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Description of key information

The results obtained from the in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions.


According to the in vitro Eye Irritation Test in isolated chicken eyes, the test item excluded ocular corrosive or severe eye irritant.


According to (Q)SAR calculation, using Eye irritation/corrosion Inclusion rules by BfR, no alert were met.


Also according to (Q)SAR calculation using Eye irritation/corrosion Eclusion rules by BfR the substance is not irritating, even it is not recommended to be used directly for prediction purposes (as SARs).


 


The test item LEVOTHYROXINE ACID is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 december 2018 - 12 february 2019
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
other: EpiSkinTMSM
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 mg
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h (± 1h)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
ca. 120
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
ca. 114
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
ca. 121
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: Optical Density (OD)
Run / experiment:
1
Value:
ca. 0.884
Vehicle controls validity:
valid
Irritation / corrosion parameter:
other: Optical Density (OD)
Run / experiment:
2
Value:
ca. 0.842
Vehicle controls validity:
valid
Irritation / corrosion parameter:
other: Optical Density (OD)
Run / experiment:
3
Value:
ca. 0.894
Vehicle controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item LEVOTHYROXINE ACID is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
Executive summary:

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes(± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1°C for 42 hours (± 1h) in an incubator with 5±1% CO2, ≥ 95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1°C in 5±1% CO2, ≥ 95% humidified atmosphere, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.In this in vitro skin irritation test using the EPISKIN model, the test item LEVOTHYROXINE ACID did not show significantly reduced cell viability in comparison to the negative control (mean value: 118 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
weight of evidence
Study period:
7 December 2018 - 23 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.03 g
Duration of treatment / exposure:
10 s
Observation period (in vivo):
30, 75, 120, 180 and 240 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.
EQUILIBRATION AND BASELINE RECORDINGS
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate numbers of eyes were selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm. Any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.
NUMBER OF REPLICATES
3
NEGATIVE CONTROL USED
NaCl (9 g/L saline)
Name: Sodium chloride
Supplier: lach:ner
Batch No.: PP/2017/00996
Retest date: 24 November 2019
Storage condition: Room temperature
Diluted with ultra-pure water (prepared by Direct Q5 water purification system) in Toxi-Coop ZRT.

POSITIVE CONTROL USED
Imidazole
Name: Imidazole
Supplier: Sigma-Aldrich
Batch No.: SLBR9142V
Retest date: October 2019
Storage condition: Refrigerator (2-8°C)
APPLICATION DOSE AND EXPOSURE TIME

OBSERVATION PERIOD
10 s
REMOVAL OF TEST SUBSTANCE
the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.
The Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole and test item treated eyes after the 30, 75, 120 and 180 minutes of observation. The Imidazole and test item treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse.

METHODS FOR MEASURED ENDPOINTS:
The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. If morphological effects were observed the classification of these findings were interpretations of the study director (e.g., pitting or loosening of the epithelium). Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE (Isolated Chicken Eye) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance

SCORING SYSTEM:
Scores were taken at any given timepoint according to the list below:
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent area; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible

DECISION CRITERIA: The mean values of the treated eyes for maximum corneal thickness change, corneal opacity, fluorescein retention and other observation (morphological effect etc.) are given below. The conclusion on eye irritancy was based on the OECD guideline on quantitative assessments.
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean maximum corneal swelling at up to 75 min
Value:
ca. 7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean maximum corneal swelling at up to 240 min
Value:
ca. 11
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Irritation parameter:
cornea opacity score
Run / experiment:
Mean maximum corneal opacity
Value:
ca. 1.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class III
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean fluorescein retention
Value:
ca. 1.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class III
Interpretation of results:
study cannot be used for classification
Conclusions:
According to the guideline OECD 438, LEVOTHYROXINE ACID has been categorized as “No prediction can be made”.
Executive summary:

In this ICET, LEVOTHYROXINE ACID did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE classes were twice II (based on corneal swelling of 11% within 240 min and opacity score of 1.2) and once III (based on thefluorescein retention of 1.7). Positive and negative controls showed the expected results. The experiment was considered to be valid. According to the guideline OECD 438, LEVOTHYROXINE ACID overall in vitroclassification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.

