L-Tyrosine, N-acetyl-3,5-diiodo-O-(4-methoxyphenyl)-, ethyl ester

Endpoint summary

Description of key information

Two in vitro studies according to OECD 442C and 442D, respectively, were performed. The results were ambigous.

Thus an in vivo study according to OECD 429 (LLNA test) was conducted. According to the results of the study the test item does not has to be classified as skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The study was performed in order to evaluate the reactivity of the test item L-Tyrosine, N-acetyl-3,5-diiodo-O-(4-methoxyphenyl)-, ethyl ester towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile and the respective peptide was incubated 22 h and 5 min for the Cys-peptide and 22 h for the Lys-peptide at 25 °C, respectively, and the peptide concentration after the incubation was measured using HPLC-UV.
Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: % Cys-peptide depletion

Value:

36.09

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: % Lys-peptide depletion

Value:

0.41

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, the DPRA prediction is “positive” with low reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item L-Tyrosine, N-acetyl-3,5-diiodo-O-(4-methoxyphenyl)-, ethyl ester possesses low skin sensitization potential.

Executive summary:

All acceptance criteria were fulfilled. Therefore, the test was considered valid. The DPRA prediction for the test itemL-Tyrosine, N-acetyl-3,5-diiodo-O-(4-methoxyphenyl)-, ethyl esterwas positive with reactivity class low according to the Cysteine 1:10/Lysine 1:50 prediction model.

No observations arousing doubts concerning the accuracy of the results and the validity of the study were made.

The mean peptide depletion in the Cys-peptide and Lys-peptide assay was 18.25 %, therefore the test item was classified with:

DPRA Prediction: Positive

Reactivity class: Low

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

The assay included a cytotoxicity range finder test (CRFT) and three independent exper-iments (experiment I, II and III) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for experiment I, II and III were determined.
Due to a technical error, experiment II was declared as invalid. This experiment is not re-ported, all documentation is kept with the raw data and will be archived at the GLP test facility.
In the experiments, the highest nominal applied concentration (15.63 µg/mL) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilu-tions thereof was prepared. Precipitation of the test item was not visible in any of the ex-periments.
DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.

Key result

Run / experiment:

other: 1st experiment

Parameter:

other: % viability

Value:

71

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No cytotoxic effect was observed at the test item concentrations 2.10 µM to 10.85 µM.

Key result

Run / experiment:

other: 1st experiment

Parameter:

other: luciferase induction

Remarks:

fold luciferase induction in comparison to the solvent control

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

In the Luciferase assay, all of the tested non cytotoxic concentrations induced a statisti-cally significant increase in luciferase induction above or equal 1.5 fold in comparison to the solvent control.

Key result

Run / experiment:

other: 2nd experiment

Parameter:

other: % viability

Value:

74

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No cytotoxic effect was observed at the test item concentrations 2.10 µM to 9.05 µM.

Key result

Run / experiment:

other: 2nd experiment

Parameter:

other: luciferase induction

Remarks:

fold luciferase induction in comparison to the solvent control

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

In the Luciferase assay, all of the tested non cytotoxic concentrations induced a statisti-cally significant increase in luciferase induction above or equal 1.5 fold in comparison to the solvent control.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test item, L-Tyrosine, N-acetyl-3,5-diiodo-O-(4-methoxyphenyl)-, ethyl ester, was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (sensitizing potential).

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

The animals were housed in IVC polycarbonate cages (5 animals per cage) suspended on stainless steel racks, in a room equipped with central air-conditioning. The room temperature was within the range of 22 ± 3°C, relative humidity was within the range of 50 - 60 %. The light regimen was set to a 12-hour light / 12-hour dark cycle. The sanitation was performed according to standard operation procedures.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10%, 25%, 50% (w/v)
The concentration of 100% could not be prepared because of mixing of 1000mg of test item with vehicle up to a volume of 1 mL was obtained a semidry substance inappropriate for application.

No. of animals per dose:

Number of animals:
5 females – negative control (vehicle)
5 females – positive control
15 females – test item
4 females - pre-screen test, plus spare animals

Details on study design:

Day 1:
Each animal was identified and the body weight was recorded. To the dorsum of each ear 25µL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6:
The body weight of each animal was recorded. 250µL of sterile phosphate-buffered saline (PBS)
containing 2 µCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected
into all test and control mice via the tail vein.
Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Key result

Parameter:

SI

Value:

1.25

Test group / Remarks:

25% group

Key result

Parameter:

SI

Value:

1.97

Test group / Remarks:

50% group

Key result

Parameter:

SI

Value:

1.97

Test group / Remarks:

100% group

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitization potential of L-Tyrosine, N-acetyl-3,5-diiodo-O-(4-methoxyphenyl)-, ethyl ester was evaluated by LLNA method, which basic underlying principle is that sensitizers induce a primary proliferation of lymphocytes in the auricular lymph nodes draining the site of chemical application.
In the present study, the test item was applied to the dorsum of each ear of five female mice (CBA/Ca) per group over three consecutive days, at three concentrations. All animals survived throughout the test period without showing any clinical signs of toxicity. Calculated SI values in treated groups remained under the value of 3, which is the threshold to consider the substance as a sensitizer. Therefore, it was not possible to calculate an EC3 value.
These results demonstrate that the test item L-Tyrosine, N-acetyl-3,5-diiodo-O-(4-methoxyphenyl)-, ethyl ester was not a skin sensitizer under the test conditions of this study.