Registration Dossier

Administrative data

Description of key information

The potential of 1,3,5-Tris-(3-mercaptopropyl)isocyanurate (target substance) to induce skin irritation/corrosion was evaluated in two suitable in vitro test methods (OECD 439 and OECD 431). Based on the results, the target substance can be considered as irritant to the skin. Therefore, classification as Skin Irrit. 2, H315 is warranted in accordance with the CLP criteria.

The potential of the target substance to induce eye irritation was assessed in a weight-of-evidence approach by using data from two suitable in vitro test methods (OECD 437 and OECD 492). Based on the results, the target substance can be considered as non-irritant to the eye and no classification is warranted in accordance with the CLP criteria.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-09-27 to 2018-11-06
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)
Version / remarks:
adopted on 28th July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was applied undiluted. 30 µL of the test item were dispensed directly atop the EpiDerm tissue.
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, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EpiDerm™-Standard Model (EPI-200-SIT, MatTek)
- Tissue batch number(s): 28659 for main experiment, 26658 for killed tissue controls

EpiDerm Kit:
The EpiDerm tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
- 1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm²); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport
- 2x 24-well plates
- 8x 6-well plates
- 1x bottle of assay medium (DMEM-based medium, Lot No.:1004118MSB)
- 1x bottle of DPBS Rinse Solution (Lot No.: 082818ISA)
- 1x 1 vial 5% SDS Solution (TC-SDS-5%)
- 25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for the first 35 +/- 1 min, afterwards the plates were placed under the sterile flow until 60 +/- 1 min incubation time of the first dosed tissue was over.
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 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. 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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after exposure and post-incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after exposure and post-incubation is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL (undiluted)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL DPBS (Gibco, Cat. No.14040-091, Lot No.: 1838067)

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL 5% sodium dodecyl sulfate (MatTek, CAS No.: 151-21-3, Lot No.: 110317TMB)

Duration of treatment / exposure:
60 ± 1 min
Duration of post-treatment incubation (if applicable):
42 h post-incubation
Number of replicates:
3 tissues per dose group
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of three tissues
Value:
30.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.709)
- Acceptance criteria met for positive control: Yes, the mean relative tissue viability of the positive control was ≤ 20% (3.5%)
- Acceptance criteria met for variability between replicate measurements: Yes, the standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.6% - 9.6%).

For detailed results see Table 1 in box "Any other information on results incl. tables".

Results of the Pre-Experiments:

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. Non specific reduction of MTT (NSMTT) was determined to be ≤ 30% (29.8%) relative to the negative control of living epidermis. However, as the coloured test material or MTT reducing substance was classified as “Irritant” i.e. mean tissue viability is ≤ 50%, no correction procedures were necessary.

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, non-specific colour (NSC) was determined to be 0%.

Results of the main experiment:

Table 1: Result of the test item 1,3,5-Tris-(3-mercaptopropyl)isocyanurate

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.795

1.851

1.547

0.092

0.101

0.114

0.606

0.562

0.569

1.738

1.814

1.510

0.095

0.101

0.114

0.584

0.512

0.497

OD570(Blank Corrected)

1.752

1.807

1.503

0.048

0.057

0.070

0.562

0.518

0.526

1.695

1.771

1.466

0.051

0.057

0.070

0.540

0.468

0.454

Mean OD570of the Duplicates (Blank Corrected)

1.723

1.789

1.485

0.050

0.057

0.070

0.551

0.493

0.490

Total Mean OD570of 3 Replicate Tissues (Blank Corrected)

1.666*

0.059

0.511

SD OD570

0.160

0.010

0.035

Relative Tissue Viability [%]

103.5

107.4

89.1

3.0

3.4

4.2

33.1

29.6

29.4

Mean Relative Tissue Viability [%]

100.0

3.5**

30.7

SD Tissue Viability [%]***

9.6

0.6

2.1

CV [% Viabilities]