Endpoint:
eye irritation, other
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, and documentation / justification is limited
Justification for type of information:
1. SOFTWARE: OECD Toolbox 4.4.1.
2. MODEL: Eye irritation/corrosion Exclusion rules by BfR - Scheme type Linear - Scheme nature: EndpointSpecific - Version: 2.0 - December 2016.
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: NC(Cc1cc(I)c(Oc2cc(I)c(O)c(I)c2)c(I)c1)C(O)=O
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL Developer: National Institute for Public Health and the Environment (RIVM), Netherlands Donator: National Institute for Public Health and the Environment (RIVM), Netherlands. The aim of this profiler is to classify chemicals with respect to its eye irritation or corrosion potential. Unlike most profilers, it determines compounds that do not exhibit an irritating / corrosive effect, namely those that are not characterized by below mentioned Risk Phrases according to Annex III of European Union Directive 67/548/EEC: Nature of special risks attributed to dangerous substances and preparations:
R34: Causes burns
R35: Causes severe burns
R36: Irritating to eyes (moderate eye irritation reversible within ≤ 21 days)
R41: Risk of serious damage to eyes (moderate but persistent eye lesions, eye corrosion)
Definition of structural classes for which separate skin irritation exclusion rules have been defined:
1. Class C - Structure contains only Carbon (C), hydrogen (H) and oxygen (O) atoms – Formula CxHyOz
2. Class CN - Structure contains only C, H, O and Nitrogen (N) atoms – Formula CxHyOzNa
3. Class CNHal - Structure contains only C, H, O, N and Halogen (Hal) atoms – Formula CxHyOzNaHalb
4. Class CNS - Structure contains only C, H, O, N and Sulphur (S) atoms – Formula CxHyOzNaSb
5. Class CHal - Structure contains only C, H, O and Hal – Formula CxHyOzHalb
Tsakovska I, Netzeva T, Worth A., (2005), Evaluation of (Q)SARs for the prediction of Eye Irritation/Corrosion Potential. Physico-chemical exclusion rules. Report for the European Chemicals Bureau, available at:
https://eurl-ecvam.jrc.ec.europa.eu/laboratories-research/predictive_toxicology/doc/Evaluation_of_Eye_Irritation_QSARs.pdf

5. APPLICABILITY DOMAIN The exclusion rules for eye irritation/corrosion are based on physico-chemical cut-off values to identify chemicals that do not exhibit eye irritation or corrosion potential. The parameters used for defining eye irritation rules are: Lipid Solubility, Octanol Water partition coefficient, Aqueous Solubility, Melting Point and Molecular Weight.
Qualifier:
no guideline required
Specific details on test material used for the study:
NC(Cc1cc(I)c(Oc2cc(I)c(O)c(I)c2)c(I)c1)C(O)=O
Irritation parameter:
other: (Q)SAR prediction
Remarks on result:
no indication of irritation
Interpretation of results:
other: not irritating
Conclusions:
The structural and parametric boundaries used to define the chemical categories, in “Eye irritation/corrosion Exclusion rules by BfR” profiler, confirmed that the structure of LEVOTHYROXINE ACID do not exhibit an eye irritation or corrosion potential.
Executive summary:

According to the “Eye irritation/corrosion Exclusion rules by BfR” profiler, LEVOTHYROXINE ACID do not exhibit an eye irritation or corrosion potential.

Endpoint:
eye irritation, other
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Justification for type of information:
1. SOFTWARE: OECD Toolbox 4.4.1.
2. MODEL: Eye irritation/corrosion Inclusion rules by BfR - Scheme type Linear - Scheme nature: EndpointSpecific - Version: 2.0 - December 2016.
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: NC(Cc1cc(I)c(Oc2cc(I)c(O)c(I)c2)c(I)c1)C(O)=O
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL Developer: National Institute for Public Health and the Environment (RIVM), Netherlands Donator: National Institute for Public Health and the Environment (RIVM), Netherlands.
The aim of this profiler is to classify chemicals with respect to its eye irritation or corrosion potential.
Tsakovska I, Saliner AG, Netzeva T, Pavan M, Worth AP. Evaluation of SARs for the prediction of eye irritation/ corrosion potential: structural inclusion rules in the BfR decision support system, European Chemicals Bureau, Institute for Health and Consumer Protection, Joint Research Centre, 21020 Ispra (VA), Italy. ivanka.tsakovska@jrc.it . SAR QSAR Environ Res. 2007 May-Jun 18 (3-4): 221-35
Saliner AG,Tsakovska I ,Payan M ,Patlewicz G,Worth AP. Evaluation of SARs for the prediction of skin irritation/corrosion potential: structural inclusion rules in the BfR decision support system. European Chemicals Bureau, Institute for Health and Consumer Protection, Joint Research Centre, Ispra (VA), Italy. ana.gallegos@jrc,it, SAR QSAR Environ Res.2007 May-Jun:18(3-4):331-42
OECD(2002), Acute Eye Irritation/Corrosion, OECD Guideline
5. APPLICABILITY DOMAIN This profiler comprised of 17 structural alerts is based on empirically derived structural inclusion rulеs. It identifies chemicals that show potential for eye irritation and corrosion. The profiler is also based on known mechanisms of action (biochemical reaction within the eye and /or conjunctival tissues). The model is applicable to organic substances with at least 95% purity and contain hetero atoms not other O, N, S, P, Si and halogen.The structural boundaries used to define the chemical classes (e.g. “Alcohol” – chemical class from “Organic functional group” profiler) or alerting groups responsible for the binding with biological macromolecules (e.g. “Aldehydes” – structural alert for protein binding), represent structural functionalities in the molecule which could be used for building chemical categories for subsequent data gap filling. They are not recommended to be used directly for prediction purposes (as SARs).
Specific details on test material used for the study:
NC(Cc1cc(I)c(Oc2cc(I)c(O)c(I)c2)c(I)c1)C(O)=O
Irritation parameter:
other: inclusion rules not met
Interpretation of results:
other: inclusion rules not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification