Phosphoric acid, C8-16-alkyl esters, reaction products with (Z)-N-9-octadecenyl-1,3-propanediamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, C8-16-alkyl esters, reaction products with (Z)-N-9-octadecenyl-1,3-propanediamine

EC Number:

291-909-3

EC Name:

Phosphoric acid, C8-16-alkyl esters, reaction products with (Z)-N-9-octadecenyl-1,3-propanediamine

Cas Number:

90506-47-1

Molecular formula:

complex substance

IUPAC Name:

Phosphoric acid, C8-16-alkyl esters, reaction products with (Z)-N-9-octadecenyl-1,3-propanediamine

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 80555E094
- Expiration date of the lot/batch: 05/12/2018
- Purity test date: 23/01/2018
- Date of receipt: 22/02/2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient

In vitro test system

Test system:

human skin model

Justification for test system used:

The EpiDerm(TM) Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The test was carried out with the reconstituted three-dimensional human skin model EpiDerm(TM) (MatTek).
- Tissue batch number: 25882
- Date of initiation of testing: 22/02/2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: incubated at 37 ± 1°C,

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper.
- Observable damage in the tissue due to washing: no damage reported.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT stock solution: 5 mg/mL MTT (Sigma; Lot MKBZ5197V) in PBS (Gibco; Lot No.: 1909266, 1877596). MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL).
- Incubation time: 3h
- Spectrophotometer used
- Wavelength: OD was measured at 570 nm

NUMBER OF REPLICATE TISSUES: 4

CONTROLS
- Negative control: Distilled water (Aqua dest.; Lot: RNBG3519, Sigma) ; 50µL applied
- Positive control: 8 N Potassium Hydroxide (KOH; CAS No.: 1310-58-3; Lot: 10357-004, NeoLab) ; 50 µL applied

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues used
- Procedure used to prepare the killed tissues: two tissues per treatment period were treated with the test item (KT) or left untreated (KU), respectively.
- N. of replicates: 2
- Method of calculation used:
NSMTT (non-specific reduction of MTT) was calculated relative to the negative control of living tissues (NK) per treatment period according to the following formula: NSMTT = [(ODKT - ODKU)/ODNK] * 100
The true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) was corrected for each treatment period according to the following formula: TODTT = ODTM - (ODKT - ODKU)

PREDICTION MODEL / DECISION CRITERIA
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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was applied undiluted. 50 μL of the test item were dispensed directly atop the EpiDerm(TM) tissue. The test item was spread to match size of the tissue.

Negative control: 50 μL of distilled water
Positive control: 50 μL of 8 N KOH

Duration of treatment / exposure:

2 treatment period: 3 min and 60 min exposure time.

Duration of post-treatment incubation (if applicable):

The plate was incubated for 3 h at 37 ± 1°C, 5.0% CO2 / 95% air.

Number of replicates:

The test was performed on a total of 4 replicates tissues, 2 replicates for each treatment period (3 min and 60 min exposure time).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3min exposure

Value:

96.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: NSMTT-corrected

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60min exposure

Value:

94.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: NSMTT-corrected

Any other information on results incl. tables

Results of 3 min Experiment:

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	1.730	1.801	1.689	1.761	0.193	0.300
	1.702	1.800	1.763	1.784	0.203	0.278
	1.784	1.800	1.796	1.771	0.227	0.287
OD570-  Blank Corrected	1.685	1.756	1.644	1.716	0.148	0.255
	1.657	1.755	1.718	1.739	0.158	0.233
	1.739	1.755	1.751	1.726	0.182	0.242
Mean OD570of 3 Aliquots (Blank Corrected)	1.694	1.755	1.705	1.727	0.163	0.243
SD OD570 of 3 Aliquots	0.042	0.001	0.055	0.012	0.017	0.011
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.724*		1.716		0.203
TODTT	-		1.659		-
SD OD570 of 2 Replicate Tissues	0.043		0.016		0.057
Mean Relative Tissue Viability [%]	100.0		99.5		11.8
Mean Relative Tissue Viability [%] - NSMTT Corrected	-		96.2		-
Coefficient Of Variation [%]^***	2.5		0.9		28.0

^*    corrected mean OD₅₇₀of the negative control corresponds to 100% absolute tissue viability.

^*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is≤30%.