9.6

17.7

6.8

* Blank-corrected mean OD570 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:
other: irritant to the skin (UN GHS category 1 or 2)
Conclusions:
In conclusion, in this in vitro skin irritation study (OECD 439), 1,3,5-Tris-(3-mercaptopropyl)isocyanurate is considered to be irritant to the skin (UN GHS category 1 or 2). Further testing is required.
Executive summary:

In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to 1,3,5-Tris-(3-mercaptopropyl)isocyanurate (94.5% purity) for 60 min and 42 h post incubation period. 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 mean relative tissue viability (% negative control) was ≤ 50% (30.7%) after 60 min treatment and 42 h post-incubation. Therefore, the test item is considered to be irritating to the skin in accordance with UN GHS "Category 1 or 2" and further testing is required.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-11-12 to 2019-01-24
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:
adopted 29th July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was applied undiluted. 50 µL of the test item were dispensed directly atop the EpiDerm tissue.

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EpiDerm 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: Reconstructed Human Epidermal Model EpiDerm™ (EPI-200, MatTek)
- Tissue batch number(s): 28668 for main experiment, 28658 for killed tissue controls

EpiDerm Kit:
The EpiDerm tissues were provided as kits (MatTek), consisting of the following components relevant for this study:
- 1x sealed plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm²); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot: 25876)
- 2x 24-well plates
- 4x 6-well plates
- 1x bottle of assay medium (DMEM-based medium; Lot: 110818TMB)
- 1x bottle of DPBS Rinse Solution (Lot: 082818ISA)
- 25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C


REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
- 3 min experiment: After 3 min of application, the first insert was removed from the 6-well plate with forceps. 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.
- 60 min experiment: After 60 min application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper
- 3 min and 60 min experiment: After the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA
See Table 1 in box "Any other information on materials and methods incl. tables".
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL undiluted test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL potassium hydroxide
- Concentration (if solution): 8 N
Duration of treatment / exposure:
3 and 60 min
Duration of post-treatment incubation (if applicable):
3 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
66.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indication of corrosion
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure
Value:
29.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no indication of corrosion
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: The mixture of 50 µL test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple.
For quantitative correction of results, the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. Therefore, two tissues per treatment period were treated with the test item (KT) or left untreated (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NC) per treatment period. NSMTT was ≤ 30% relative to the negative control of living epidermis. In the 3-min experiment NSMTT was 19.0%, in the 60- min experiment 22.8%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative & positive control: The controls confirmed the validity of the study. The mean OD570 nm of the two negative control tissues was >0.8 and ≤ 2.8 for each exposure period (2.016, 1.883). The mean relative tissue viability (% negative control) of the positive control was < 15% (6.2%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was < 30% (0.7% - 13.9%).

For detailed results see Table 2 and 3 in box "Any other information on results incl. tables".

Table 2: Results of the 3-min experiment

Name

Negative Control

Positive Control

Test Item

Replicate Tissue

1

2

1

2

1

2

Absolute OD570

1.995

2.067

0.344

0.357

1.730

1.719

1.891

2.057

0.361

0.340

1.758

1.712

2.008

2.080

0.341

0.359

1.774

1.725

Mean Absolute OD570

2.016****

0.350

1.736

OD570
(Blank Corrected)

1.951

2.023

0.300

0.314

1.687

1.675

1.847

2.013

0.317

0.297

1.715

1.668

1.965

2.037

0.297

0.316

1.731

1.681

Mean OD570of 3 Aliquots (Blank Corrected)

1.921

2.024

0.305

0.309

1.711

1.675

SD OD570 of 3 Aliquots

0.064

0.012

0.011

0.010

0.022

0.007

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.973*

0.307

1.693

TODTT

 -

 -

1.320

SD OD570of 2 Replicate Tissues

0.073

0.003

0.025

Mean Relative Tissue
Viability [%]

100.0

15.6

85.8

Mean Relative Tissue Viability [%]
- NSMTT Corrected

 -

 -

66.9

Coefficient Of Variation [%]***

3.7

0.9

1.5

* corrected mean OD570 of 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%.

Table 3: Results of the 60-min experiment

Name

Negative Control

Positive Control

Test Item

Replicate Tissue

1

2

1

2

1

2

Absolute OD570

1.871

1.889

0.155

0.162

0.903

1.094

1.904

1.888

0.158

0.154

0.923

1.107

1.845

1.900

0.155

0.163

0.919

1.113

Mean Absolute OD570

1.883****

0.158

1.010

OD570
(Blank Corrected)

1.827

1.846

0.112

0.118

0.860

1.050

1.860

1.844

0.115

0.110

0.879

1.063

1.802

1.857

0.111

0.120

0.875

1.069

Mean OD570of 3 Aliquots (Blank Corrected)

1.830

1.849

0.113

0.116

0.871

1.061

SD OD570 of 3 Aliquots

0.029

0.007

0.002

0.005

0.010

0.010

Total Mean OD570of 2 Replicate Tissues (Blank Corrected)

1.839*

0.114

0.966

TODTT

 -

 -

0.546

SD OD570 of 2 Replicate Tissues

0.014

0.002

0.134

Mean Relative Tissue Viability [%]

100.0

6.2**

52.5

Mean Relative Tissue Viability [%]
- NSMTT Corrected

 -

 -

29.7

Coefficient Of Variation [%]***

0.7

2.1

13.9

* corrected mean OD570 of 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%.

**** The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8

Interpretation of results:
other: non-corrosive in accordance with UN GHS
Conclusions:
In this study under the given conditions the test item showed no corrosive effects. The mean relative tissue viability (% negative control) was greater than 50% (66.9%) after a three minute treatment and greater than 15% (29.7%) after 60 min treatment. The test item is therefore classified as non-corrosive in accordance with the UN GHS classification system.
Executive summary:

In a primary skin corrosion study conducted according to OECD testing guideline 431, two EpiDerm tissues per dose group were exposed to 30 µL of undiluted 1,3,5-Tris-(3-mercaptopropyl)isocyanurate (94.5% purity) for 60 min and 3 min each and cytotoxicity was measured in comparison to the concurrent negative controls. Irritation was scored by the method of mean relative tissue viability. The negative and positive controls confirmed the validity of the study. The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was greater than 15% (29.7%) after 60 min treatment and greater than 50% (66.9%) after 3 min treatment. Based on the results, the test substance is classified as “non-corrosive“ in accordance with the UN GHS classification system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-01-19 to 2019-02-14
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:
adopted 09 October 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was used as provided by the sponsor
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany
- Number of animals: no data
- Characteristics of donor animals (e.g. age, sex, weight): no data
- Storage, temperature and transport conditions of ocular tissue: On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Indication of any antibiotics used: no data
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
Duration of treatment / exposure:
10 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
2 h at 32 ± 1 °C
Number of animals or in vitro replicates:
3 corneas each for the test item, negative control and positive control
Details on study design:
SELECTION AND PREPARATION OF CORNEAS / QUALITY CHECK OF THE ISOLATED CORNEAS
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 (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.

TREATMENT METHOD: closed chamber

APPLICATION DOSE AND EXPOSURE TIME
750 μL of the test substance or the control substance was introduced into the anterior chamber. After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The epithelium was washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red).

POST-EXPOSURE INCUBATION:
The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C. Also, each cornea was observed visually and pertinent observations were recorded. 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. 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 was determined.

METHODS FOR MEASURED ENDPOINTS:
- The optical density was determined at 490 nm (OD490) using a spectrophotometer (Jenway 6405 UV/VIS).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA:
- IVIS ≤ 3: UN GHS No Category
- IVIS > 3 to ≤ 55: No prediction can be made
- IVIS > 55: Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
mean (triplicates)
Value:
23.13
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: According to the UN GHS, this IVIS does not allow to make any prediction
Other effects / acceptance of results:
All 3 corneas treated with 1,3,5-Tris-(3-mercaptopropyl)isocyanurate showed slight opacity of the tissue and were spotted with test item residues. 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. For details on the results see box "Any other information on results incl. tables".

Table 1: In vitro Irritation Score (IVIS)

Cornea No. Test Item Corrected Opacity Value  Corrected OD490
Value 
IVIS
1 Negative Control 0.18 0.004

-0.16

2 -0.07 0.005
3 -0.79 0.004
MV -0.23 0.004
1 Positive Control 35.52 1.438

56.27

2 36.19 1.078
3 31.98 1.826
MV 34.56 1.447
1 Test Item
24.67 0.010

23.13

2 15.97 0.013
3 28.02 0.028
MV 22.88 0.017

MV = mean value

Interpretation of results:
other: mean in vitro irritation score does not allow to make any prediction
Conclusions:
In conclusion, based on the mean in vitro irritation score of 23.13 obtained in the bovine corneal opacity and permeability assay (OECD 437), no prediction can be made regarding the classification of 1,3,5-Tris-(3-mercaptopropyl)isocyanurate.
Executive summary:

The eye irritation potential of the test substance was investigated in the bovine corneal opacity and permeability assay (OECD 437). 750 µL of the test item (purity: 94.5%) was applied undiluted to the corneas as supplied. A mean in vitro irritation score of 23.13 was determined. 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. According to the UN GHS criteria, this mean in vitro irritation score does not allow to make any prediction regarding classification of 1,3,5-Tris-(3-mercaptopropyl)isocyanurate.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-15 to 2019-05-21
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:
adopted 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was applied undiluted. 50 µL (83.3 µL/cm²) of the test item were dispensed directly atop the EpiOcular™ tissue.
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method: This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
Duration of treatment / exposure:
30 min ± 2 min at 37 ± 2 °C, 5% CO2, 95% air
Duration of post- treatment incubation (in vitro):
- Post exposure post-soak plate: 12 ± 2 min at room temperature
- Post exposure post-treatment plate: 120 ± 15 min at 37 ± 2 °C
Number of animals or in vitro replicates:
2 tissues per dose group
Details on study design:
- Details of the test procedure used:
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% air. 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% air 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% air 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% air. 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% air.

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% air.

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 24-well “extraction plates“, containing 2 mL of isopropanol. 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 pierced and the liquid within each insert was decanted into the well from which it was taken.

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.

- RhCE tissue construct used, including batch number:
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo. The EpiOcular™ tissues were provided as kits (e.g. OCL-200-EIT; MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing 24 inserts with EpiOcular™ tissues on agarose (Lot No.: 27097 for main experiment, 27085 for killed tissue controls)
1x bottle EpiOcularTM assay medium (Lot No.: 032519ISA)
1x bottle Ca2+/Mg2+-free DPBS buffer (Lot No.: 010719ISB)

- Doses of test chemical and control substances used:
Negative Control: 50 µL Aqua dest., Positive Control: 50 µL methyl acetate, Test Item: 50 µL (undiluted)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
Exposure: 30 ± 2 min at 37 ± 2 °C, 5.0% CO2/95% air.
Post exposure post-soak plate: 12 ± 2 min at room temperature
Post exposure post-treatment plate: 120 ± 15 min at 37 ± 2 °C, 5.0% CO2/95% air

- Number of tissue replicates used per test chemical and controls: 2 tissues per group

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm ± 30 nm

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
Mean tissue viability (% negative control) <= 60 %: Irritant (I): No prediction can be made
Mean tissue viability (% negative control) > 60%: Non-Irritant (NI): UN GHS “No Category”

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
Historical control data were generated from 2014-2018:
Absolute OD570 ± 30 nm NC: Mean: 1.697; SD: 0.275, n= 50
Relative Viability PC [%]: Mean: 24.9, SD: 12.9, n= 50
SD Viability NC, PC, test item [%]: Mean: 6.6, SD: 7.2, n= 216

Test Acceptance Criteria:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%
Irritation parameter:
other: Relative Tissue Viability (%)
Run / experiment:
Mean of replicates
Value:
98.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (98.7% NSMTT-corrected). For detailed information please refer to Table 1 in box "Any other information on results incl. tables".

