[No public or meaningful name is available]

Endpoint summary

Description of key information

An acute dermal Irritation study, according to OECD 404 (GLP, Klimisch 1) was performed. In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

A Reconstructed Human Cornea-like Epithelium (RhCE) Test according to OECD 492 (GLP, Klimisch 1) was performed.

In this study under the given conditions the test item showed no irritant effects in both experiments. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”.

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:

November 18, 2021 to November 26, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2021

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

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.

Vehicle:

unchanged (no vehicle)

Details on test system:

The test was carried out with the reconstituted three-dimensional human skin model EpiDerm
(MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have
been cultured to form a multi-layered, highly differentiated model of the human epidermis. The NHEK
are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm
epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and
highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered
stratum corneum analogous to patterns found in vivo.

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Amount/concentration applied:

30 µl (47 µL/cm²)

Duration of treatment / exposure:

60 min exposure

Duration of post-treatment incubation (if applicable):

42 h post-incubation

Number of replicates:

The test was performed on a total of 3 tissues per dose group.

Details on study design:

PRE-EXPERIMENTS
To check the non-specific MTT-reducing capability of the test item 30 μL of the test item was mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator. The test material sedimented. The mixture did not turn blue/purple. Thus, the additional test with freeze-killed tissues and the quantitative corrections were not necessary.
To check the colouring potential of the test item 30 μL of the test item was mixed per 300 μL aqua dest. and/or per 300 μL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min.
As the test item was classified as non-irritant and colouring was detected by unaided eye-assessment, the light absorption of the chemical in water and in isopropanol in the range of 570 ± 30 nm was measured. Since there was no absorption in the relevant range, the additional test with viable tissues and the quantitative corrections were not necessary.
The test item showed no non-specific reduction of MTT and no relevant colouring potential after
mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of
possible false-negative results were necessary.

EXPERIMENTAL PROCEDURE
Upon receipt of the EpiDermTM, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL assay medium per well. The surface was dried using a sterile cotton tip and the plates were incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 for 60 ± 5 min. Subsequently the tissues were transferred into new wells containing 0.9 mL pre-warmed assay medium per well and were incubated for 18 ± 3 h in a humidified incubator at 37 ± 1 °C, 5.0% CO2.
After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue and occurred sequentially for the other tissues, e.g. in one-minute intervals. After dosing of all tissues, all plates were incubated for 25 ± 1 min under the sterile flow and for the remaining time of 35 ± 1 min transferred to the incubator.
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.
The inserts were placed in prepared new 6-well plates containing 0.9 mL pre-warmed fresh assay
medium per well and the tissue surface was dried using a sterile cotton tip. The plates were postincubated 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.
After this post-incubation period the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a prepared 24-well plate containing 300 μL pre-warmed MTT medium. This plate was incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%.
After the MTT incubation period, the tissues were rinsed three times with DPBS and afterwards placed on blotting paper to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature for at least 2 h with gentle shaking on a plate shaker.
Before using the extracts, the plate had been shaken for at least 15 min on a plate shaker and the
inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 μL aliquots per each tissue were transferred into a 96-well plate. 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.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

71.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The relative mean
tissue viability after 60 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In the present study the skin irritant potential of the test item was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as nonirritant.
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 test item has no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary.
The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (71.7%) after 60 min treatment and 42 h post-incubation.

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:

December 21, 2021 to February 10,2022

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)

Version / remarks:

18 Jun 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL (83.3 µL/cm2) of the test item were dispensed directly atop the EpiOcular™ tissue.

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per dose group.

