Registration Dossier

Administrative data

Description of key information

For this endpoint, three Weight of Evidence in chemico/in vitro assays (a DPRA, a h-CLAT and an ARE-test), and a Weight of Evidence in vivo Open Epicutaneous Test (without GLP or OECD, however similar to OECD 406) are available to evaluate the skin sensitising potential of the test item Frambinon methyl ether. Additionally, a Weight of Evidence DEREK QSAR-, QSAR Toolbox-, and a Danish QSAR Database evaluation were carried out on Frambinon Methyl Ether.

In the DPRA the test item was considered as non-senstizer. In the h-CLAT and the ARE assay the test item was positive, but regarded as inconclusive. Therefore, the combined results of the three in chemico/in vitro tests resulted in a negative outcome, as two out of three assays were regarded negative according to the evaluation critera and the test item is considered to be a non skin sensitiser.

In the in vivo Open Epicutaneous Test Frambinon methyl ether was found to be a non-sensitiser up to a concentration of 5%.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
4.2.2016 - 8.4.2016
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))
Version / remarks:
4 February 2015
Deviations:
yes
Remarks:
Adaptations in the report structure and phrasing. These deviations did not influence the quality or integrity of the present study
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
TEST-SUBSTANCE PREPARATION
- Stock solution: 100 mM
- Vehicle: acetonitrile
- Reason for the vehicle: The test substance is soluble in acetonitrile.

CONTROLS
- Positive control: cinnamic aldehyde (100 mM solution in acetonitrile)
- Co-elution control: buffer and test substance without the peptide
- Reference controls were set up in parallel to sample preparation in order to verify the validity of the test run

PEPTIDES
- Synthetic peptides:
-- Cysteine- (C-) containing peptide: Ac-RFAACAA
-- Lysine- (K-) containing peptide: Ac-RFAAKAA

- STOCK SOLUTION:
-- C-containing peptide: 0.667 mM of peptide in pH 7.5 phosphate buffer
-- K-containing peptide: 0.667 mM of peptide in pH 10.2 ammonium acetate buffer
- Ratios
- C-containing peptide: ratio of 1:10 (0.5 mM peptide, 5mM test substance)
- K-containing peptide: ratio of 1:50 (0.5 mM peptide, 25 mM test substance)

EXPERIMENTAL PROCEDURE
- Replicates: 3 for each peptide
- Determination remaining non-depleted peptide concentration: HPLC at 220nm: HPLC analysis
started 24±2 hours after sample preparation and the analysis time was less than 30 hours.
- Calibration samples: samples of a known peptide concentration are measured in parallel

PREPARATIONS SAMPLES
- Calibration sample was prepared from the peptide stock solution in 20% acetonitrile in the respective
buffer using serial concentration: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 or 0.000 mM peptide
- Test-substance samples: samples were incubated at 25°C +/- 2.5°C in the dark for 24 +/- 2 hours and
visually investigated for any precipitate that may occur during the exposure period.
- Preparation of the reference controls: in triplicated in the same way as the test-substance samples described
above but with acetonitrile instead of the test subtance.
- Preparation of co-elution control: in the same way as the test-substance sampled described above
but without the peptide, instead buffer was used.

MEASUREMENT PEPTIDE CONCENTRATIONS
- Method: HPLC Agilent 1200 Series
- Wavelength: 220 nm
- Detection: Diode Array Detector

DATA EVALUATION
- The concentration of cysteine and lysine peptide was determined in each sample calculating the concentration of peptide using linear calibration curves derived from the standard solutions
- Calculation of the peptide depletion: percent peptide depletion = [ 1 - (peptide peak area in the replicate injection / mean peptide peak area in reference control C)] * 100
- Mean peptide depletion for each peptide: C - containing peptide depletion of a test substance (%) =
mean (C - containing peptide depletion of samples 1-3) (%)
- Mean peptide depletion (%): (C - containing peptide depletion (%) + K - containing peptide depletion (%)) / 2

