Registration Dossier

Administrative data

Description of key information

Skin sensitisation (similar to OECD TG 406): sensitising

Skin sensitisation (DEREK Nexus prediction): sensitising (EC3 = 17%)

Skin sensitisation (DPRA, OECD 442C): negative, no indication for sensitisation

Skin sensitisation (KeratinoSens, OECD 442D): negative, no indication for sensitisation

Skin sensitisation (h-CLAT, OECD 442E): positive, indication for sensitisation

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 24, 1980 - February 21 , 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Challenge was done with a slightly irritating concentration and a challenge control was not included.
Justification for type of information:
Buehler test is available from 1980
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Study performed prior to introduction of guideline.
Deviations:
yes
Remarks:
irritant concentration (5%) used for challenge, the negative control group is not challenged with the test material and therefore irritation and sensitisation is difficult to distinguish; 15 instead of 20 animals used in treatment group.
GLP compliance:
no
Remarks:
pre-GLP
Type of study:
Buehler test
Justification for non-LLNA method:
A Bueler test from 1980 is available, which can be used for the assessment of skin sensitisation.
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Murphy Breeding Laboratories
- Females (if applicable) nulliparous and non-pregnant: not specified
- Weight: 200 - 300 grams
- Housing: singly in wire mesh cages suspended above the droppings
- Diet: Purina Guinea Pig Chow, ad libitum
- Water (e.g. ad libitum): The animals were maintained on medicated water containing 4% of sulfaethoxypyridazine (6.25% S.E.Z. , American Cyanamid) for four days. At the end of this period they were furnished with non-medicated water ad libitum
- Acclimation period: minimum 4 days

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Route:
epicutaneous, occlusive
Vehicle:
other: SDA39C
Concentration / amount:
5% v/v
Day(s)/duration:
3 exposures (once a week) of 6 hours per day
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: SDA39C
Concentration / amount:
5% v/v
Day(s)/duration:
6 hours (2 weeks after last induction)
Adequacy of challenge:
other: same concentration as used for induction (slightly irritant) and therefore skin sensitisation results may also be due to skin irritation.
No. of animals per dose:
Dose-selection: 4
Test group: 15
Positive control: 10
Negative control: 5
Details on study design:
RANGE FINDING TESTS:
Prior to the induction phase of the study the test material was applied to four guinea pigs to determine the highest non-irritating concentration which could be applied for the induction and the primary challenge. For this purpose the test substance was tested as 10%, 5%, 2.5% and 1.3% v/v solutions in SDA39C. On the day before applications were made the backs of the guinea pigs were clipped with electric clippers. The four concentrations were tested the following day on each guinea pig (0.4 mL on each patch).

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 3 weeks
- Test groups: 15 animals
- Control group: 10 animals for positive control, 5 for negative control
- Site: upper left quadrant of the back
- Frequency of applications: once/week
- Duration: 6 hours
- Concentrations: test material as a 5% v/v solution in SDA39C for test group and 100% SDA39C for negative control group

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 1
- Exposure period: 6 hours
- Test groups: 15 animals
- Control group: 10 animals for positive control, 5 for negative control
- Site: lower left quadrant of the back
- Concentrations: test material as a 5% v/v solution in SDA39C for test group and 100% SDA39C for negative control group
- Evaluation (hr after challenge): 24 and 48 hrs
Challenge controls:
Not included
Positive control substance(s):
yes
Remarks:
DNCB, for information on concentration see Positive control results.
Positive control results:
At induction and challenge the positive control was tested as a 10% v/v solution in SDA39C as described in the report. However 0.1% positive control is also mentioned in the report. In view of DNCB being a strong sensitizer the latter seems applicable.

Results:
For the positive control animals one animal showed severe erythema (grade 3), in six animals moderate erythema was observed (grade 2), and three animals developed slight confluent or moderate patchy erythema (grade 1) at the 24-hour reading. At the 48-hour reading two cases of severe erythema (grade 3) were noted, three moderate erythema (grade 2) and five cases of slight confluent or moderate patchy erythema (grade 1) was noted at the 48-hour reading.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5% v/v
No. with + reactions:
9
Total no. in group:
15
Clinical observations:
Five animals with moderate erythema (grade 2), four animals with slight confluent or moderate patchy erythema (grade 1) and six slightly patchy erythema (grade +/-)
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5% v/v
No. with + reactions:
9
Total no. in group:
15
Clinical observations:
Two animals with moderate erythema (grade 2), seven animals with slight confluent or moderate patchy erythema (grade 1), five animals developed slightly patchy erythema (+/-) and one had no reaction (grade 0).
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.1%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
One animal showed severe erythema (grade 3), in six animals moderate erythema was observed (grade 2), and three animals developed slight confluent or moderate patchy erythema (grade 1).
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Two cases of severe erythema (grade 3) were noted, three moderate erythema (grade 2) and five cases of slight confluent or moderate patchy erythema (grade 1).
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation

A preliminary irritation test was performed with 10, 5, 2.5 and 1.3% test substance in SDA 39C. At 10%, 2/4 animals exhibited slight confluent or moderate patchy erythema (grade 1) and 1/4 animals exhibited slightly patchy erythema (grade +/-). At 5%, 2/4 animals exhibited slightly patchy erythema (grade +/-), while at 2.5% and 1.3% no effects were observed.

Induction and challenge concentrations were 5% test material in SDA 39C.

Under the conditions of the study, 5% test material in vehicle SDA 39C produced 9/15 positive responses (1 and 2 grades) at the 24 and 48 hour readings of the test animals. When considering ± responses (slightly patchy erythema) also as a positive result, 15/15 animals responded at the 24 hour reading and 14/15 at the 48 hour reading. In view of the irritation seen during the preliminary test at 5% and the fact that the challenge is also conducted at 5% the sensitization effects may be difficult to separate from irritation. Though the results indicate some sensitization, also because similar scores were seen at 24 and 48 hours, the severity of the sensitisation is inconclusive.

Interpretation of results:
other: Skin sensitising, Category 1
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
The results of this Buehler assay with Dimeth Cyclormol indicate that the material is a sensitiser in view of the positive results in at least 9/15 animals. Although this result may be confounded with irritancy the substance is considered a skin sensitiser because the erythema remained after 48 hours. The skin sensitisation potency is however inconclusive.
Executive summary:

A Buehler test was performed in accordance with a method similar to OECD TG 406 (performed prior to guideline introduction). The study was rated Klimisch 2, firstly because irritant concentrations were used during the challenge. Secondly because the animals in the challenge control (non-induced) animals were exposed to the vehicle instead of the 5% test substance concentration. The irritant effects cannot be distinguished from the skin sensitisation effects. A preliminary irritation test was performed with 10, 5, 2.5 and 1.3% test substance in SDA 39C. At 10%, 2/4 animals exhibited slight confluent or moderate patchy erythema and 1/4 animals exhibited slightly patchy erythema. At 5%, 2/4 animals exhibited slightly patchy erythema, while at 2.5% and 1.3% no effects were observed. Induction and challenge concentrations selected were 5% test material in SDA 39C, which were applied under occlusion. The challenge concentration of 5% test material produced 9/15 positive responses (grades 1: slight confluent or moderate patchy erythema and grades 2: moderate erythema) at the 24 and 48 hour readings of the test animals. When considering ± responses (slightly patchy erythema) also positive, 15/15 animals responded positive at the 24 hour reading and 14/15 at the 48 hour reading. The positive reactions in most animals and being consistent at 48 hours the substance is considered a sensitiser. The potency remains inconclusive because Buehler tests are not developed to present potency and because the irritation of the substance may have confounded the skin sensitisation.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
3 March 2017 - 10 March 2017
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))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
15/03/2017
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
Information from in vitro/in chemico test method(s) are recognised according to Article 13(3) of regulation (EC) No 1907/2006.
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:
Synthetic peptide containing cysteine: Ac-RFAACAA-COOH, lot number 1556171, purity 95% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).
Synthetic peptide containing lysine: AC-RFAAKAA-COOH, lot number 1556172 , purity 94% (by HPLC), supplied by AnaSpec,stored frozen (-10°C to -30°C).

Positive control: cinnamic aldehyde lot number MKBR2427V, purity > 95%.

Preparation of peptide stock solutions:
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (for cysteine, 100 mM phosphate buffer pH 7.5, for lysine 100 mM ammonium acetate buffer pH 10.2).

Preparation of peptide calibration standards:
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Reference (Stability) Controls and Precision Controls:
Reference (stability) controls and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile. These were injected throughout the analytical run to confirm consistency of peptide response throughout each analytical run.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls:
A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials. Cysteine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in more acetonitrile and cysteine peptide stock solution. The final sample concentration was 5 mM of the test substance, 0.5 mM cysteine.
In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 5 mM with 0.5 mM cysteine.
The co-elution control sample contained 5 mM of the test substance in phosphate buffer solution. An additional control sample of 5 mM of the test substance in acetonitrile was also prepared so as to positively identify peak(s) of the test item in the co-elution control so confirming that the test item had not evaporated away during incubation and injection of the samples on the HPLC.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls:
A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials. Lysine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in lysine peptide stock solution. The final sample concentration was 25 mM of the test substance, 0.5 mM lysine.
In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 25 mM with 0.5 mM lysine. The co-elution control sample contained 25 mM of the test substance in ammonium acetate buffer solution. An additional control sample of 25 mM of the test substance in acetonitrile was also prepared so as to positively identify peak(s) of the test item in the co-elution control so confirming that the test item had not evaporated away during incubation and injection of the samples on the HPLC.

Incubation:
The appearance of the test substance, positive control samples and co-elution controls in the HPLC vials was documented following preparation with the vials then placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis:
The concentration of both the cysteine and lysine peptides in the presence of the test substance and the associated positive controls were quantified by HPLC using UV detection.
Equipment: HPLC Waters Alliance 2695 separation module and 2487 dual wavelength detector.
Column: Agilent Zorbax SB C18, 3.5 µm, 100 × 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30 °C
Sample temperature: 25 °C
Mobile phase A: 0.1% trifluoroacetic acid in water
Mobile phase B: 0.085% trifluoroacetic acid in acetonitrile
Flow rate: 0.35 mL/minute
Detector wavelength: UV, 220 nm
Injection volume: 2 μL
Run time: 30 minutes
Approximate retention time (cysteine): 11 minutes
Approximate retention time (lysine): 7 minutes

Calculations:
The peak area response for each peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:

% peptide depletion = 100 - [(Peptide peak area in replicate depletion samples x 100) / (Mean peptide peak area of reference (stability) control samples)]

Positive control results:
71.8% depletion (SD = 0.43%, n = 3) and 58.4% depletion (SD = 0.45%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.
Key result
Parameter:
other: cysteine depletion, %
Run / experiment:
Main experiment
Value:
-0.786
Vehicle controls validity:
valid
Remarks:
stability and precision controls
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
Concentrations of the positive control are outside of the range established during the validation study. No impact expected on the data reporting as the individual and overall mean depletion values are within the acceptance criteria (60.8-100%).
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: lysine depletion, %
Run / experiment:
main experiment
Value:
-0.875
Vehicle controls validity:
valid
Remarks:
stability and precision controls
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, criteria for reference (stability) controls and precision controls of both peptides were met (CV 0.87%, n = 6 and CV 0.45%, n = 6, for cysteine and lysine, respectively, at 0.50 mM).
- Acceptance criteria met for positive control: yes, 71.8% depletion (SD 0.43%, n = 3) and 58.4% depletion (SD 0.45%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.
- Acceptance criteria met for variability between replicate measurements: yes, SD 0.60% and 0.68%, respectively, for cysteine and lysine depletion by the test item.

TEST SUBSTANCE RESULTS:

Mean depletion of -0.786% and -0.875% was observed for the test substance with cysteine and lysine peptides, respectively. With the test substance not reacting with the cysteine nor lysine peptide it is classed as “no to minimal reactivity”, hence the DPRA prediction is negative.
No co-elution peaks were observed in either the Cysteine or Lysine assays.
Interpretation of results:
other: DPRA was negative
Conclusions:
It can be concluded that this DPRA test is valid and with effectively no depletion of either Cysteine nor Lysine peptide in the presence of the test item, the test item was classified in the "no or minimal reactivity" class based on the DPRA prediction model and is thus negative in the DPRA.
Executive summary:

In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. Solutions of the test substance were successfully analysed by the validated DPRA analytical method. In the Cysteine reactivity assay, Cysteine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM Dimeth Cyclormol solution in more acetonitrile and Cysteine peptide stock solution. The final sample concentration was 5 mM of Dimeth Cyclormol, 0.5 mM Cysteine. In the Lysine reactivity assay, Lysine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM Dimeth Cyclormol solution in Lysine peptide stock solution. The final sample concentration was 25 mM of Dimeth Cyclormol, 0.5 mM Lysine. The individual peptide depletion results for Dimeth Cyclormol were -0.786% (Cysteine) and -0.875% (Lysine). The mean result is therefore considered zero. All analytical acceptance criteria of the test were met. No co-elution peaks were observed in either the Cysteine or Lysine assays. With effectively no depletion of either peptide in the presence of the test item, Dimeth Cyclormol was classified in the “no to minimal reactivity” class based on the DPRA prediction model and was thus considered to be negative in the DPRA.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
01 May, 2017 - 19 May, 2017
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)
Version / remarks:
February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
Information from in vitro/in chemico test method(s) are recognised according to Article 13(3) of regulation (EC) No 1907/2006.
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:

Controls:
Positive control: ethylene dimethacrylate glycol, 7.81-250 µM, tested in triplicate, final concentration DMSO of 1%
Negative control: DMSO (vehicle) (1% in exposure medium)
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values

Number of replicates: two independent experiments, each concentration tested in triplicate for the luciferase activity measurements, one parallel replicate for MTT cell viability assay.

Test system:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25, the passage number used was 21 in experiment 1 and 25 in experiment 2) and are employed for routine testing using the appropriate maintenance medium. Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator.

Test item preparation:
No correction was made for the composition/purity of the test item.
The test item was dissolved in DMSO to a final concentration of 200 mM (clear colourless solution). The 100-fold dilution in DMEM of the 200 mM stock showed no precipitation (final concentration 2000 μM). This concentration was selected as highest concentration for the main assay (highest concentration required by the guideline). The stock and spike solutions were diluted 25-fold with exposure medium (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.98 μM (final concentration DMSO of 1%). All formulations formed a clear solution at the start and end of the incubation period. All concentrations of the test item were tested in triplicate. Two independent experiments were performed.

Media:
Basic medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
Maintenance medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 µg/ml).
Exposure medium
Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

Treatment of cells:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours in a humid atmosphere of 80 - 100% (actual range 59 – 86 %) at 37.0 ± 1.0°C (actual range 36.5 – 37.5°C), in the presence of 5% ± 0.5%CO2.

Luciferase acitivity measurement:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity assessment:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed (by adding 10% SDS solution to each well) overnight. After shaking, the absorption is measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Data analysis:
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.