Details on study design:

Pre-Experiments
To check the non-specific MTT-reducing capability of the test item 50 µL of the test item were mixed per 1 mL MTT medium and incubated for 3 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% humidity.
As the mixture turned blue/purple, the test item was presumed to have reduced MTT. The part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues if the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value. For quantitative correction of results, two killed tissues were treated with 50 µL of the test item (KT) and one tissue was treated with 50 µl of the negative control (Aqua dest.; KU), respectively. All steps were performed exactly as described in the chapter below. NSMTT was calculated relative to the negative control of living tissues (NC) according to the following formula:
NSMTT [%] = [(ODKT - ODKU)/ODNC] * 100
If the viability difference of the two identically treated killed test item treated tissues (KT) was > 20% the killed control was considered as non-qualified.
If NSMTT was ≤ 60% relative to the negative control of living tissues it was used for quantitative correction of the results by calculation of the killed control corrected viability (KCCV) which was considered for the classification of the test item according to the following formula:
KCCV [%] = viabilityTM – NSMTT
If NSMTT was > 60% relative to the negative control of living tissues the results obtained should be taken with caution as this is the cut-off used to distinguish classified from not classified test items.
If uncorrected ODKT of the tissue extracts fell outside the linear range of the spectrophotometer the test item was considered as incompatible with the test method.

To check the colouring potential of the test item 50 µL of the test item were mixed per 1 mL Aqua dest. and per 2 mL isopropanol each in a 6-well plate. The water solution was incubated for at least 1 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% humidity. The isopropanol solution was shaken on a plate shaker for 2 to 3 h. After the respective incubation period, 2 x 200 µL aliquots per test solution were transferred into a 96-well plate, using 200 µL Aqua dest. and isopropanol as respective blanks and OD was measured in a range of 570 ± 30 nm without reference wavelength in a plate spectrophotometer.
The mixture showed an ODnet < 0.08. Thus, the additional test with viable tissues and the quantitative corrections were not necessary.
The test item showed non-specific reduction of MTT but no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

Experimental Procedure
Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% humidity. Then the inserts were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% humidity for 16 - 24 h.
After the overnight incubation the tissues were pre-treated with 20 µL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% humidity to mimic the wet conditions of the human eye.
 
Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. Then the 6-well plate(s) were incubated for 30 ± 2 min at 37 ± 2 °C, 5.0% CO2 / 95% humidity. At the end of the exposure period, the test item and control substances were removed by extensively rinsing the tissue with DPBS. Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper. After rinsing, the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 5 mL fresh pre-warmed assay medium per well and incubated for 12 ± 2 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 120 ± 15 min at 37 ± 2 °C, 5.0% CO2 / 95% humidity.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h ± 15 min at 37 ± 2 °C, 5.0% CO2 / 95% humidity.
After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract only the bottom of the tissues. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out after storage overnight in the dark at 2 - 8 °C. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

Data Analysis
Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities obtained after treatment compared to the negative control tissues concurrently treated with Aqua dest. The test item is considered to be irritant to the eye but it cannot be differentiated between UN GHS “Category 1” or “Category 2”, if the relative tissue viability is less or equal to 60%. The test item is considered to be non-irritant in accordance with UN GHS “No Category” if relative tissue viability is higher than 60%

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Experiment 2

Value:

74.5

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Experiment 1

Value:

65.6

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects in both experiments. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”

Executive summary:

The potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium.

In the present study was applied topically to the EpiOcular tissue for 30 min followed by 12 min post-soaking incubation after removal of the test item. After a 120 min post-treatment period cytotoxic effects were determined via MTT reduction assay.

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest.

The test item showed non-specific MTT-reducing potential. Therefore, additional killed tissue controls were treated with the test item to determine the non-specific reduction of MTT (NSMTT) and the results were corrected to the true MTT metabolic conversion (TOD_TT). The test item showed no water-colouring potential.

In the first experiment, the test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (65.6%).

Since the result is very close to what is considered to be the borderline range (mean percent tissue viability equal to 60±5%), a second experiment was conducted to verify the result.

In the second experiment, the test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (74.5% NSMTT- corrected).

As the test item showed no irritant effects in both experiments, the test item can be considered as non-irritant.

The controls confirmed the validity of the study. The mean absolute OD₅₇₀ of the two negative control tissues was > 0.8 and < 2.8 (1.893 [exp. 1] and 2.072 [exp. 2]). The mean relative tissue viability (% negative control) of the positive control was < 50% (43.5% [exp. 1 and 2]). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (10.6% [exp. 1] and 3.9% [exp. 2]).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Based on the available data, the substance does not need to be classified and labelled according to Regulation (EC) No.1272/2008 (CLP).