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Administrative data

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

Referenceopen allclose all

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:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
The study was performed in order to evaluate the reactivity of the test item L-Tyrosine, N-acetyl-3,5-dinitro- towards cysteine (Cys-) and lysine (Lys-) containing peptides. The test item was incubated for 22 h at 25 °C together with Cys- and Lys-peptides, respectively. 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
Parameter:
other: % Lys-peptide depletion
Run / experiment:
Experiment 4
Value:
6.55
Key result
Parameter:
other: % Cys-peptide depletion
Run / experiment:
Experiment 7
Value:
25.32
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In conclusion, the DPRA prediction is “positive” with ≥ low reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model in case of co-elution from the DB-ALM protocol n° 154. It can be stated that in this study and under the experimental conditions reported, the test item L-Tyrosine, N-acetyl-3,5-dinitro- possesses a skin sensitisation potential.
Executive summary:

All acceptance criteria were fulfilled; therefore, the test was considered valid.

The experiments 1 to 4 and 6 were not valid for the Cys-peptide assay, because the mean peptide concentration of the reference control C with acetonitrile/water (50/50 %, v/v) was not in the given range. Reason for this was the wrong sample preparation in the first six experiments.

For the Lys-peptide assay in the experiments 1 to 3 the mean peptide concentration of the positive control 2,3-Butanedione was not in the given range.

Experiment 4 was valid for the Lys-peptide assay and the results are reported here.

The fifth experiment was invalid for the Cys-peptide, because of a technical error during the HPLC measurement and thereby missing values for the positive control, the test item and some reference controls.

The seventh experiment was performed with the correct sample preparation and a valid result for the Cys-peptide could be obtained. The invalid experiments are not reported in this report, but the raw data are kept in the test facility in the GLP- archive.

Under the experimental conditions reported here, the test item shows co-elution with the respective peptide and no peak is detectable at 258 nm in both assays.

Co-elution means, the test item itself absorbs at 220 nm and has the same retention time as the Cys-peptide or Lys-peptide, respectively. Nevertheless, according to the DB-ALM protocol n° 154, the peaks could be integrated. But the baseline is not flat so the values will be considered estimates because the “area under the curve” cannot be determined with complete certainty. It is possible that the peak area appears to be larger than it really is, therefore the calculated percent depletion may be underestimated. So, the DPRA classification was made with the estimated values andcannot stand alone, for assessment it is necessary to combine the results with other complementary data and information.

In conclusion, under the experimental conditions reported here and with the estimated values, the DPRA-prediction is positive and the reactivity class ≥ Low.

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

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:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
The assay included a cytotoxicity range finder test (CRFT) and two independent experi-ments (experiment I and II) 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 con-centrations for the two experiments were determined.
In the experiments, the highest nominal applied concentration (2000 µM) 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
Parameter:
other: % viability
Run / experiment:
1st experiment
Value:
> 89
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No cytotoxic effect was observed at all test item concentrations.
Key result
Parameter:
other: luciferase induction
Remarks:
fold luciferase induction in comparison to the solvent control
Run / experiment:
1st experiment
Value:
<= 1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: % viability
Run / experiment:
2nd experiment
Value:
> 91
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No cytotoxic effect was observed at all test item concentrations.
Key result
Parameter:
other: luciferase induction
Remarks:
fold luciferase induction in comparison to the solvent control
Run / experiment:
2nd experiment
Value:
<= 1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item, L-Tyrosine, N-acetyl-3,5-dinitro-, was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential).
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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
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 at least of 30 % and did not exceed 70 %, the aim was 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:
three concentrations (25 %, 50 % and 100 % w/v)
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 25 µL 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. 250 µL 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:
1.1
Test group / Remarks:
25% group
Key result
Parameter:
SI
Value:
1.46
Test group / Remarks:
50% group
Key result
Parameter:
SI
Value:
2.07
Test group / Remarks:
100% group
Interpretation of results:
GHS criteria not met
Conclusions:
The skin sensitization potential of L-Tyrosine, N-acetyl-3,5-dinitro- 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 (25 %, 50 % and 100 % w/v). 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-dinitro- was not a skin sensitizer under the test conditions of this study.

Justification for classification or non-classification