Equation 1: Fold induction= (Lsample - Lblank)/(Lsolvent - Lblank)
Where:
Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and no treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative) control

The EC1.5 is calculated by linear interpolation according to Equation 2, and the overall EC1.5 is calculated as the mean of the individual repetitions.

Equation 2: EC1.5 = (Cb - Ca) x [(1.5 - Ia) / (Ib - Ia)] + Ca
Where:
Ca is the lowest concentration in μM with > 1.5 fold induction
Cb is the highest concentration in μM with < 1.5 fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5 fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells)

Viability is calculated by Equation 3:
Equation 3: Viability = 100 x [(Vsample - Vblank) / (Vsolvent - V blank)]
Where:
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control
Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.

In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

Data intepretation:
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 µM).
• The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
• Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

Positive control results:
Experiment 1: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 52.66 and the EC1.5 14.1 µM.
Experiment 2: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.53 and the EC1.5 24.1 µM.
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
1.19
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
1.22
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
The test substance showed toxicity (IC30 values of 559 μM and 524 μM and IC50 values of 685 μM and 660 μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.19-fold and 1.22-fold in experiment 1 and 2 respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes. The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 μM (14 μM and 24 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (52.7-fold and 4.53-fold in experiment 1 and 2, respectively). The difference in Imax between the two experiments is not expected to have an impact on the results of the test item since all acceptance criteria of the tests were passed and the tests were thus fully valid.
- Acceptance criteria met for variability between replicate measurements: yes. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% in each repetition (respectively 16.5% and 9.6% for experiment 1 and 2).

Table 1. Overview luminescence induction and cell viability of the test substance in Experiments 1 and 2

Concentration (μM)

0.98

1.95

3.91

7.81

15.6

31.3

62.5

125

250

500

1000

2000

Exp 1 luminescence

0.82

0.82

0.79

0.79

0.86

0.84

0.80

0.88

0.88

1.19

0.00

0.01

Exp 1

Viability (%)

98.9

99.8

108.7

104.2

104.0

108.6

124.2

127.2

125.8

79.4

0.1

0.3

Exp 2 luminescence

0.63

0.74

0.86

0.90

0.95

0.92

0.98

1.07

1.17

1.22

0.02

0.03

Exp 2

Viability (%)

95.0

87.7

91.6

113.8

96.3

85.6

98.1

96.7

89.4

73.5

-0.1

0.0

  

Table 2. Overview of luminescence induction and cell viability positive control Ethylene dimethacrylate glycol in Experiments 1 and 2

Concentration (μM)

7.81

15.6

31.3

62.5

125

250

Exp 1 luminescence

1.14

1.59***

2.08***

3.39***

7.73**

52.66***

Exp 1

Viability (%)

111.3

120.2

140.8

133.9

109.4

73.7

Exp 2 luminescence

1.10

1.24

1.72***

2.27***

2.76***

4.53***

Exp 2

Viability (%)

82.9

90.9

87.8

103.8

100.7

92.7

** p<0.005 Students t-test

*** p<0.001 Students t-test

Table 3. Overview of EC1.5, Imax, IC30 and IC50 values

 

EC1.5 (μM)

Imax

IC30 (μM)

IC50 (μM)

Test item experiment 1

NA

1.19

559.3

685.4

Test item experiment 2

NA

1.22

523.8

659.6

Pos. control experiment 1

14.1

52.66

NA

NA

Pos. control experiment 2

24.1

4.53

NA

NA

 

NA = Not applicable

Interpretation of results:
other: KeratinoSens assay was negative
Conclusions:
In a GLP-compliant guideline study, the test substance did not cause a biologically relevant induction in luciferase activity in Keratinosens assay. Based on this, the test substance is classified as negative under the experimental conditions in this assay.
Executive summary:

The GLP-compliant in vitro KeratinoSens test (ARE-Nrf2 luciferase reporter assay) was performed in accordance with OECD guideline 442D to assess the skin sensitising potential of the test substance. Two independent experiments were performed. The test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). Ethylene dimethacrylate glycol was used as a positive control. The acceptance criteria were met in both experiments.

The test substance showed toxicity (IC30 values of 559 μM and 524 μM and IC50 values of 685 μM and 660 μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.19-fold and 1.22-fold in experiment 1 and 2, respectively. In conclusion, the current study was valid and the test substance is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction; no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) were observed at test concentrations of ≥ 1000 µM.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
February 27, 2017 - May 05, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD guideline 442E: In vitro skin sensitization: human Cell Line Activation Test (h-CLAT)
Version / remarks:
July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
September 14, 2015
Type of study:
other: human Cell Line Activation Test (h-CLAT)
Justification for non-LLNA method:
Information from in vitro/in chemico test method(s) are recognised according to Article 13(3) of regulation (EC) No 1907/2006.
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
Solvent: test substance was solved in culture medium.
Positive control: DNCB in DMSO diluted with culture medium to a final concentration of 2 and 3 μg DNCB/mL, (DMSO final concentration 0.2%)

Cell line: THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers. The cell density should not exceed 1 × 10^6 cells/mL. The passage numbers of the used THP-1 cells was 13 in both XTT assays and 18, 19 and 21 in the h-CLAT for runs 1, 2 and 3, respectively.
Culture medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) was used to culture the cells during the assay.
Preparation and seeding of THP-1 cells: On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 μL with a cell density of 0.9 - 1 × 10^6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate. For the main experiment (h-CLAT) 0.9- 1 × 10^6 cells/well in a volume of 500 μL was seeded in a 24-well plate before the treatment.

