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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is classified as skin irritation and the available information indicates that it should be classified as eye irritation (Category 2)

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10/07/2018

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:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: PCAS, Batch No. 80555E094
- Expiration date of the batch: 05 December 2018
- Production date of the batch: 05 December 2017
- Purity test date: 23 January 2018

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

Test system:

human skin model

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EpiDermTM (MatTek)
- Tissue batch number: Lot No.: 28615
- Date of initiation of testing: 17/04/2018

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: Then the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the 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 (VWR; Lot 0977C002) in PBS (Gibco; Lot No.: 1943446). MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL).
- Incubation time: 3 h ± 5 min
- Spectrophotometer: OD was measured at 570 nm with a filter band pass of maximum ± 30 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570nm
- Filter: filter band pass of maximum ± 30 nm without reference wavelength

NUMBER OF REPLICATE TISSUES: Triplicate

CONTROLS
- Negative control: DPBS
- Positive control: 5% SDS solution

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues used
- Procedure used to prepare the killed tissues: two killed tissues were treated with 30 μL of the test item (KT) and two killed tissues were left untreated as a control (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 (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after exposure is less or equal to 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied (volume): 30 μL (undiluted)

VEHICLE
applied as such.

NEGATIVE CONTROL
- Amount applied (volume): 30 μL DPBS
- Concentration: Dulbecco’s phosphate buffered saline used as such (DPBS; Gibco, Cat. No. 14040-091, Lot No.: 1838067).

POSITIVE CONTROL
- Amount applied (volume): 30 μL
- Concentration: 5% sodium dodecyl sulfate

Duration of treatment / exposure:

Total of 60min.
After dosing of all tissues, all plates were transferred to the incubator for 35 ± 1 min. Afterwards all plates were removed from the incubator and placed under the sterile flow for the remaining time until the 60 ± 1 min incubation time of the first dosed tissue was over.

Duration of post-treatment incubation (if applicable):

Total of 42h post-incubation.
The plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h.

Number of replicates:

The tissues were treated with each dose group in triplicate.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min of exposure

Value:

3.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Result of the Test Item ARCOT 3135/384

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.860 1.847	1.838 1.822	1.785 1.773	0.100 0.105	0.103 0.110	0.104 0.106	0.210 0.203	0.214 0.221	0.233 0.230
OD570 (Blank Corrected)	1.816 1.803	1.793 1.777	1.740 1.729	0.056 0.060	0.059 0.065	0.060 0.061	0.165 0.159	0.170 0.177	0.189 0.185
Mean OD570of the Duplicates (Blank Corrected)	1.810	1.785	1.735	0.058	0.062	0.061	0.162	0.173	0.187
Total Mean OD570of 3 Replicate Tissues (Blank Corrected)	1.777*			0.060			0.174
SD OD570	0.038			0.002			0.013
Relative Tissue Viability [%]	101.9	100.5	97.6	3.3	3.5	3.4	9.1	9.8	10.5
Mean Relative Tissue Viability [%]	100.0			3.4**			9.8
SD Tissue Viability [%]***	2.2			0.1			0.7
CV [% Viabilities]	2.2			3.3			7.2

 

^* Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

^** Mean relative tissue viability of the three positive control tissues is ≤20%.

^*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18% 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (9.8%) after 60 min treatment and 42 h post-incubation. The controls confirmed the validity of the study.
The test item is therefore classified as “irritant” in accordance with UN GHS “Category 2”.

Executive summary:

In the present study the skin irritant potential of ARCOT 3135/384 was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. 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 60 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

The mixture of 30 μL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 30 μL of the test item per 300 μl aqua dest. and per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%. No absorption was measured in the relevant range.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (9.8%) after 60 min treatment and 42 h post-incubation.