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Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 August 2018 - 30 August 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
The study was conducted to GLP and standard giudeline with all criteria met

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to 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)
Version / remarks:
Deviation is considered to not affect the outcome or the integrity of the study.
GLP compliance:
yes (incl. QA statement)

Test material

Reference substance name:
Reaction products of triphenyl phosphite, oleyl alcohol and alcohols, C10-16
EC Number:
Molecular formula:
Not applicable - UVCB substance
Reaction products of triphenyl phosphite, oleyl alcohol and alcohols, C10-16
Test material form:
Details on test material:
Although the test item used within this test contains different identifiers as compared to the subtance that is being registered, the tested substance is within the boundary composition of the substance being registered. Therefore, this particular batch of the tested substance, can be considered as the same substance as (Z,Z)-di-9-octadecenyl phosphonate. It can be considered that all tests conducted on Reaction products of triphenyl phosphite, oleyl alcohol and alcohols, C10-16, Batch 2955, are considered relevant and reliable for (Z,Z)-di-9-octadecenyl phosphonate.
Specific details on test material used for the study:
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

- Storage condition of test material: At room temperature in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

- Treatment of test material prior to testing: no

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ).
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (28th August 2018) of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.

Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (about 16 - 24 hours).

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): undiluted

- 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
Duration of treatment / exposure:
30 minutes
Observation period (in vivo):
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Each group tested in duplicate
Details on study design:
- Details of the test procedure used

Pre Test Assessments

The test items ability to directly reduce MTT was assessed. For this purpose approximately 50 µl of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. The result indicated that the test item did not have MTT reducing effects. An additional test with freeze-killed tissues did not have to be performed.

The photometric properties of the test item after contact with water and isopropanol were assesed. For this purpose each 50 µL of the test item were added to 1.0 ml of water and to 2 ml isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for at least one hour, the isopropanol mixture or for 2 to 3 hours at room temperature. The result indicated that the test item did not dye the water or isopropanol and would therefore not interfere with the photometric assessment of MTT during the main test.

Main Test

After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes.

After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 µL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.

At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).

After rinsing, the tissues were immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.

At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 ml of warm Assay Medium. The tissues are incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

After post-treatment incubation of 120 minutes the MTT assay was performed.

At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 ml of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.

The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

- RhCE tissue construct used, including batch number: EpiOcular kit (Lot: 23574). Supplied by MatTek Coorp., Slovakia.

- Doses of test chemical and control substances used: 50 µL applied topically.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Tissues exposed for 30 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH. Tissues were immersed for 2 seconds during the rinsing process. Post exposue incubation was 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2.

- Justification for the use of a different negative control than ultrapure H2O (if applicable): N/A

- Justification for the use of a different positive control than neat methyl acetate (if applicable): N/A

- Description of any modifications to the test procedure: N/A

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): N/A

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per test group (test item, positive and negative control). 1 blank tissue prepared with no treatment.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm

- Description of the method used to quantify MTT formazan: See above.

- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): N/A

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The percent viability of each of the two relating tissues for each control and test item relative to the negative control (100% control) were calculated. The difference of the viability between duplicate tissues was calculated. If the difference is >20% the test is considered as non-qualified. The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model. If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes

- Complete supporting information for the specific RhCE tissue construct used: Yes

- Reference to historical data of the RhCE tissue construct: Yes

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Yes

- Positive and negative control means and acceptance ranges based on historical data: Yes

- Acceptable variability between tissue replicates for positive and negative controls: Yes

- Acceptable variability between tissue replicates for the test chemical: Yes

Results and discussion

In vitro

Irritation parameter:
other: Spectrophotometric mean relative absorbance of MTT solution at 570 nm
Run / experiment:
Mean value
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
- Visible damage on test system: No


- Acceptance criteria met for negative control: The negative control OD is > 0.8 and < 2.5 (1.927)
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (46.58%)
- Range of historical values if different from the ones specified in the test guideline: n/a

Any other information on results incl. tables

Table 1: Assessment of Colour Interference with the MTT endpoint

Treatment Group

OD 570 nm Well 1

OD 570 nm Well 2

Mean OD of 2 Wells

Mean OD of 2 Wells blank corrected


Evaluation Mean OD570 (blank corrected)
> 0.08

Blank Aqua Deion.





Test Item + Aqua Deion.






Blank Isopropanol





Test Item+ Isopropanol






The mean OD was < 0.08 and therefore viable tissues were not necessary in the main experiment

Table 2: Assessment of Colour Interference with the MTT endpoint

Test Group


Tissue No.

Well 1 [OD570]

Well 2 [OD570]

Mean [OD570] (Well 1 and well 2)

Mean [OD570] blank corr. (Well 1 and well 2)

Mean [OD570] of T1 and T2

Tissue viabil.* [%]

rel. viabil. of T1 and T2**

Diff. of viabil. between T1 and T2 [p.p.]











Negative Control






























Positive Control





























Test Item





























Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
In conclusion, it can be stated that in this study and under the experimental conditions reported, AD-464 does not possess an eye irritating potential.
Executive summary:

OECD 492 (2018) - This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test.

Additional tests with viable or freeze-killed tissues were not performed, since the colourless test item did not dye water or isopropanol, and did not prove to be a MTT reducer.

Tissues of the human cornea model EpiOcular™were treated with the test item, the positive and the negative control for 30 minutes each in duplicate.

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 46.58%, thus the validity of the test system is ensured.

Relevant irritating effects were not observed following 30 minutes incubation with the test item. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 104.35 % compared with the value of the negative control (threshold for irritancy: ≤ 60%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item does not possess an eye irritating potential.