Dose finding assay: The doses investigated in the main experiment (h-CLAT) were determined with two XTT tests, instead of flow cytometry recommended by the guideline. The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl)-(3,4-tetrazolium)–bis-(4–methoxy–6-nitro)-benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. Two independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT). CV75 is defined as the concentration of toxicant required to reduce the relative absorbance to 75% of the solvent control and is calculated as:
CV75 = Conc.>75 - {[(Conc.>75 - Conc.<75) x (%>75 - 75)]/(%>75 - %<75)}, where:
a) Conc.>75 = maximal measured concentration with the % of solvent control > 75%
b) Conc.<75 = minimal measured concentration with the % of solvent control < 75%
c) %>75 = relative absorpbance at a) in %
d) %<75 = relative absorpbance at b) in %
Test item preparation: immediately prior to start the substance was dissolved in culture medium. The maximum concentration of test item was 1250 μg/mL in culture medium, as tested by a solubility test. For the XTT test (dose finding assay) eight concentrations of the test item were analysed. Therefore, dilutions were prepared by 1:2 serial dilutions from 1250 μg/mL in culture medium.
XTT treatment: The XTT labelling mixture consists of two components, a XTT buffer solution and the substrate solution. Both components were mixed right before application at a ratio of 1:100. After the cell seeding, 100 µL of the test item dilutions, the medium and solvent controls, respectively were added to the cells. All dose groups were tested in 7 replicates for each XTT test. At the end of the incubation period of 24 ± 1 hours, the cell cultures were microscopically evaluated for morphological alterations.
XTT Labelling and Measurement: At the end of the incubation period, 50 µL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Acceptability criteria of XTT assay:
The XTT test is considered to be acceptable if it meets the following criteria:
• mean absorbance of the medium control is ≥ 0.5
• mean viability of the solvent control is ≥ 90% in comparison to the medium control

Main test:
The test item was tested in three independent runs
Test item preparation: Two independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT).
Eight final concentrations (µg/mL) were used for the test item in the main experiment (h CLAT). The highest concentration used was 1.2 × CV75 and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.
Treatment of the cells: Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24+/-1 hours. Each concentration of the test item, medium control, positive and DMSO control was tested in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).
Staining of the cells: The triplicates of each test item-treated and not test item treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). All solutions were kept light protected at 2-8 °C or on ice during the staining and analysis procedure. The cells were gently mixed by hand and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
Sample preparation for measurement: After staining with the antibodies, the cells were washed twice (2-8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-aminoactinomycin D (7-AAD) solution were added.
Flow cytometry acquisition: The expression of cell surface antigens (CD54, CD86) was analyzed by flow cytometry. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
Acquisition: A total of 10,000 living cells were analyzed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50 % (due to diffuse labelling of cytoplasmic structures that are generated due to cell membrane destruction).
Data analysis and interpretation:
The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:
RFI (%) = 100 x [(MFI of test item treated cells - MFI of test item treated isotope control cells) / (MFI of solvent control cells - MFI of solvent isotope control cells)], where MFI is geometric mean fluorescent intensity
The cell viability is calculated as follows:
Cell viability (%) = 100 x (Mean cytotoxicity of solvent control cells / Mean cytotoxicity of the test item treated cells), where Mean cytotoxicity is the mean of geometric mean (7-AAD) isotype control, geometric mean (7-AAD) CD54 and geometric mean (7-AAD) CD86.
Acceptability criteria of the h-CLAT assay:
The study is considered as valid, if the following criteria are met:
• Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
• In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
•The cell viability of at least 4 doses in each experiment should be ≥50%.
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such a case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90%.

Evaluation of results:
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs:
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA.

Test chemicals with a Log Pow > 3.5 tend to produce false negative results. Therefore negative results with test chemicals with a Log Pow > 3.5 should not be considered (according to the OECD guideline). However, positive results obtained with test chemicals with a Log Pow > 3.5 could still be used to support the identification of the test chemical as a skin sensitiser.
Positive control results:
The RFI value for CD54 of the positive control (3 µg/mL DNCB) in the second run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the RFI values for CD54 and CD86 of the positive control (2 µg/mL DNCB) in the same run exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. The RFI values of the positive controls (DNCB) for CD54 and CD86 of the first and third run exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. All valid runs meet the acceptance criteria.
Key result
Parameter:
other: % RFI
Remarks:
CD86 Antibody
Run / experiment:
1
Value:
> 150
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
First run
Key result
Parameter:
other: % RFI
Remarks:
CD54 Antibody
Run / experiment:
1
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
First run
Key result
Parameter:
other: % RFI
Remarks:
CD86 Antibody
Run / experiment:
2
Value:
< 150
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Second run
Key result
Parameter:
other: % RFI
Remarks:
CD54 Antibody
Run / experiment:
2
Value:
< 200
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Second run
Key result
Parameter:
other: % RFI
Remarks:
CD86 Antibody
Run / experiment:
3
Value:
> 150
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
Third run
Key result
Parameter:
other: % RFI
Remarks:
CD54 Antibody
Run / experiment:
3
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
Third run
Other effects / acceptance of results:
OTHER EFFECTS:
- Cytotoxic effects (cell viability < 75%) were observed following incubation with the test item at the highest tested concentration 1250 µg/mL in the first test and starting with the concentration of 625 µg/mL in the second test up to the highest tested concentration (1250 µg/mL). The mean CV75 value of both XTT tests was calculated as 554 µg/mL.
The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT):
186, 223, 267, 321, 385, 462, 554 and 665 µg/mL