ACCEPTANCE CRITERIA
acceptance criteria of the run
- The standard calibration curve should have an r2 > 0.99.
- The mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD)
for the positive control replicates is <14.9%
- The mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is <11.6%
- The mean peptide concentration of the three reference controls A replicates is 0.50±0.05 mM
- The coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is <15.0%

acceptance criteria of the results of the test item
- The maximum SD for the test chemical replicates is <14.9% for the cysteine PPD
- The maximum SD for the test chemical replicates is <11.6% for the lysine PPD
- The mean peptide concentration of the three reference controls C replicates in the appropiate solvent is 0.50 ± 0.05 mM
Positive control results:
Cinnamic aldehyde showed highly reactivity towards the synthetic peptides
Key result
Run / experiment:
other: C-containing peptide (%)
Parameter:
other: peptide depletion
Value:
1.68
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: minimal reactivity
Key result
Run / experiment:
other: K-containing peptide (%)
Parameter:
other: peptide depletion
Value:
1.1
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: minimal reactivity
Other effects / acceptance of results:
The mean peptide depletion of the positive control was 72.27%.

Results of the Cysteine Peptide Depletion

Sample

Peak area at 220 nm

Peptide concentration
(
mM)

Peptide depletion
(%)

Mean peptide depletion
(%)

SD of peptide depletion (%)

CV of peptide depletion (%)

Positive control

906.7542

0.1345

71.54

72.27

0.90

1.24

892.3105

0.1323

71.99

851.7282

0.1261

73.27

Test item

3199.5308

0.4847

0.00*

1.68

2.06

122.45

3152.0117

0.4775

1.07

3059.0840

0.4633

3.99

*Value was set to 0.00 due to negative depletion

 

 

Results of the Lysine Peptide Depletion

Sample

Peak area at 220 nm

Peptide concentration
(
mM)

Peptide depletion
(%)

Mean peptide depletion
(%)

SD of peptide depletion (%)

CV of peptide depletion (%)

Positive control

1438.2354

0.1957

58.85

63.95

4.50

7.04

1140.3182

0.1549

67.37

1201.1472

0.1632

65.63

Test item

3457.4512

0.4727

1.08

1.10

0.04

3.93

3458.0591

0.4728

1.06

3455.2009

0.4724

1.15

Interpretation of results:
other: CLP/EU GHScriteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards the peptides.
The test item (might = BERICHT) SHOULD be considered as “non-sensitiser”.

The data generated with this method should be considered in the context of an integrated approach such as IATA, combining the result with other complementary information..
Executive summary:

In the current study the skin sensitisation effect of the test item was assessed in an "in chemico" assay according to OECD 442C and under GLP, without significant deviations.

This method quantifies the remaining concentrations of cysteine- or lysine-containing peptides after 24 hours incubation with the test item at 25 +/-2.5 °C. The percentage of depletion of cysteine- and lysine-peptides is calculated and used in a prediction model to assign the test chemical to one of four reactivity classes and in this way discriminate between sensitisers and non-sensitisers.

The test item was dissolved at a 100 mM concentration in acetonitrile and 3 samples of the test item were incubated with a typical concentration of 0.667 mM of cysteine in a pH 7.5 phosphate buffer or 0.667 mM of lysine in a pH 10.2 ammonium acetate buffer. Incubation was done for 24 hours in the dark at 25 +/- 2.5°C. The remaining non-depleted concentrations were determined by HPLC with gradient elution and UV-detector at 220 nm.

The mean C-peptide depletion, caused by the test substance was determined to be 1.68%.

The mean K-peptide depletion, caused by the test substance was determined to be 1.10%.

Based on the results and the prediction model, it is concluded that the test substance shows minimal chemical reactivity

in the DPRA test under the current conditions. The test item might SHOULD be considered as “non-sensitiser”.

The data generated with this method should be considered in the context of an integrated approach.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27.02.2017-23.03.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 442E
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
purity: 99.8%
Details on the study design:
TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line (ATCC TIB-202)

TEST-SUBSTANCE PREPARATION
- Concentrations: experiment 1 and 2: 774.14, 645.12, 537.60, 448.0, 373.33, 311.11, 259.26 and 216.05 μg/mL
- Stock: diluting the highest soluble concentration seven times
- Vehicle: DMSO

CONTROLS
- Solvent control: 0.2% DMSO (v/v) incell culture medium
- Positive control: 2,4-dinitro-chloro-benzene (DNCB); 4.0 μg/mL in 0.2% DMSO in culture medium

