Registration Dossier

Administrative data

Description of key information

LLNA results:

In conclusion, under the conditions of the present assay, sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate, tested in a suitable vehicle, was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay.

Keratinosens:

Under the experimental conditions of this study, the test item, ISOMETHOL, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

DPRA:

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item ISOMETHOL, was considered to have no or minimal peptidereactivity, though with limitations due to its precipitation with the cysteine peptide.

HCLAT:

Under the experimental conditions of this study, the test item, ISOMETHOL, was found to be positive in the h-CLAT test method.

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
Study period:
July 2018
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)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The objective of this study was to evaluate the potential of the test item, ISOMETHOL, to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.
The reactivity of the test item was evaluated in vitro : it is an alternative method in order to avoid animal study

Principle
This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.
Details on study design:
Methods
The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Three independent runs were performed.
Positive control results:
Name : Cinnamic Aldehyde (CA)
Synonym : trans-Cinnamaldehyde

For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of 2, to obtain a total of five concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 μM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.
Key result
Parameter:
other: Imax
Run / experiment:
1
Value:
2.19
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5
Run / experiment:
1
Value:
658.31
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Imax
Run / experiment:
2
Value:
1.26
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: IC30
Run / experiment:
2
Value:
898.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Imax
Run / experiment:
3
Value:
1.52
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
First run
All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid.
The evaluation criteria for a positive response are met in this run.

Second run
All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.
The evaluation criteria for a negative response are met in this run.

Third run
Since non-concordant results were obtained in the first two runs, a third run was performed.
All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid. In this run, an outlier luminescence value was removed from the data analysis of the negative control wells (the second replicate of the third plate), therefore 17 negative control values were taken into consideration for the analysis instead of 18.
The evaluation criteria for a negative response are met in this run.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item, ISOMETHOL, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.
Executive summary:

For each run, the test item was prepared in pre-warmed water for injections at 100 mM. The formulation was then heated at 60°C for up to 1h30 and sonicated for 10 minutes prior being filtered through a 0.22 μm filter.

Each run was considered to be valid since all acceptance criteria were fulfilled for the positive and negative controls. In the third run, an outlier luminescence value was removed from the data analysis of the negative control wells (the second replicate of the third plate), therefore 17 negative control values were taken into consideration for the analysis instead of 18.

First run

This run was performed using the following concentrations: 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.3, 63, 125, 250, 500 and 1000 μM in culture medium containing 1% DMSO and 1% water.

At these tested concentrations:

. no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

. no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%), therefore no IC30 or IC50 was calculated,

. a statistically gene-fold induction above the threshold of 1.5 was noted at concentration of 1000 μM,

. the Imax was 2.19 and the calculated EC1.5 was 658.31 μM.

The evaluation criteria for a positive response are met in this run.

Second run

Due to results obtained in the first run (i.e. statistically significant gene-fold induction > 1.5 at the highest tested concentration only), a narrower range of concentrations was used in the second run (i.e. dilution factor of 1.41): 22.83, 32.20, 45.40, 64.01, 90.25, 127.26, 179.4, 253, 357, 503, 709 and 1000 μM in culture medium containing 1% DMSO and 1% water.

At these tested concentrations:

. no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

. a decrease in cell viability (i.e. cell viability < 70%) was noted at 1000 μM, the corresponding IC30 was 898.70 μM, no IC50 was calculated since the cell viability was > 50% in this run,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was < 1.5.

The evaluation criteria for a negative response are met in this run.

Third run

Since non-concordant results were obtained in the first two runs, a third run was performed.

The same concentrations as those used in the second run were used in this third run and the following results were obtained:

At these tested concentrations:

. no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,

. no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%), therefore no IC30 or IC50 was calculated,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was 1.52.

The evaluation criteria for a negative response are met in this run.

No geometric mean IC30 or IC50 was calculated since the cell viability was > 70% in two out of three runs.

The evaluation criteria for a negative response are met in two out of three runs, the final outcome is therefore negative. This negative result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2018
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
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.
The reactivity of the test item was evaluated in chemico : it is an alternative method in order to avoid animal study
Details on study design:
Methods

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.
Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).
Positive control results:
Name: Cinnamaldehyde
The positive control was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in acetonitrile at 100 mM. The formulation was colorless limpid solution and was used just after its preparation.
Key result
Parameter:
other: percent cysteine depletion
Run / experiment:
1
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: percent cysteine depletion
Run / experiment:
2
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: percent cysteine depletion
Run / experiment:
3
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE CRITERIA
The run was considered valid if the following criteria were fully met:
 the calibration curve should have a coefficient of determination (r2) >= 0.99,
 the mean peptide concentrations of the reference control A samples should be within ± 10% of the nominal concentration,
 the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
 for the cysteine peptide, the mean percent depletion value should be between 60.8 and 100% with a SD < 14.9%,
 for the lysine peptide, the mean percent depletion value should be between 40.2 and 69.0% with a SD < 11.6%,
 the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0%.

The test item’s results were considered valid if the following criteria were fully met:
 the mean peptide concentrations of the reference control C samples prepared in the appropriate solvent (milli-Q water) should be within ± 10% of the nominal concentration,
 the maximum SD for the test item replicates should be < 14.9% for the percent cysteine depletion value and < 11.6% for the percent lysine depletion value.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item ISOMETHOL, was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation with the cysteine peptide.
Executive summary:

Results

The test item was dissolved at 100 mM inmilli-Q water after up to 1h12 of heating at 60°C.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

 

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the cysteine peptide. However an interferent peak was observed at the lysine retention time in the co-elution sample prepared with the lysine peptide. As a result, the peptide reactivity of the test item was evaluated according to the cysteine 1:10-only prediction model using the formula described in § Data analysis and calculation.

 

The mean of the percent cysteine depletion was equal to 0%. Accordingly, the test item was considered tohave no or minimalpeptide reactivity. Therefore, the DPRA prediction is considered as negative and the test item may haveno potential to cause skin sensitization.

 

Since precipitate was observed at the end of the incubation with the cysteine peptide, the peptide depletion may be underestimated. Therefore, the conclusion on the lack of reactivity cannot be drawn with sufficient confidence.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September - October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD guideline No. 442E: "In vitro skin sensitization assays addressing the Key Event on activation of dendritic cells on the adverse outcome pathway for Skin sensitization”
Version / remarks:
25 June 2018.
Qualifier:
according to
Guideline:
other: DB-ALM Protocol No. 158: human Cell Line Activation Test (h-CLAT)
Version / remarks:
July 2015
GLP compliance:
yes
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
The objective of the study was to determine the ability of the test item ISOMETHOL to induce an increase in cell surface markers expression in THP-1 cells using the h-CLAT test method.
The reactivity of the test item was evaluated in vitro : it is an alternative method in order to avoid animal study
Details on study design:
The study was divided in two successive phases. First, a Dose-Range Finding assay (DRF) was performed to assess test item toxicity. Secondly, based on cytotoxicity data obtained from the DRF, a concentration series was tested in successive runs in the main tests.
At each phase, all information relating to test item concentrations and run identification were given by the Study Director in the study files and no study plan amendment was issued for that purpose.

Positive control results:
2,4-Dinitrochlorobenzene (DNCB)
Nickel Sulfate (NiSO4)
Positive controls preparation
The positive control DNCB was prepared at the concentration of 8 µg/mL in DMSO as follows:
 on the treatment day, the required quantity of DNCB was mixed with DMSO at the concentration of 2 mg/mL,
 this solution was then 250-fold diluted in cRPMI in order to obtain a 8 µg/mL DNCB stock solution.

The positive control NiSO4 was prepared at the concentration of 200 µg/mL in 0.9% NaCl as follows:
 on the treatment day, the required quantity of NiSO4 was mixed with 0.9% NaCl at the concentration of 10 mg/mL,
 this solution was then 50-fold diluted in cRPMI in order to obtain a 200 µg/mL NiSO4 stock solution.

Both positive control stock solutions were prepared within 4 hours before use, and kept at room temperature and protected from light until use.
Key result
Parameter:
other: RFI for CD86
Run / experiment:
A
Value:
166
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: concentration 833.3µg/L
Key result
Parameter:
other: RFI for CD86
Run / experiment:
A
Value:
160
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: concentration 1000 µg/L
Key result
Parameter:
other: RFI for CD54
Run / experiment:
A
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: concentration 401.9 µg/L
Key result
Parameter:
other: RFI for CD54
Run / experiment:
A
Value:
331
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: concentration >= 578.7 µg/L
Key result
Parameter:
other: RFI for CD54
Run / experiment:
B
Value:
209
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: concentration 833.3 µg/L
Other effects / acceptance of results:
All acceptance criteria were reached in each run. The study was therefore considered as validated.

Individual run interpretation:
A run conclusion is positive if at least one of the conditions below is met:
 RFI of CD86 is ≥ 150 in at least one tested concentration leading to ≥ 50% viability,
 RFI of CD54 is ≥ 200 in at least one tested concentration leading to ≥ 50% viability.


Run A:
 at post-treatment observation, slight to strong test item precipitate was observed at concentrations ≥ 401.88 µg/mL, with a lower cell density at concentrations ≥ 833.33 µg/mL,
 RFI CD86 exceeded the positivity threshold at concentrations ≥ 833.3 µg/mL,
 RFI CD54 exceeded the positivity threshold at the concentration of 401.9 µg/mL and at concentrations ≥ 578.7 µg/mL.

This run A was therefore considered positive.

Run B:
 at post-treatment observation, slight to moderate test item precipitate was observed at concentrations ≥ 694.44 µg/mL,
 RFI CD86 did not exceed the positivity thresholds at any tested concentrations,
 RFI CD54 exceeded the positivity threshold only at the concentration of 833.3 µg/mL.

The run B was therefore considered positive for RFI CD54.

The following results were obtainedin both DRF assays:

at post-treatment observations, strong test item precipitate was observed at the highest tested concentration in the DRF1 and at concentrations    500 µg/mL in the DRF2, associated with a low cell density at the highest concentration in both DRF assays. No cell morphology modification was observed at any tested concentrations,

flow cytometry measurement after Propidium Iodide staining revealed no cell viability decrease below 75% at any tested concentrations.Therefore, no CV75 value was calculated.

.   Based on the results from both DRF assays, no mean CV75 was calculated, and the maximum concentration tested in the main test was therefore1000µg/mL.

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the experimental conditions of this study, the test item, ISOMETHOL, was found to be positive in the h-CLAT test method.
Executive summary:

Results

 Solubility assessment

The test item was found soluble in 0.9% NaCl at the concentration of 100 mg/mL afterheating (1 hour and 30 minutes at 60°C) and sonication (10 minutes).

 Dose-Range Finding

During both DRF assays, no decrease in cell viability (i.e. cell viability < 75%) was noted in test item‑treated wells. No mean CV75 value was therefore calculated, and the highest tested concentration retained for the main test was 1000 µg/mL.

 


Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
january-may 2019
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)
Version / remarks:
22 July 2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%).
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Species and strain: CBA/CaOlaHsd mice
Source: Envigo
San Pietro al Natisone (UD)
Zona Industriale Azzida, 57,
33049 Italy
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD No. 429 Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non-pregnant
Age of animals at starting: 10 weeks old (age-matched, within one week)
Body weight range at starting: 18.6 – 20.7 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: at least 21 days
Note: In the Preliminary Experiment, mice of 11 weeks of age (21.8-22.7 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II polypropylene / polycarbonate
Bedding/Nesting: Bedding and certified nest building material was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 18.4 – 25.9°C
Relative humidity: 22 - 76 %
Ventilation: 15-20 air exchanges/hour
The minimum /maximum values of temperature and relative humidity were recorded twice every day during the acclimatization and experimental phases.