The test item was tested in 3 independent runs. The highest concentration of all three runs was excluded from the evaluation since the cell viability was below 50%. The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively in at least one dose of 2 out of 3 independent run data. Two out of three runs were POSITIVE relating to the prediction model used in the h-CLAT test method, therefore the test item is considered to have a skin sensitisation potential.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative/solvent control: yes
In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
Cell viability of the DMSO control was more than 90% in comparison to the medium control.
For both medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control was > 105%.
- Acceptance criteria met for positive control: yes
The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%. (The RFI value for CD54 of the positive control (3 µg/mL DNCB) in the second run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the RFI values for CD54 and CD86 of the positive control (2 µg/mL DNCB) in the same run exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%)
- Acceptance criteria met for test chemical: yes
For the test chemical, the cell viability was more than 50% in at least four tested concentrations in each run.

Results of the XTT assay:

1st test:

    Microscopic Evaluation Photometric Evaluation            
Test Group Concentration Cytotoxicity

Mean

Absorbance*

Standard Deviation

Chem.

Blank

Mean Absorbance

Chemical Blank

Absorbance in % of Solvent Control**
  [µg/mL]            
Medium Control - no 0.651 0.036 0.226 0.425 103.64
Solvent Control - no 0.634 0.023 0.223 0.410 100.00
Test Item 9.8 no 0.657 0.020 0.225 0.432 105.32
  19.5 no 0.668 0.022 0.221 0.448 109.11
  39.1 no 0.621 0.041 0.222 0.399 97.36
  78.1 no 0.661 0.030 0.220 0.441 107.50
  156.3 no 0.671 0.020 0.225 0.446 108.66
  312.5 no 0.732 0.034 0.222 0.510 124.36
  625 no 0.540 0.029 0.222 0.317 77.39
  1250 yes 0.244 0.008 0.224 0.019 4.74

Shaded test groups:          cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

*            mean absorbance (absolute)of 7wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 96.5%.

The CV75 value of the first XTT test: 645.6 µg/mL

2nd test:

 

Microscopic Evaluation

Photometric Evaluation


Test Group

Concen-tration
[µg/mL]

Cytotoxicity

Mean Ab-sorbance*

Standard-Deviation

Chem. Blank

Mean Ab-sorbance – Chemical Blank

Absorbance in % of Solvent Control**

Medium Control

-

no

0.613

0.013

0.221

0.392

95.10

Solvent Control

-

no

0.631

0.027

0.219

0.412

100.00

Test Item

9.8

no

0.642

0.028

0.216

0.425

103.20

19.5

no

0.679

0.033

0.216

0.463

112.20

39.1

no

0.691

0.043

0.216

0.475

115.27

78.1

no

0.679

0.036

0.216

0.463

112.27

156.3

no

0.632

0.035

0.217

0.415

100.60

312.5

no

0.601

0.051

0.217

0.384

93.08

625

no

0.445

0.037

0.217

0.228

55.38

1250

yes

0.246

0.015

0.216

0.031

7.51

Shaded test groups:       cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

*       mean absorbance (absolute) of 7 wells

**       relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 105.1%.

The CV75 value of the second XTT test: 462.4 µg/mL

The mean CV75 value of both XTT tests: 554 µg/mL

Results of the h-CLAT test:

1st test run:

  Concentration (µg/mL) RFI (%) RFI (%) Cell Viability (%)
    CD 54 Antibody CD 86 Antibody  
Medium Control - 100 100 100
DMSO Control - 100 100 100

Positive Control (DNCB)

2.0

317.4*

526.7*

61.1

 

3.0

356.3*

447.9*

55.6

Test Item

186

123.7

129.2

89.3

 

223

145.6

120.4

87.6

 

267

147.3

116.8

86.4

 

321

154.4

117.5

85.3

 

385

176.9

126.3

82.1

 

462

194.7

121.2

78.3

 

554

232.5*

192.0*

60.1

 

665

270.4*

354.0*

39.5

Shaded test groups:          cell viability below 50%, are excluded from the evaluation

*   RFI value of CD86 or CD54 fulfilled the positive criteria (CD86150% and CD54200%).


2nd test run:

 

Concentration (µg/mL)

RFI (%)

RFI (%)

Cell Viability (%)

 

 

CD 54 Antibody

CD 86 Antibody

 

Medium Control

-

100

100

100

DMSO Control

-

100

100

100

Positive Control (DNCB)

2.0

250.3*

374.8*

57.4

 

3.0

195.4#

307.4*

57.7

Test Item

186

88.6

70.6

107.5

 

223

113.6

76.1

102.7

 

267

120.7

98.5

94.0

 

321

123.6

124.4

96.8

 

385

120.0

115.4

79.2

 

462

125.7

96.5

90.9

 

554

142.1

126.4

80.9

 

665

260.0*

658.7*

36.3

Shaded test groups:          cell viability below 50%, are excluded from the evaluation

#CD54 RFI value of the positive control (3.0 µg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%).

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86150% and CD54200%).

3rd test run:

 

Concentration (µg/mL)

RFI (%)

RFI (%)

Cell Viability (%)

 

 

CD 54 Antibody

CD 86 Antibody

 

Medium Control

-

100.0

100.0

100.0

DMSO Control

-

100.0

100.0

100.0

Positive Control(DNCB)

2.0

309.0*

464.0*

64.8

 

3.0

345.5*

453.2*

76.6

Test Item

186

100.7

103.8

94.5

 

223

98.6

106.0

95.8

 

267

112.2

118.0

99.7

 

321

120.9

117.3

94.9

 

385

146.8

131.6

92.9

 

462

208.6*

161.7*

87.2

 

554

252.5*

266.2*

70.4

 

665

352.5*

659.4*

33.5

Shaded test groups:          cell viability below 50%, are excluded from the evaluation

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).


Interpretation of results:
other: h-CLAT was positive
Conclusions:
In a GLP-compliant guideline study, the test substance was found to be positive in the in vitro h-CLAT test, indicating a possible skin sensitizing potential of the substance.
Executive summary:

The GLP-compliant in vitro Human Cell Line Activation Test (h-CLAT) was performed in accordance with OECD guideline 442E to assess the skin sensitising potential of the test substance dissolved in culture medium when administered to THP-1 cells for 24 +/- 1 hours. The highest test item concentration for the main experiment (h-CLAT) of the test substance was previously determined by two XTT tests. The mean CV75 value of both XTT tests was calculated as 554 µg/mL. Based on this result the following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT): 186, 223, 267, 321, 385, 462, 554 and 665 µg/mL. The test substance was tested in 3 valid independent runs. All valid runs meet the acceptance criteria, e.g. the values obtained for controls. The highest concentration of all three runs was excluded from the evaluation since the cell viability was below 50%. The RFI of CD86 and CD54 was greater than 150% and 200%, respectively in at least one dose of 2 out of 3 independent run data. Two out of three runs were positive relating to the prediction model used in the h-CLAT test method, therefore the test item is considered to be positive in the h-CLAT, indicating a possible skin sensitizing potential of the substance.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Summary

The skin sensitization of Dimeth Cyclormol was tested in a Buehler test, in a battery of in vitro/in chemico assays (DPRA, Keratinosens and h-CLAT) and the in silico model Derek Nexus was used.

The Buehler test showed skin sensitisation but the results are partly confounded by irritation because the animals were challenged with slight irritant concentrations. To further support or defer the skin sensitisation in vitro assays were performed. DPRA and KeratinoSens produced a negative outcome but the h-Clat was positive overall indicating a negative result when using the Bauch et al (2012) testing strategy. To further characterize the skin sensitisation also a prediction by Derek Nexus was made, which marked the test substance having an terpenoid type of structure (double bond with a methyl group) being a structural alert for skin sensitisation. This model predicted an LLNA EC3 value of 17%.

In conclusion: The substance is considered a skin sensitiser based on the Buehler. The skin sensitisation potency is derived with Derek Nexus which predicts an LLNA EC3 of 17%.

The available data for Dimeth Cyclormol are summarised below starting with the Buehler test and the Derek Nexus prediction, which are defining the final results. Thereafter the executive summaries of the in vitro tests are presented.

 

Key study: Skin sensitisation in vivo - Buehler test (OECD TG 406)

A Buehler test was performed in accordance with a method similar to OECD TG 406 (performed prior to guideline introduction). The study was rated Klimisch 2, firstly because irritant concentrations were used during the challenge. Secondly because the animals in the challenge control (non-induced) animals were exposed to the vehicle instead of the 5% test substance concentration. The irritant effects cannot be distinguished from the skin sensitisation effects. A preliminary irritation test was performed with 10, 5, 2.5 and 1.3% test substance in SDA 39C. At 10%, 2/4 animals exhibited slight confluent or moderate patchy erythema and 1/4 animals exhibited slightly patchy erythema. At 5%, 2/4 animals exhibited slightly patchy erythema, while at 2.5% and 1.3% no effects were observed. Induction and challenge concentrations selected were 5% test material in SDA 39C, which were applied under occlusion. The challenge concentration of 5% test material produced 9/15 positive responses (grades 1: slight confluent or moderate patchy erythema and grades 2: moderate erythema) at the 24 and 48 hour readings of the test animals. When considering ± responses (slightly patchy erythema) also positive, 15/15 animals responded positive at the 24 hour reading and 14/15 at the 48 hour reading. The positive reactions in most animals and being consistent at 48 hours the substance is considered a sensitiser. The potency remains inconclusive because Buehler tests are not developed to present potency and because the irritation of the substance may have confounded the skin sensitisation.