MEDIUM
- Culture medium: RPMI 1640: with L-glutamine, 25mM HEPES (Gibco) + 10% FBS + Penicillin/Streptomycin + 2-mercaptoethanol (Gibco)
- FACS Buffer: Phosphate Buffered Saline (DPBS) + 0.1% BSA
-Blocking buffer: FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction)
- Reagent for cytotoxicity test: Propidium iodide (Sigma)

EXPERIMENTAL PROCEDURE
- Replicates: 3
- Experiments: 2
- Exposure period: 24 ± 0.5 hours
- Preparation of cells: THP-1 cells were thawed and cultured in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, streptomycin and 2 -mercaptethanol

ANALYSIS
- FACS: cell staining and flow cytometric analysis 24 ± 0.5 hours after exposure

DATA EVALUATION
- CV75 calculation: Relative survival rate is calculated by linear extrapolation. This value is the substance concentration at which cell viability is 75% compared to the vehicle control.
- Relative cell viability: % relative cell viability = (number of living cells / total number of acquired cells) * 100
- Relative fluorescence intensity: RFI (%) = ( MFI of chemical-treated cells - MFI of chemical-treated isotype control cells) / (MFI of vehicle control cells - MFI of vehicle isotype control cells) * 100

EVALUATION RESULTS
- Positive result: A test is considered to be positive when the dendritic cells are activated meaning that CD86 expression is increased ≥150% and/or CD54 expression increased ≥ 200% in relation to the vehicle control in at least 2 independent experiments (cell vialbility ≥ 50%)
- Negative result: A test is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (=5000 μg/mL for the vehicle culture medium or 2000 μg/mL for 0.2% DMSO in culture medium).

ACCEPTANCE CRITERIA
- Cell viability of vehicle control cells must yield at least 90%
- The cell viability of at least four tested doses of the test item in each run is > 50%
- In the positive control (DNCB), RFI (relative fluorescence intensity)
values of both CD86 and CD54 should exceed the positive criteria (RFI CD86 ≥ 150% and CD54 ≥
200%) and cell viability should be ≥ 50%.
- The RFI values of the solvent control is not >= 150% for CD86 and not >= 200% for CD54
- The MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control is >105%

Remarks on result:
positive indication of skin sensitisation

Results Experiment 1

Sample Conc (mg/ml) Cell Viability (%) CD86 Cell Viability (%) CD54 RFI (%) CD 86 RFI (%) CD54
Medium Control   96.9 97.9 105 82
DMSO Control 0.20% 98.1 97.6 100 100
DNCB 4 85.3 85.4 729 546
Test Item 774.14 67.5 66.8 242 237
645.12 80 80.3 202 193
537.6 88.3 88.6 220 166
448 89.5 89.9 168 142
373.33 93 93.6 159 90
311.11 93.5 93.6 169 111
259.26 94.7 94.8 166 86
216.05 95.9 95.9 140 53

Results Experiment 2

Sample Conc (mg/ml) Cell Viability (%) CD86 Cell Viability (%) CD54 RFI (%) CD 86 RFI (%) CD54
Medium Control   97 97 100 63
DMSO Control 0.20% 97.1 97.2 100 100
DNCB 4 85.8 86.4 423 641
Test Item 774.14 58.5 58.2 223 356
645.12 80.4 81.2 187 264
537.6 84.9 85.6 165 218
448 86.8 88 161 206
373.33 91 91.4 140 173
311.11 91.6 91.6 153 164
259.26 93.5 93.7 151 183
216.05 93.4 94.3 151 131
Interpretation of results:
other: limited relevance for classification
Conclusions:
According to the study report, the test item upregulated the cell surface marker in two independent runs and, therefore, could be considered a skin sensitiser.

The data generated with this method should be considered in the context of an integrated approach (IATA).

OECD Guideline No. 442e in its initial considerations and limitations is read as:
 8. …Therefore, data generated with the h-CLAT method should be considered in the context of integrated approaches, such as IATA, and combined with other complementary information e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods, including read-across from chemical analogues.

