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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:
2017 July to 2017 September
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Supplier: Borica Co., Ltd.
- Lot number: 110422Y
- Expiration date of the lot/batch: 2017-Dec-12
- CAS number: 101033-44-7

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm (EPI-200-MatTek Corporation) was used as reconstructed human epidermal model. It consisted of normal human-derived epidermal keratinocytes which have been cultured to form a multi-layered highly differentiated model of the human epidermis. It consisted of organized basal, spinous and granular layers and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200) RECONSTRUCTED HUMAN EPIDERMIS
- Tissue batch number(s): 25837
- Date of initiation of testing: 2017-Jul-13


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 microliter neat test material


VEHICLE
- Not used


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 microliter Sterile Dulbecco's phosphate buffered saline (DPBS)
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 microliter Sodium dodecyl sulphate (SDS)
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 +/- 1 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, >=95% RH)
Duration of post-treatment incubation (if applicable):
42 +/- 4 h hours post-treatment incubation.
Number of replicates:
3 repliecate per run.
Vehicle:
unchanged (no vehicle)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
ca. 90.85
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Table 3. Mean and SD of cell viability measurements and of viability precentages after 60 min (+/- 1 min) of application and 42 h (+/-4 h) post-incubation.

 Name  Code  Mean of OD  SD of OD  Mean of viability (%)  SD of viability (%)  CV (%)  Classification
 DPBS  NC  1.322  0.150  100.0  11.34  11.34  Non-irritant
 SDS 5%  PC  0.054  0.014  4.085  1.07  26.26  Irritant
 Test item  TA3 01.201  0.086  90.847  6.50  7.15  Non-irritant

Predition model: test items that reduce the viability to 50% or below are irritant (I); test items with a percentage viability above 50% are considered to be non-irritant (NI).

Notes:

NC: Negative control

PC: Positive control

TA3: Tetrakis [[2,2',2"-nitrilotris[ethanolato]](1-)-N,O]zirconium

Interpretation of results:
GHS criteria not met
Conclusions:
A test item is considered to be an irritant (I) to skin in accordance with UN GHS Category 2 or EU R38 if the skin model viability after exposure and post-treatment incubation is ≤50%.
A test item may be considered as a non-irritant (NI) if the skin model viability after exposure and post-treatment incubation is > 50%.
The percentage of viability obtained with the test item Tetrakis [[2,2',2"-nitrilotris[ethanolato]](1-)-N,O]zirconium was 90.85%, therefore it was considered as non-irritant to the skin.
Executive summary:

The skin irritation potential of Tetrakis [[2,2',2"-nitrilotris[ethanolato]](1-)-N,O]zirconium was assessed in vitro according to OECD Test Guideline 439: In Vitro Skin irritation: Reconstructed Human Epidermis Test Method.

After 60 +/- 1 min exposure on the surface of the EpiDerm™ reconstructed human epidermis and a 42 +/- 4 h post-exposure incubation time, viability of the tissues was assessed and compared to the negative control. The percentage of viability obtained was 90.85% and therefore the target substance was classified as non-irritant to the 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:
2017 July to 2017 September
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Supplier: Borica Co., Ltd.
- Lot number: 110422Y
- Expiration date of the lot/batch: 2017-Dec-12
- CAS number: 101033-44-7
Species:
human
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 microliter
- Concentration (if solution): neat

VEHICLE
- Not used
Duration of treatment / exposure:
30 minutes +/- 2 min
Duration of post- treatment incubation (in vitro):
12 +/- 2 min post-treatment immersion and 2 hours +/- 15 min post-treatment incubation.
Number of animals or in vitro replicates:
3 replicates per run.
Details on study design:
- Details of the test procedure used
Prior to the study, the required compatibility checks confirmed that the test item interfered with MTT and therefore additional freeze killed negative controls (NC-FK) and freeze killed test item controls (TA-FK) were included in the assay to determine the background level of interference (manifesting as altered viability) produced by the test item. The level of viability measured in the interference controls was subtracted from the standard test viability value to give an adjusted final viability. No water colouration was observed and therefore colorant control was not required.

After pre-wetting tissues with 20 microliter PBS (Sterile Dulbecco’s phosphate buffered saline) for 30 +/- 2 min, single topical application of 50 microliter of neat test item or of reference items to the surface of the EpiOcular™ models for 30 +/- 2 min, followed by a 12 +/- 2 min post-treatment immersion, and 2 hr +/- 15 min post-treatment incubation, prior to the MTT endpoint; three tissues per condition (n = 3).


