	1		0.300	0.293	0.290	0.256	0.211	0.005	10.70	30.6
	2		0.205	0.202	0.203	0.165		0.002
Test item	1		2.073	1.994	2.013	1.989	1.923	0.041	97.62	4.8
	2		1.867	1.911	1.908	1.857		0.024
Blank	-	60	0.038	0.038	0.038
Negative control	1		2.209	2.157	2.162	2.138	2.016	0.028	100.00	8.5
	2		1.959	1.928	1.912	1.895		0.024
Positive control	1		0.125	0.120	0.115	0.082	0.069	0.005	3.41	27.1
	2		0.095	0.094	0.093	0.056		0.001
Test item	1		2.088	2.051	2.059	2.028	2.017	0.020	100.04	0.8
	2		2.037	2.063	2.033	2.006		0.016

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study the test substance is not considered to be corrosive.

Executive summary:

OECD 431 (2018) - The skin corrosivity potential of the test item was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.

The test item passed the MTT- and the colour interference pre-tests and all 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 (10.70%) and for the 1 hour exposure period (3.41%) thus confirming the validity of the test system and the specific batch of tissue models.

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 97.62 and 100.04 %, respectively.

 

Under the conditions of this study the test substance is not considered to be corrosive to the skin according to the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixture.