Registration Dossier

Administrative data

Description of key information

Skin irritation: OECD 439, not irritating to the skin
Eye irritation: OECD 437, not irritating to the eye

Respiratory irritation: no data available

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 1, 2016 - December 20, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to guideline
Guideline:
other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Designation: Art. 108219
Synonym: Tris(hydroxymethyl)-aminomethananhydrochlorid
Trade name: TRIS-HCL Emprove Bio Bulk
CAS-No.: 1185-53-1
Batch: TD16007119
Purity: 100.2% (Titration with perchloric acid)
Appearance: Colourless crystals
Expiry date: June 30, 2021
Storage: +15 to +25°C, store in tightly closed containers, protected from moisture
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Justification for test system used:
standard model
Vehicle:
unchanged (no vehicle)
Remarks:
No vehicle used in this study; The test item was applied neat to the tissues.
Details on test system:
CELL CULTURE
- Supplier: EpiSkin/SkinEthic Laboratories, Lyon, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 16-RHE-122
- Expires: November 28, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL: 16 mg of solid test material
NEGATIVE CONTROL: 16 µL (Dulbecco`s Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (5% aqueous solution of sodium dodecyl sulfate in deionised water)
Duration of treatment / exposure:
42 min (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Experiment 1 / Run 1
Value:
85.95
Vehicle controls validity:
not applicable
Remarks:
The test item was applied neat to the tissues
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:
Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
The negative control OD values were 2.450, 2.192 and 2.313 and, thus, in the range of ≥0.8 and ≤3.0.

Acceptability of the Positive and Negative Control:
After treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) the mean viability value was 1.13% (standard deviation: 2.95%) and, thus, lower than the historically established threshold of 3.12%.
After treatment with the negative control (DPBS-buffer) the mean OD was 2.318 (standard deviation: 5.57%) and, thus, higher than the historically established threshold of 1.436.

Variability of the Data:
The standard deviation between the three tissues replicates treated with the test item was 11.38% and, thus, ≤18%.
The standard deviations between the three tissue replicates of the negative control and the positive control were 5.57% and 2.95%, respectively, and, thus, ≤18%.

The study met all acceptance criteria.


 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 42  2.318 100 
 Positive Control 42

0.026

1.13

 Test Material

42

1.993

85.95

Interpretation of results:
GHS criteria not met
Conclusions:
This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the conditions of the present study, the test item TRIS-HCL is not considered to possess an irritant potential to skin (UN GHS: No Category).
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the conditions of the present study, the test item TRIS-HCl is not considered to possess an irritant potential to skin.

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:
November 1, 2016 - January 6, 2017
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 Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Designation: Art. 108219
Synonym: Tris(hydroxymethyl)-aminomethananhydrochlorid
Trade name: TRIS-HCL Emprove Bio Bulk
CAS-No.: 1185-53-1
Batch: TD16007119
Purity: 100.2% (Titration with perchloric acid)
Appearance: Colourless crystals
Expiry date: June 30, 2021
Storage: +15 to +25°C, store in tightly closed containers, protected from moisture


PREPARATION OF THE TEST MATERIAL
The test item Art. 108219 (TRIS-HCL Emprove Bio Bulk) was prepared as a 20% (w/v) solution in a 0.9% sodium chloride solution. The stability in the vehicle was not investigated. The test item preparation was administered within <1 hour after preparation.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL: 750 µL (i.e. 150mg/750µL)

NEGATIVE / VEHICLE CONTROL: 750 µL

Designation: 0.9% sodium chloride solution
Supplier: B. Braun Melsungen AG, Germany
Batch: 15464012
Released until: October 2018


POSITIVE CONTROL: 750 µL

Designation: Art. 814223
Synonym: Imidazole
Supplier: Merck KGaA, Germany
Batch: S6746923
Purity (GC) 99.8% (a/a)
Released until: August 31, 2018
Imidazole was dissolved with 0.9% sodium chloride solution to a concentration of 20% (w/v)
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
in vitro: triplicate design
Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1 / Experiment 1
Value:
0.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.4 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 – 3.4).
- Acceptance criteria met for positive control: After treatment with the positive control (20% Imidazole) the calculated IVIS was 111.4 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS 78.8 – 134.7). Therefore, the study fulfilled the acceptance criteria.

Opacity
Permeability
IVIS
per cornea
per group
(mean value)
SD
Negative control
0.9% NaCl Solution
0.5
0.000
0.500
0.4
0.2
0.2
0.001
0.515
0.2
-0.002
0.170
Positive control
20% Imidazole solution
76.1
2.854
118.910
111.4
9.5
80.6
2.270
114.650
54.2
3.102
100.730
Test item
Art. 108219
1.8
-0.001
1.785
0.8
0.9
0.1
-0.001
0.085
0.4
0.001
0.415


Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test itemTRIS-HCL did not show an eye hazard potential. The test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 437. Under the conditions of the present study, the test itemTRIS-HCL did not show an eye hazard potential. The test item is not requiring classification for eye irritation or serious eye damage (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

Skin irritation:

In an in-vitro skin irritation study according to OECD 439 the potential of the test item TRIS HCl to induce skin irritation in an in vitro human skin model was investigated (reference 7.3.1 -1). The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.  Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. After treatment with the negative control (DPBS-buffer) the mean OD was 2.318 (study acceptance criterion: >1.436). Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.13% (study acceptance criterion: <3.12%). Thus, the acceptance criteria were met. Following treatment with the test item TRIS HCl, the tissue viability was 85.95% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin.Under the conditions of the present study, the test item TRIS HCl is not considered to possess an irritant potential to skin (UN GHS: No Category).

There is further supporting in-vivo data from the source substance Trometamol (CAS 77-86-1, EC 201-064-4) available, which is considered suitable for read-across using the analogue approach.

In a supporting study performed according to OECD 404 on rabbits, 0.5 g Trometamol (moistened with water) was applied to shaved skin and covered with a semi-occlusive dressing for 4 hours (reference 7.3.1 -2). No erythema or edema was observed in any of the animals 1, 24, 48 and 72 hours after exposure ended.

Eye irritation:

In an in-vitro eye irritation study according to OECD 437 the potential of the test item TRIS HCl to induce serious eye damage in the BCOP assay was investigated (reference 7.3.2 -1). The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item TRIS HCL as a 20% (w/v) solution in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the dissolved test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an in vitro Irritancy Score (IVIS). After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.4 (study acceptance criteria range: -1.4 – 3.4). Treatment with the positive control (20% Imidazole) revealed an IVIS of 111.4 (study acceptance criteria range: 78.8 – 134.7). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with TRIS HCL was 0.8 and, thus, lower than 3,i.e.according to OECD 437 the test item is not requiring classification for eye irritation or serious eye damage. Under the conditions of the present study, the test item TRIS HCl did not show an eye hazard potential. The test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

There is further supporting data from the source substance Trometamol (CAS 77-86-1, EC 201-064-4) available, which is considered suitable for read-across using the analogue approach.

In a study performed according to OECD 405, 0.1 g of Trometamol was instilled into one eye of each of 3 rabbits (reference 7.3.2 -2). The eyes remained unwashed and were assessed for irritation 1, 24, 48 and 72 hours after the instillation. Slight to moderate effects on the conjunctiva (score 1-2) and chemosis (score 1-2) were observed from the 1-hour reading time point in 3/3 rabbits. The maximum mean scores for both effects on the conjunctiva and chemosis were 1.33 for 1/3 rabbits, and 0.33 for 2/3 rabbits, respectively. The irritation effects had cleared completely in 2/3 rabbits within 48 hours and in the remaining rabbit with the highest mean scores within 72 hours. No damage to the iris or cornea was observed at any reading time point.

Justification for classification or non-classification

The available data on skin and eye irritation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.