Since the cell viability at 774.14 µg/mL was decreased below 75 % in both experiments 1 and 2, the relative fluorescence Intensity (RFI) at these concentrations should not be considered reliable. Considering only values with a viability of >=75 %, it is concluded that CD 54 stayed below the threshold limit of 200 in experiment 1 and should be evaluated as negative. In experiment 2, the values increased above 200 (206, 218, and 264 at 448.0, 537.6, and 645.12 µg/mL, respectively). The according positive control (DNCB) was noted with a RFI of 546 and 641 in the two experiments. CD54 thus showed no consistent positive response in both experiments.
CD86 was noted with values above the threshold limit of 150 in experiment 1 at concentrations between 259.26 and 645.12 µg/mL with RFI values between 159 and 220 (at 537.6 µg/mL), showing a rather weak dose-response. In experiment 2, all RFI values were found to be between 151 and 187 for the same concentration rage. At 373.33 µg/mL, the RFI was 140 and thus below the positivity threshold. The according positive control (DNCB) showed RFI values of 729 and 423, resp., in experiments 1 and 2. The plateau-like dose-response curves of the test item in the lower concentration range and the small increase in RFI values above their according threshold values (CD54: 200, CD86: 150) led to the overall interpretation of the study as inconclusive.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24.05.2016 - 01.08.2016
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)
Version / remarks:
4 February 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany
Type of study:
activation of keratinocytes
Details on the study design:
TEST SYSTEM
- Cell line: Human transgenic keratinocyte cell line derived from HaCaT cells
- Source: KeratinoSens (Givaudan, Switzerland)

TEST-SUBSTANCE PREPARATION
- Concentrations: 2000, 1000, 500, 250, 125, 61.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.98 μg/mL
- Stock solution: 200 mM
- Vehicle: 1% (v/v) DMSO

CONTROLS
- Blank control: 1% DMSO (AppliChem) without cells
- Negative control: 1% DMSO in test item exposure medium
- Positive control: cinnamic aldehyde (CAS 104-55-2, Sigma Aldrich) dissolved in 1% DMSO resulting in a concentration range of 0.4 - 6.4 mM

MEDIUM
- Maintenance medium: Dulbecco's Modified Eagle Medium (GlutaMAX) with 1.0 g/L D-glucose and Na-pyruvate supplemented with 10% FBS, 1% geneticin (500 μg/mL final conc.)
- Assay medium: Dulbecco's Modified Eagle MEdium (GlutaMAX) with 1.0 g/L D-glucose and Na-pyruvate supplemented with 10% FBS
- Test item exposure medium: Dulbecco's Modified Eagle MEdium (GlutaMAX) with 1.0 g/L D-glucose and Na-pyruvate supplemented with 1% FBS

EXPERIMENTAL PROCEDURE
- Replicates: 3
- Experiments: 3
- Exposure period: 48 hours at 37 ± 1°C and 5% CO2
Luciferase assay:
- Washing cells: once with DPBS
- Buffer: passive lysis buffer
- Incubation: 20 min, room temperature, in darkness
MTT assay:
- Incubation: 4 hours at 37 ± 1°C and 5% CO2

ANALYSIS
- Absorbance measurement: OD at a wavelength of 600 nm

DATA EVALUATION
- % relative cell viability (%) = (absorbance of test substance treated cells - absorbance of blank) / (absorbance of mean vehicle control treated cells - absorbance of blank) * 100
- Luciferase fold induction = (test substance treated cells - background without cells) / (mean vehicle control treated cells - background without cells)

ACCEPTANCE CRITERIA
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 in at least one of the test concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 μM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO is < 20% in each repetition which consists in 6 wells

EVALUATION
The test item is considered positive in accordance with UN GHS Category1 if the following conditions were met in at least two independently prepared test repetitions:
- Imax is > 1.5 fold increased and statistical significant (p < 0.05) compared to the negative control
- cell viability > 70% at the lowest concentration with an induction of luciferase activity > 1.5
- EC1.5 value is < 1000 μM
- an apparent overall dose-response for luciferase induction
Positive control results:
luciferase activity induced at a concentration of 64 μM was between 2 and 8
Run / experiment:
other: 1
Parameter:
other: fold induction
Remarks:
at 1000 μM
Value:
2.05
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
other: 2
Parameter:
other: fold induction
Remarks:
at 1000 μM
Value:
2.38
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
other: 3
Parameter:
other: fold induction
Remarks:
at 1000 μM
Value:
2.13
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
The controls confirmed the validity of the study