- RhCE tissue construct used, including batch number
EpiOcular™ EIT (OCL-200) Reconstructed Human Ocular Epithelium
Supplier: MatTek Corporation
Keratinocyte Strain: 4F1188
Lot number: 23799
Description of the RhCE tissue: The EpiOcular™ tissue model (OCL200-MatTek Corporation) is composed of stratified human keratinocytes in a three-dimensional structure, reflecting the morphology and function of the human corneal epithelium found in vivo.


- MTT test conditions
Spectrometer: BMG LabTech FluoStar Optima
Wavelength for O.D. measurements: 570 nm


- Positive and negative control means and acceptance ranges based on historical data
1. Mean OD570 of the negative control (treated with sterile water) tissues is > 0.8 and < 2.5.
2. Mean of the positive control relative percentage viability is below 50% of negative control viability after 30 +/- 2 minutes exposure.


- Acceptable variability between tissue replicates for positive, negative controls and test items
The SD between three tissues replicates should not exceed 18% in the same run (for negative and positive control tissues and tissues of test items).



Irritation parameter:
other: Tissue viability
Run / experiment:
1 run, 3 replicates per run
Value:
ca. 72.62
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Table 3. Mean and SD of viability measurements and of viability percentages after 30 +/- 2 min of application and 2 h +/- 15 min post-incubation of test and reference items.

 Consition  Mean of OD  SD of OD  mean of viability %  SD of viability (%)  CV %  Classification
 NC  1.657  0.016  100.00  0.938  0.938  Non-irritant
 PC  0.522  0.115  31.479   6.964  22.122  Irritant
 TA  1.204 0.233  72.624  14.058  19.358  Non-irritant

Note:

NC: Negative control (sterile H2O)

PC: neat methyl acetate

TA: Tetrakis[[2,2',2''-nitrilotris[ethanolato]](1 -)-N,O]zirconium

Interpretation of results:
GHS criteria not met
Conclusions:
A Test item is considered to be irritant to the eye (i.e. would require labelling as either GHS 1 or 2) if they eye model viability after exposure and post-treatment incubation is ≤ 60%.
A test item is considered as a non-irritant to the eye (i.e. would not require a warning label in the European chemical classification systems) if the eye model viability after exposure and post-treatment incubation is > 60%.
The current assay is not intended to differentiate between GHS class 1 and GHS class 2 or R36 and R41 (degree of stromal damage).
The percentage of viability obtained with test item target substance was 72.62%, therefore it is considered as Non-Irritant to the eye.
Executive summary:

The eye irritation potential of Tetrakis [[2,2',2"-nitrilotris[ethanolato]](1-)-N,O]zirconium was assessed in vitro according to OECD Test Guideline 492 (Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labeling for eye irritation or serious eye damage).

After a 30 +/-2 minute exposure on the surface of EpiOcular™ reconstructed ocular epithelium and 2 h +/- 15 mins post-incubation time, the viability of the tissues was assessed and compared to a negative control. The percentage viability was 72.62 % and therefore the target substance was classified as Non-Irritant to the human eye.

 

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin and eye irritation tests have been conducted on the target substance using alternative in vitro methods – OECD 439, reconstructed human epidermis, and OECD 492, reconstructed human cornea-like epithelium. The target substance was found non-irritating in the two tests.

 

As the target substance hydrolyses rapidly (half-life < 30 minutes) the intrinsic properties are related to hydrolysis products –2,2',2''-Nitrilotriethanol (triethanolamine, TEA).

 

Triethanolamine is not irritating to skin and eyes. A group of 64 humans was tested with 1% TEA (vehicle unknown). Testing was conducted by 24-hour semi-occlusive patches to the upper arm 3 times per week for 3 weeks. Fourteen days later challenge patches were applied to the opposite arm. During the induction period, signs of irritation ranged from "moderate" (on anly a very small number of occasions) to mild or no irritation (approximately evenly distributed between these two). At challenge, 14 days later (graded at 48 and 96 hours) there were some signs of mild irritation, but overall no evidence of sensitisation. Some other reports of skin sensitisation amongst humans exist, albeit at a low incidence amongst the populations studied. However, due to poor reporting it remains unclear whether or not these reports are in fact skin irritation rather than sensitisation. (OECD SIDS 1995). 

 

Based on above findings, the target substance is therefore considered not irritating to skin and eyes, according to CLP regulations. In addition, the two in vitro studies serve as evidence that the toxicological properties of the target substance closed related to the hydrolysis products.

Justification for classification or non-classification

Based on results of alternative in vitro methods, OECD 439 and OECD 492, tested on the target substance, the results indicated non-irritating to skin and eyes.

The hydrolysis product, triethanolamine, have also been found non-irritating to skin and eyes. Therefore, the target substance was classified as non-irritant to skin and eyes according to CLP regulation.