Table 1: Main Results

Name

Negative control

Positive control

Test Item

Replicate Tissue

1

2

1

2

1

2

OD570values
(blank-corrected)

1.345

1.427

0.512

0.450

1.327

1.595

1.434

1.456

0.527

0.448

1.343

1.614

Mean of the Duplicates (Blank corrected)

1.390

1.442

0.520

0.449

1.335

1.605

Mean OD

1.416*

0.484

1.470

SD of mean OD

0.037

0.050

0.191

Relative Tissue viability [%]

98.2

101.8

36.7

31.7

94.3

113.4

Relative Tissue Viability

Difference [%]***

3.7

5.0

19.1

Mean Relative Tissue Viability [%]

100.0

34.2**

103.8

 Mean Relative Tissue Viability [%] -NSMTT corrected

 -  -  98.7

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

** mean relative tissue viability of the positive control is < 50%

*** relative tissue viability difference of replicate tissues is < 20%

Table 2: Acceptance Criteria
  Value Cut-off pass/fail
Mean absolute OD570 NC 1.460 0.8 < NC < 2.5 pass
Mean relative viability PC [%] 34.2 < 50% pass
Max difference of % viability [%] 19.1 < 20% pass

NC: negative control; PC: positive control

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions, the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”.
Executive summary:

In the present study the eye irritation potential of 1,3,5-Tris-(3-mercaptopropyl)isocyanurate (94.5% purity) was analysed according to OECD 492 using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. Hereby, 50 µL of the test item was applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 30-min exposure and 120-min post-incubation period and compared to those of the concurrent negative controls. The test item showed reduction of MTT as compared to the control. Therefore, killed tissue controls were performed for quantitative correction of results (NSMTT was determined to be 5.2%). The controls confirmed the validity of the study. The test item showed no irritant effects. The mean relative tissue viability of two replicates (% negative control) was > 60% (98.7% after NSMTT correction). Therefore, the test item is considered to be non-irritating to the eye in accordance with UN GHS “No Category”.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The potential of 1,3,5-Tris-(3-mercaptopropyl)isocyanurate (target substance) to induce skin irritation/corrosion was evaluated in two suitable in vitro test methods (OECD 439 and OECD 431). Based on the results, the target substance must be considered as irritant to the skin. Therefore, classification as Skin Irrit. 2, H315 is warranted in accordance with the CLP criteria.

The potential of the target substance to induce eye irritation was evaluated in two suitable in vitro test methods (OECD 437 and OECD 492). The bovine corneal opacity and permeability assay (BCOP, OECD 437) allows classification as “not classified” or “Eye Dam 1, H318” based on the In Vitro Irritancy Score (IVIS). Results which are between the two categories are identified as “No prediction can be made”. Based on the results obtained from the BCOP test, no prediction can be made regarding the classification of the target substance. Therefore, to further assess the eye irritant potential of the target substance, a second in vitro eye irritation test was conducted, namely the EpiOcular Eye Irritation test (OECD test guideline 492). In this test, the target substance showed no irritant effects. By assessing the results from both in vitro eye irritation tests, it can be concluded that no classification is warranted. This conclusion is in line with the decision scheme on classification presented in the “Guidance document on an IATA for serious eye damage and eye irritation” (OECD Series on Testing and Assessment No. 263, 2017).

Justification for classification or non-classification

Based on the results from the in vitro skin irritation/corrosion tests, the target substance can be considered as irritant to the skin. Therefore, classification as Skin Irrit. 2, H315 is warranted.

Based on the results obtained from two in vitro eye irritation/corrosion tests, the target substance can be considered as non-irritant to the eye and no classification is warranted in accordance with the CLP criteria.