Results of the cytotoxicity measurements

  Concentration (μM) Mean Cell Viability (%)
Mean SD
Solvent Control   100.00 0
Positive Control 4.0 105.7 10.9
8.0 109.3 20.1
16.0 115.8 18.8
32.0 125.2 13.9
64.0 123.5 16.5
Test Item 0.98 103.1 4.6
1.95 96.4 2.1
3.91 100.3 6.5
7.81 97.2 9.5
15.63 106.5 7.1
31.25 105.5 16.1
62.50 102.6 14.1
125.00 105.6 12.0
250.00 107.1 14.1
500.00 112.3 17.4
1000.00 122.0 8.4
2000.00 120.3 5.4

Induction of luciferase activity - overall induction

  Concentration (μM) Fold induction
Experiment 1 Experiment 2 Experiment 3 Mean SD
Solvent Control   1.00 1.00 1.00 1.00 0.00
Positive Control 4.0 1.15 1.11 1.28 1.18 0.09
8.0 1.30 1.24 1.40 1.31 0.08
16.0 1.55 1.49 1.69 1.58* 0.10
32.0 2.29 1.99 2.57 2.28* 0.29
64.0 5.07 4.26 5.07 4.80* 0.47
Test Item 0.98 1.22 1.11 1.01 1.11 0.10
1.95 1.11 1.09 1.01 1.07 0.06
3.91 1.12 0.77 0.96 0.95 0.18
7.81 1.19 1.17 0.93 1.10 0.14
15.63 1.16 1.12 0.98 1.09 0.10
31.25 1.19 1.08 0.93 1.06 0.13
62.50 1.28 1.33 1.13 1.25 0.11
125.00 1.49 1.34 1.29 1.37 0.10
250.00 1.91 1.49 1.59 1.66* 0.22
500.00 1.90 2.11 1.86 1.95* 0.13
1000.00 2.05 2.38 2.13 2.19* 0.17
2000.00 2.04 2.28 2.08 2.13* 0.13

* = significant induction according to Student's T-test, p < 0.05

Interpretation of results:
other: limited relevance for classification
Conclusions:
According to the study report, the test item induced luciferase activity in the KeratioSens cell line in at least two independent runs and therefore could be considered a sensitiser.

The data generated with this method should be considered in the context of an integrated approach (IATA).

The paragraphs nos. 12 and 13 of Guideline OECD no. 442 d, indicate that there could be the possibility that this test may be interpreted as wrongly positive:

§ 12.Test chemicals that do not act as a sensitiser but are nevertheless chemical stressors may lead on the other handto false positive results (14). Furthermore, highly cytotoxic test chemicals cannot always be reliablyassessed. Finally, test chemicals that interfere with the luciferase enzyme can confound the activity ofluciferase in cell-based assays causing either apparent inhibition or increased luminescence (22).
 
§ 13.In addition to supporting discrimination between skin sensitisers and non-sensitisers, theKeratinoSensTM assay also provides concentration-response information that maypotentially contribute tothe assessment of sensitising potency when used in integrated approaches such as IATA (19).However,further work preferably based on reliable human data is required to determine how KeratinoSensTM resultscan contribute to potency assessment (24) and to sub-categorisation of sensitisers according to UN GHS(1).

 14. EURL-ECVAM (2014). Recommendation on the KeratinoSensTM assay for skin sensitisationtesting, 42 pp. Available at: http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvamrecommendations/recommendation-keratinosens-skin-sensitisation.
 19. Jaworska J., Dancik Y., Kern P., Gerberick F., Natsch A., 2013. Bayesian integrated testingstrategy to assess skin sensitization potency: from theory to practice. J Appl Toxicol. 33, 1353-1364.
 22. Thorne N., Inglese J., Auld D.S. (2010). Illuminating Insights into Firefly Luciferase and OtherBioluminescent Reporters Used in Chemical Biology. Chemistry and Biology 17, 646-657.
 24. ECETOC (2003). Contact sensitization: Classification according to potency. European Centre for Ecotoxicology & Toxicology of Chemicals (Technical Report No. 87).

It should be taken into consideration that the test substance showed no clear dose-response relationship. Although luciferase induction was larger than 1.5fold over nearly a ten-fold concentration range, the values only increased from 1.7 to 2.1 fold. In comparison, the positive control showed a much steeper dose-response curve. Also, the test substance caused an increase in cell viability (measured as metabolic capacity to reduce MTT) at the positive concentrations (up to 123.1 % and 126.6 % in experiments 1 and 2). This increase in cell number may have contributed to the higher luciferase activity even in the absence of specific cell activation. Since for the test item the increase in Luciferase activity was mild, showed only a shallow dose-response relationship, which correlated with an increase in cell viability, the biological relevance of the positive test results are considered questionable.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

For the skin sensitization endpoint three in chemico/in vitro tests, as well as a study in Guinea pigs and a study in humans are available on Frambinon® methyl ether. In addition, several QSAR predictions were performed with the chemical structure. All information was considered in a weight-of-evidence approach.

 

DPRA test

In the Direct Peptide Reactivity Assay (DPRA) (OECD 442C, Symrise 2016, study #2015449) the reactivity of the test item towards synthetic cysteine (C) or lysine (K) containing peptides was evaluated. Incubation of the synthetic peptides with 3-methylbutylisovalerate was done for 24 hours and the remaining concentrations of cysteine- or lysine-containing peptides were determined.

The mean C-peptide depletion, caused by the test substance was determined to be 1.68 %.

The mean K-peptide depletion, caused by the test substance was determined to be 1.1 %.

Based on the results and the prediction model, it is concluded that the test substance shows no chemical reactivity in the DPRA test under the current conditions. Therefore, the test item can be considered to have no skin sensitising properties in this study.

Since peptide-binding is the hallmark for low-molecular weight skin sensitizers, the clear negative result in the assay is attributed a high weight of evidence.

 

ARE (LuSens) test

In the ARE Reporter (LuSens) assay (OECD 442D, Symrise 2016, study #2015450) the keratinocyte activating potential of the test item was evaluated. The test substance was incubated with a luciferase reporter cell line (LuSens cell line) for 48 hours and the antioxidant response element (ARE) dependent luciferase activity was measured, in parallel to a MTT assay to assess the cytotoxicity.

A dose-response for luciferase activity induction was observed for each individual run a well as for an overall luciferase activity induction. The controls confirmed the validity of the study.

Under the condition of this study the test item induced the luciferase activity in the transgenic KeratinoSens cell line in at least two independent experiment runs. Therefore, the test item was considered to have shown a sensitising property in this study by the study authors.

However, it should be taken into consideration that the test substance showed no clear dose-response relationship. Although luciferase induction was larger than 1.5fold over nearly a ten-fold concentration range, the values only increased from 1.7 to 2.1 fold. In comparison, the positive control showed a much steeper dose-response curve. Also, the test substance caused an increase in cell viability (measured as metabolic capacity to reduce MTT) at the positive concentrations (up to 123.1 % and 126.6 % in experiments 1 and 2). This increase in cell number may have contributed to the higher luciferase activity even in the absence of specific cell activation. Since for Frambinon® methyl ether the increase in Luciferase activity was mild, showed only a shallow dose-response relationship, which correlated with an increase in cell viability, the biological relevance of the positive test results are considered questionable. Therefore, this result is attributed a medium weight of evidence.

 

h-CLAT test

The potential of the test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the human dendritic Cell Line Activation Test (h-CLAT) (OECD 442E, Symrise 2016, study #2015451). The test substance dissolved in DMSO was incubated with the human promonocytic cell line THP-1 for 24 hours at 37°C and the membrane markers expression was measured by flow cytometry through staining with FITC labeled anti-human-CD86/anti human CD54 antibody and propidium iodide.

Since the expression of both cellular marker exceeded the threshold in two independent experiments (CD86: 202 and 187 %; CD54: 193 % and 264 % in experiment 1 and 2, respectively) the test item was considered positive by the study authors.

However, since the cell viability at 774.14 µg/mL was decreased below 75 % in both experiments 1 and 2, the relative fluorescence Intensity (RFI) at these concentrations should not be considered reliable. Considering only values with a viability of >=75 %, it is concluded that CD 54 stayed below the threshold limit of 200 in experiment 1 and should be evaluated as negative. In experiment 2, the values increased above 200 (206, 218, and 264 at 448.0, 537.6, and 645.12 µg/mL, respectively). The according positive control (DNCB) was noted with a RFI of 546 and 641 in the two experiments. CD54 thus showed no consistent positive response in both experiments.