Results of NSMTT control of 3 min Experiment:

NSMTT	KU		KT		Negative Control
Tissue	1          2		1          2		1	2
absolute OD570 -values	0.267	0.227	0.282	0.321	1.730	1.801
	0.267	0.229	0.290	0.319	1.702	1.800
	0.268	0.225	0.291	0.323	1.784	1.800
OD570(Blank Corrected)	0.222	0.182	0.237	0.276	1.685	1.756
	0.222	0.184	0.245	0.274	1.657	1.755
	0.223	0.180	0.246	0.278	1.739	1.755
mean OD570  (mean of 3 aliquots)	0.222	0.182	0.243	0.276	1.694	1.755
total mean OD570 (mean of the replicate tissues)	0.202		0.260		1.724
SD OD570(of the replicate tissues)	0.029		0.023		0.043
NSMTT [%]	3.3				-
Relative Tissue Viability [%]	-				98.2	101.8
Mean Relative Tissue Viability [%]	-				100.0
SD Tissue Viability [%]	-				2.5
CV [% Viabilities]	-				2.5

Results of 60 min Experiment:

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	1.763	2.027	1.779	1.901	0.096	0.176
	1.786	2.030	1.840	1.889	0.099	0.182
	1.793	2.003	1.841	1.912	0.102	0.181
OD570-  Blank Corrected	1.718	1.982	1.734	1.856	0.051	0.131
	1.741	1.985	1.795	1.844	0.054	0.137
	1.748	1.958	1.796	1.867	0.057	0.136
Mean OD570of 3 Aliquots (Blank Corrected)	1.736	1.975	1.775	1.856	0.054	0.135
SD OD570 of 3 Aliquots	0.016	0.015	0.036	0.011	0.003	0.003
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.856*		1.815		0.094
TODTT	-		1.746		-
SD OD570 of 2 Replicate Tissues	0.169		0.057		0.057
Mean Relative Tissue Viability [%]	100.0		97.8		5.1**
Mean Relative Tissue Viability [%] - NSMTT Corrected	-		94.1		-
Coefficient Of Variation [%]^***	9.1		3.1		60.3

^*    corrected mean OD₅₇₀of the negative control corresponds to 100% absolute tissue viability.

^**mean relative tissue viability of the 60 min positive control < 15%

^*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is≤30%.

Results of NSMTT control of 60 min Experiment:

NSMTT	KU		KT		Negative Control
Tissue	1          2		1          2		1	2
absolute OD570 -values	0.217	0.279	0.271	0.368	1.763	2.027
	0.222	0.280	0.272	0.359	1.786	2.030
	0.224	0.282	0.271	0.375	1.793	2.003
OD570(Blank Corrected)	0.172	0.234	0.226	0.323	1.718	1.982
	0.177	0.235	0.227	0.314	1.741	1.985
	0.179	0.237	0.226	0.330	1.748	1.958
mean OD570  (mean of the aliquots)	0.176	0.235	0.226	0.322	1.736	1.975
total mean OD570 (mean of the replicate tissues)	0.206		0.274		1.856
SD OD570(of the replicate tissues)	0.042		0.068		0.169
NSMTT [%]	3.7				-
Relative Tissue Viability [%]	-				100.7	114.6
Mean Relative Tissue Viability [%]	-				107.6
SD Tissue Viability [%]	-				9.8
CV [% Viabilities]	-				9.1

Test acceptance criteria:

	Value	Cut off	pass/fail
Mean Absolute OD570 nmNK (3 min Experiment)	1.769	0.8 ≤ NK ≤ 2.8	pass
Mean Absolute OD570 nmNK (60 min Experiment)	1.900	0.8 ≤ NK ≤ 2.8	pass
Mean Relative Tissue Viability [%] of PC (60 min experiment)	5.1	< 15%	pass
CV [%](in the range of 20 – 100% viability)	0.9 – 9.1	≤ 30%	pass

Historical data:

	Mean	SD	n
OD570of NK (3 min Experiment)	1.895	0.313	10
OD570of NK (60 min Experiment)	1.867	0.261	11
Relative Tissue Viability [%] of PC (60 min experiment)	6.1	1.99	11
CV [%](in the range of 20 – 100% viability)	7.3	6.6	31

Historical control data were generated from 2015 - 2016.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no corrosive effects. The test item is classified as “non-corrosive“.

Executive summary:

In the present study the skin corrosivity potential of ARCOT 3135/384 was analysed. Since corrosive chemicals are cytotoxic after a short time exposure to the stratum corneum of the epidermis the cytotoxic effects of the test item on EpiDerm™, a reconstituted three-dimensional human epidermis model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 min and 60 min exposure period and compared to those of the concurrent negative controls.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was≥50% (96.2%, NSMTT-corrected) after 3 min treatment and≥15% (94.1%, NSMTT-corrected) after 60 min treatment.