Animals received food (The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study) and tap water from the municipal supply from 500 mL bottles. Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study.
Vehicle:
dimethyl sulphoxide
Concentration:
three groups received sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate (formulated in DMSO) at 10% (w/v), 5% (w/v) and 2% (w/v) concentrations,
- the negative control group received the vehicle (DMSO) only,
- the positive control group received 25 % (w/v) HCA (dissolved in DMSO).
No. of animals per dose:
4 animals per group
Details on study design:
Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD No. 429 Guideline, the best vehicle for the test item was DMSO (Dimethyl sulfoxide). The highest achievable concentration was 10% (w/v). The formulations at 5% (w/v) and 2% (w/v) in DMSO were also suitable for treatment. The formulations appeared to be suspension by visual examination.
The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose) 10% and 5% (w/v) in DMSO. Based on the observations recorded in the preliminary test, the 10% (w/v) dose was selected as top dose for the main test.
The test item formulations were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring the incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25% (w/v) in the relevant vehicle (DMSO) using CBA/CaOlaHsd mice.
No mortality cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 3.9 was noted for HCA in the main experiment). This value was considered to confirm the appropriate performance of the assay.
Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range. Each treated and control group included 4 animals.
Key result
Parameter:
SI
Value:
0.6
Test group / Remarks:
group 1 : test item 10% (w/v) in DMSO
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Key result
Parameter:
SI
Value:
0.6
Test group / Remarks:
group 2: test item 5% (w/v) in DMSO
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
Group 3: test item 2% (w/v) in DMSO)
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Cellular proliferation data / Observations:
EVALUATION OF THE RESULTS
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
Interpretation of Results
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Acceptability of the test
The Local Lymph Node Assay is considered valid if it meets the following criteria:
- the DPN value of the negative (vehicle) control group falls within the range of historical laboratory control data,
- the positive control substance produces a significant lymphoproliferative response increases (SI>3),
- each treated and control group includes at least 4 animals,
- the test item does not cause serious systemic or local toxicity.

The test item was solid, which was formulated in DMSO for the main test. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate is a non-sensitizer. The size of lymph nodes was in good correlation with this conclusion. Based on the observed results, the test item does not need classification according to the GHS or CLP.

CLINICAL OBSERVATION

No mortality or systemic toxicity was observed during the main study. Minimal amount of test item residue was observed on the ears of all animals at 10% (w/v) dose group on Days 2-3. No test item residue was observed in the 5% and 2% (w/v) dose group. There were no indications of any irritancy at the site of application.

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, under the conditions of the present assay, sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate, tested in a suitable vehicle, was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay.
The following classification/labelling is triggered:
Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017: none.
Executive summary:

The aim of this study was to determine the skin sensitisation potential of sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate following dermal exposure in mice. The study was performed with vertebrate animals as the applied regulatory in vitro alternative tests have been run and further testing in vivo was required for classification. Therefore, an in vivo study was run to provide reliable information about the skin sensitisation potential of the test item for regulatory acceptance.

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD No. 429 Guideline, the best vehicle for the test item was DMSO (Dimethyl sulfoxide). The highest achievable concentration was 10% (w/v). The formulations at 5% (w/v) and 2% (w/v) in DMSO were also suitable for treatment. The formulations appeared to be suspension by visual examination.

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose) 10% and 5% (w/v) in DMSO. Based on the observations recorded in the preliminary test, the 10% (w/v) dose was selected as top dose for the main test.

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate (formulated in DMSO) at 10% (w/v), 5% (w/v) and 2% (w/v) concentrations,

- the negative control group received the vehicle (DMSO) only,

- the positive control group received 25 % (w/v) HCA (dissolved in DMSO).

The test item formulations were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring the incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

No mortality or systemic toxicity was observed during the main study. Minimal amount of test item residue was observed on the ears of all animals at 10% (w/v) dose group on Days 2-3. No test item residue was observed in the 5% and 2% (w/v) dose group. There were no indications of any irritancy at the site of application. No test item related effects were observed on the mean body weight changes in the main study.

The stimulation index values were 0.6, 0.6 and 0.7 at concentrations of 10% (w/v), 5% (w/v) and 2% (w/v), respectively.

The result of the positive control substance (α-Hexylcinnamaldehyde, HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response (SI = 3.9) in harmony with historical control data was noted for the positive control chemical, this result confirmed the validity of the assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

First in vitro studies was performed: two studies were negatives and one study was positive. Further information was necessary.

The in vivo (LLNA) study was performed with vertebrate animals as the applied regulatory in vitro alternative tests have been run and further testing in vivo was required for classification. Therefore, an in vivo study was run to provide reliable information about the skin sensitisation potential of the test item for regulatory acceptance.

Justification for classification or non-classification

According to the in vivo study (LLNA), Sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate is non-sensitizer.

The following classification/labelling is triggered: Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017: none.