Supporting Information: Skin sensitisation including potency modelled by Derek Nexus:

QMR summary: DEREK (Derek Nexus: 5.0.1, Nexus: 2.1.0) predicts the Skin sensitisation EC3 in an LLNA. The model is based on LLNA data. The applicability domain is presented two-fold: First, the model presents the presence and absence of a structural alert for skin sensitisation and; Secondly it predicts an LLNA EC3 based on the available information in the model. The uncertainty from the prediction can be derived from available analogues experimental and predicted values. The MoA is, among others, based on electrophilic properties of the structural alerts of the substances in the training set.

QPR summary: Dimeth Cyclormol is predicted to be considered a skin sensitiser. An LLNA EC3 of 17% is predicted. This is based on the terpenoid structural alert of which the training set has several substances: Limonene being one of them. The uncertainty of the prediction is limited in view of the model description available, the substance being in the applicability domain and the presence of similar structures on which the prediction is based. The Mode of action is presented as follows: “Terpenoids have been reported as moderate to weak sensitizers in the murine local lymph node assay (LLNA) and in human patch tests. They are considered to sensitisers by forming antigenic adducts with skin proteins after their activation to hydroperoxides by air oxidation, which can react with protein directly via a radical thiol-ene reaction, or indirectly after their secondary oxidation to alpha,beta-unsaturated epoxides or alpha,beta unsaturated carbonyl derivatives”.

DPRA (OECD TG 442C)

In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. Solutions of the test substance were successfully analysed by the validated DPRA analytical method. In the Cysteine reactivity assay, Cysteine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM Dimeth Cyclormol solution in more acetonitrile and Cysteine peptide stock solution. The final sample concentration was 5 mM of Dimeth Cyclormol, 0.5 mM Cysteine. In the Lysine reactivity assay, Lysine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM Dimeth Cyclormol solution in Lysine peptide stock solution. The final sample concentration was 25 mM of Dimeth Cyclormol, 0.5 mM Lysine. All analytical acceptance criteria of the test were met

The individual peptide depletion results for Dimeth Cyclormol were -0.786% (Cysteine) and -0.875% (Lysine).  No co-elution peaks were observed in either the Cysteine or Lysine assays. With effectively no depletion of either peptide in the presence of the test item, Dimeth Cyclormol was classified in the “no to minimal reactivity” class based on the DPRA prediction model and was thus considered to be negative in the DPRA.

KeratinoSens  (OECD TG 442D):

The GLP-compliant in vitro KeratinoSens test (ARE-Nrf2 luciferase reporter assay) was performed in accordance with OECD guideline 442D to assess the skin sensitising potential of the test substance. Two independent experiments were performed. The test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000μM (2-fold dilution series). Ethylene dimethacrylate glycol was used as a positive control. The acceptance criteria were met in both experiments.

The test substance showed toxicity (IC30 values of 559μM and 524μM and IC50 values of 685μM and 660μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.19-fold and 1.22-fold in experiment 1 and 2, respectively. In conclusion, the current study was valid and the test substance is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction; no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) were observed at test concentrations of ≥ 1000 µM.

h-CLAT ( OECD TG 442)

The GLP-compliant in vitro Human Cell Line Activation Test (h-CLAT) was performed in accordance with OECD guideline 442E to assess the skin sensitising potential of the test substance dissolved in culture medium when administered to THP-1 cells for 24 +/- 1 hours. The highest test item concentration for the main experiment (h-CLAT) of the test substance was previously determined by two XTT tests. The mean CV75 value of both XTT tests was calculated to be 554 µg/mL. Based on this result the following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT): 186, 223, 267, 321, 385, 462, 554 and 665 µg/mL. The test substance was tested in 3 valid independent runs. All valid runs meet the acceptance criteria, e.g. the values obtained for controls. The highest concentration of all three runs was excluded from the evaluation since the cell viability was below 50%. The RFI of CD86 and CD54 was greater than 150% and 200%, respectively in at least one dose of 2 out of 3 independent run data. Two out of three runs were positive relating to the prediction model used in the h-CLAT test method, therefore the test item is considered to be positive in the h-CLAT, indicating a possible skin sensitizing potential of the substance.

Additional available data:

A HRIPT study (IFF, 1980) is available with the test substance tested at 2.5% v/v in SDA 39C. None of the 53 participants showed evidence of sensitisation under the test conditions.

Justification for classification or non-classification

Based on the evaluation of all available data, the substance can be considered a skin sensitizer based on a Buehler test with an estimated LLNA EC3 value of 17% using Derek Nexus model resulting in a classification for skin sensitisation (Skin Sens. 1B / H317: May cause an allergic reaction) in accordance with the EU CLP (EC/1272/2008 and its updates).