CD86 was noted with values above the threshold limit of 150 in experiment 1 at concentrations between 259.26 and 645.12 µg/mL with RFI values between 159 and 220 (at 537.6 µg/mL), showing a rather weak dose-response. In experiment 2, all RFI values were found to be between 151 and 187 for the same concentration rage. At 373.33 µg/mL, the RFI was 140 and thus below the positivity threshold. The according positive control (DNCB) showed RFI values of 729 and 423, resp., in experiments 1 and 2. The plateau-like dose-response curves of the test item in the lower concentration range and the small increase in RFI values above their according threshold values (CD54: 200, CD86: 150) led to the overall interpretation of the study as inconclusive. Therefore, this result is attributed a medium weight of evidence.

 

In vivo Guinea pig study

The Open Epicutaneous Test (OET) was used to evaluate the skin sensitizing effect of the test substance (Klecak et al., 1985, Curr. Probl. Derm. 14, 152-171). The study was carried out in 1985 and thus not according to GLP or OECD, however, it is similar to the version of OECD 406 which came into force in 1992. Various test concentrations were used and per group at least 6 guinea pigs were included. The maximum concentration used was 5 % and the test item was found to be a non-sensitiser as no skin reactions were observed in the study. Due to the limitations in the study method (open application vs. semi-occluded application in the Buehler method) and in reporting, this study is attributed a medium weight of evidence.

 

Human data

In a human maximisation study (Kligman, 1973, unpublished report to RIFM) in 25 male volunteers, the test substance was applied at 5 % in petrolatum to the volunteers' forearms for 5 alternate-day 48-hour patches under occlusion. The patch sites were pre-treated for 24 hours with 5 % aqueous sodium lauryl sulfate (SLS) under occlusion. After a 10-day rest period, challenge patches were applied under occlusion to fresh sites for 48 hours. Before the challenge applications were made, the sites were pre-treated for 1 hour with 10 % aqueous SLS. Reactions were read at 48 hours and 72 hours. No skin reactions were observed (0/25) in any subject. Therefore the substance showed no evidence of skin sensitization in humans. While the number of subjects is limited, the conditions of exposure were rather drastic, i.e. were highly promoting an induction of sensitization. Therefore, the negative result of the test is attributed a high weight of evidence.

 

In the PubMed and Toxnet literature databases, no case reports or other publications reporting skin sensitizing effects of the test item (searched by CAS number on 27th March 2018). Since the use of the substance in consumer products is limited and the substance is not part of a standard patch test series used in dermatologic examinations, this result is attributed a low weight of evidence.

 

QSAR predictions

The test item structure did not match any structural alerts or examples for skin sensitisation in Derek (Software used: Derek Nexus: 6.0.1 knowledge base 2018 1.1). Additionally, the test item structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. Based on the DEREK QSAR prediction, the substance is considered to be a non-sensitizer.

 

In an analogous substance approach, the test item structure was predicted to be negative for skin sensitization (Software used: OECD QSAR Toolbox 4.1 TPRF v4.1). Based on the OECD Toolbox QSAR prediction, the substance is considered to be a non-sensitizer.

 

In a set of QSAR tools (CASE Ultra, Leadscope and SciQSAR, available via the Danish EPA (QSAR database accessed 23.3.2018:http://qsar.food.dtu.dk), the test item was considered negative for skin sensitization and within the applicability domain for all QSARs. Based on the Danish QSAR Database prediction, the substance is considered to be a non-sensitizer.

 

The consistent prediction as non-skin sensitizer in several QSARs is attributed a high weight of evidence.

 

 

Conclusion

Three pieces of evidence that were given a high weight indicated that the substance is not a skin sensitizer and of pieces of evidence given a medium weight, one suggested no and two suggested a sensitization potential of the substance. Overall, it is concluded that Frambinon® methyl ether has no skin sensitizing properties in humans (or experimental animals) and therefore does not need to be classified as skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Overall, the weight of the evidence indicates that the test item has no skin sensitizing properties in humans (or experimental animals) and therefore does not need to be classified as skin sensitizer in accordance with CLP EU regulation No. 1272/2008.