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Description of key information

The substance was tested for skin and eye irritation according to OECD TG 439 and TG 437. Both studies were carried out under GLP and both studies did not identify any irritation potential.

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:
February 2020 - October 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
18 June 2019, corrected 26 June 2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: The EpiDerm tissues were provided as kits (e.g. EPI-200-SIT, MatTek)
Source strain:
other: not applicable
Details on animal used as source of test system:
not applicable
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:
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 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.
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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Dose Groups
1. Negative control 30 µL DPBS
2. Positive control 30 µL 5% SDS solution
3. Test Item 30 µL (undiluted)
The test was performed on a total of 3 tissues per dose group
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.
Number of replicates:
3 tissues per dose group
Irritation / corrosion parameter:
% tissue viability
Value:
97.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.
The mixture of 30 µL test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple.
For quantitative correction of results, 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. NSMTT was calculated relative to the negative control of living tissues (NC) according to the following formula:
NSMTT [%] = [(ODKT - ODKU)/ODNC] * 100 = 1.2
Mean ODKT = 0.063
Mean ODKU = 0.043
Mean ODNC = 1.679
NSMTT was ≤ 30% (1.2%) relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was therefore corrected according to the following formula:
TODTT = ODTM – (ODKT – ODKU) = 1.632
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 equaled 0%.
The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (97.2%, NSMTT-corrected) after 60 min treatment and 42 h post-incubation.
The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was  0.8 and ≤ 2.8 (1.724). The mean relative tissue viability (% negative control) of the positive control was < 20% (4.4%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.5% - 17.6%).

Table 1. Result of the Test Item Reaction mass of S-(6-((3-(triethoxysilyl)propyl)thio)hexyl)ethylthioester and Hexamethylene-1,6-dithio-bis((3-triethoxysilyl)propane)

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.839

1.935

1.369

0.114

0.114

0.128

1.745

1.845

1.454

1.847

1.949

1.406

0.112

0.122

0.130

1.781

1.882

1.476

Mean Absolute OD570

1.724****

0.120

1.697

OD570
(Blank Corrected)

1.794

1.889

1.323

0.068

0.068

0.082

1.700

1.799

1.408

1.802

1.904

1.360

0.067

0.076

0.085

1.735

1.837

1.430

Mean OD570of the Duplicates
(Blank Corrected)

1.798

1.896

1.342

0.067

0.072

0.084

1.717

1.818

1.419

Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)

1.679*

0.074

1.652

TODTT

-

-

1.632

SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)

0.296

0.008

0.207

Relative Tissue Viabilities [%]

107.1

113.0

79.9

4.0

4.3

5.0

102.3

108.3

84.5

Mean Relative Tissue Viability [%]

100.0

4.4**

98.4

Mean Relative Tissue Viability [%] – NSMTT Corrected

-

-

97.2

SD of Relative Tissue
Viability [%]***

17.6

0.5

12.4

CV [% Viabilities]

17.6

11.1

12.6

 

*     Blank-corrected mean OD570of 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%.

**** The mean absolute OD570of the negative control is≥ 0.8 and ≤ 2.8.


  Table 2. Result of the NSMTT control

Name

KU

KT

Negative Control

Tissue

1

2

1

2

1

2

3

Absolute OD570

0.090

0.088

0.121

0.096

1.839

1.935

1.369

0.089

0.088

0.122

0.096

1.847

1.949

1.406

OD570
(Blank Corrected)

0.044

0.043

0.075

0.050

1.794

1.889

1.323

0.044

0.043

0.076

0.050

1.802

1.904

1.360

Mean OD570of the Duplicates
(Blank Corrected)

0.044

0.043

0.076

0.050

1.798

1.896

1.342

Total Mean OD570of the 2 or 3 Replicate Tissues (Blank Corrected)

0.043

0.063

1.679

SD of Mean OD570of the 2 or 3 Replicate Tissues (Blank Corrected)

0.001

0.018

0.296

NSMTT [%]

1.2

-

Relative Tissue Viability [%]

-

107.1

113.0

79.9

Mean Relative Tissue Viability [%]

-

100.0

SD of Relative Tissue
Viability [%]

-

17.6

CV [% Viabilities]

-

17.6

Table3:  Quality Criteria

 

Value

Cut off

pass/fail

Mean Absolute OD570 nmNC

1.724

0.8 ≤ NC ≤2.8

pass

Mean Relative Viability [%] PC

4.4

< 20%

pass

SD Viability[%]

0.5 – 17.6

≤ 18%

pass

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”.
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:
April 2020 - December 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
09 October 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µL of the test substance or the control substance was introduced into the anterior chamber.
Duration of treatment / exposure:
10 minutes incubation at 32  1 °C
Number of animals or in vitro replicates:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with ethanol 100%
Details on study design:
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32  1 °C
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI 1640 medium. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32  1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).
Irritation parameter:
in vitro irritation score
Value:
0.45
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
Interpretation of results:
GHS criteria not met
Conclusions:
According to the evaluation criteria the test item is classified into UN GHS No Category.
Executive summary:

The eye irritancy potential of Reaction mass of the test substance was investigated in the bovine corneal opacity and permeability assay.

None of the corneas treated with the test substance showed any opacity of the tissue.

The following meanin vitroirritation score was calculated:

0.45

Therefore the test item was classified intoUN GHS No Category.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification

The mean relative tissue viability was > 50% in a valid test for skin irritation according to OECD TG 439 and under GLP. The test item is therefore classified as “non-irritant” in accordance with UN GHS "No Category".

The eye irritancy potential was investigated in the bovine corneal opacity and permeability assay.None of the corneas treated with the test substance showed any opacity of the tissue. The mean in vitro irritation score was calculated to amount 0.45. The test item is therefore classified into UN GHS "No Category".

The mean relative tissue viability (% negative control) was > 50% (99 ± 16%) in a valid test for skin irritation according to OECD TG 439 and under GLP. The test item was therefore classified as “non-irritant” in accordance with UN GHS “No Category”. A mean in vitro irritation score of 2.06 was determined in a valid test for eye irritation according to OECD TG 437 and under GLP. According to the evaluation criteria the test item is accordingly classified into UN GHS “No Category”.