Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 431-480-3 | CAS number: -
Endpoint summary
Administrative data
Description of key information
Skin sensitization (LLNA, OECD TG 429): not sensitizing
Skin sensitization (DPRA, OECD TG 442C): positive
Skin sensitization (KeratinoSens, OECD TG 442D): inconclusive
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 Apr 2018 to 15 May 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- The study was performed according to the OECD guideline No.429 (July 2010) and in compliance with the GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- OECD Guideline 429. Skin Sensitization: Local Lymph Node Assay, July 2010.
EC No 640/2012 Part B. Skin Sensitization: Local Lymph Node Assay, July 2012. - Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- mouse local lymph node assay (LLNA)
- Species:
- mouse
- Strain:
- CBA:J
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS:
Condition: Inbred, SPF-Quality
Source: Janvier, Le Genest-Saint-Isle, France
Number of Animals: 20 Females (nulliparous and non-pregnant). Five females per group.
Age at the Initiation of Dosing: Young adult animals (approximately 10 weeks old) were selected.
Weight at the Initiation of Dosing: 18.4 to 23.5 g.
ENVIRONMENTAL CONDITIONS:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing
Target temperatures of 18 to 24°C
relative target humidity of 40 to 70%
Daily mean temperature: 22 to 23°C
Mean relative humidity of 41 to 51%
A 12 hour light/12 hour dark cycle was maintained.
Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms. - Vehicle:
- dimethylformamide
- Concentration:
- For the preliminary test the concentrations were 5,10, 25, and 50% of the test item.
For the main test the concentrations were 0, 2, 5 and 10% of the test item. - No. of animals per dose:
- For the preliminary test: 4 females/dose
For the main test: 5 females/dose, 5 females for the negative control and 5 females for the positive control
see details in table results - Details on study design:
- Pre-screen Test
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Initially, two test item concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (5% and 10%) at a later stage.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 9-11 weeks (at initiation of treatment) the application method may have been different (see tables) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.
Main Study:
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.
Induction - Days 1, 2 and 3:
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
Irritation:
Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing). According to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.
Erythema and eschar formation:
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4
ANALYSIS:
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures, including all amendments.
Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3) (reference 1). - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Positive control results:
- In the positive control group given HCA at the concentration of 25%, a moderate increase in cellularity and stimulation index exceeding the thresold value of 3 (SI=3.5 +/-0.6%) was noted.
The study is therefore considered as valid - Key result
- Parameter:
- SI
- Value:
- ca. 1.8
- Variability:
- +/-0.2
- Test group / Remarks:
- 10%
- Key result
- Parameter:
- SI
- Value:
- 2.2
- Variability:
- +/- 0.3
- Test group / Remarks:
- 5 %
- Key result
- Parameter:
- SI
- Value:
- 1.4
- Variability:
- +/- 0.1
- Test group / Remarks:
- 2%
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity (see Appendix 3).
Based on these results,test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments). - Executive summary:
The objective of this study was to evaluate whether test item induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this study plan.
The study was carried out based on the guidelines described in:
· OECD, Section 4, Health Effects, No.429 (2010),
· EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay"
· EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.
Test item concentrations selected for the main study were based on the results of a pre-screen test. The 50% and 25% test item concentration did not meet the selection criteria. The 10% and 5% test item concentration did meet the selection criteria and therefore the 10% concentration was selected as highest concentration for the main study.
In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 5 or 10% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (N,N-dimethylformamide). Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.
The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 5% and three animals treated at 10% which were considered to be enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 727, 1166 and 982 DPM, respectively. The mean DPM/animal value for the vehicle control group was 538 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% were 1.4, 2.2 and 1.8, respectively.
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 03 Jan 2018 to 12 Jan 2018
- 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:
- no
- GLP compliance:
- yes
- Type of study:
- direct peptide reactivity assay (DPRA)
- Details on the study design:
- Synthetic peptides:
Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA COOH) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.
Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in the table below.
:
Preparation of Co-elution Control, Test Item and Positive Control Samples
Sample Replicates Sample code Preparation
Co-elution control (CC) 1 CClys-208992/A 750 µL Ammonium acetate buffer pH 10.2
250 µL 208992/A test solution (100 mM)
Cinnamic aldehyde (PC) 3 PClys-1 to PClys-3 750 µL Stock solution of 0.667 mM SPCL
250 µL Cinnamic aldehyde solution (100 mM in ACN)
Test item 208992/A 3 208992/A-lys-1 to 750 µL Stock solution of 0.667 mM SPCL
208992/A-lys-3 250 µL 208992/A test solution (100 mM)
Sample Incubations
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 25 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC PDA analysis the samples were visually inspected for precipitation. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature.
HPLC-PDA Analysis
SPCC and SPCL peak areas in the samples were measured by HPLC PDA. Sample analysis was performed using the following system:
System 1 (used for Cysteine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
• LC Column oven 300 (Thermo Scientific)
• Surveyor PDA detector (Thermo Scientific)
System 2 (used for Lysine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
• Column Oven #151006 (Grace, Worms, Germany)• Surveyor PDA detector (Thermo Scientific)
All samples were analyzed according to the HPLC-PDA method presented in Table 1 (Appendix 1). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 (Appendix 1).
Data Evaluation
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion= [1-((Peptide Peak Area in Replicate Injection (at 220 nm))/(Mean Peptide Peak Area in Reference Controls (at 220 nm)))]×100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90% - Key result
- Parameter:
- other: SPCC depletion %
- Value:
- 13.1 %
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Parameter:
- other: SPCL depletion %
- Value:
- 0.6 %
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Parameter:
- other: Mean of SPCC and SPCL depletion %
- Value:
- 6.9 %
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Interpretation of results:
- Category 1 (skin sensitising) based on GHS criteria
- Conclusions:
- In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The substance was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed upon addition of the test item to the SPCC and SPCL peptide solutions, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the percentages of SPCC and SPCL depletion might be underestimated.
- Executive summary:
The objective of this study was to determine the reactivity of test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.
The study procedures described in this report were based on the most recent OECD guideline.
Isopropanol was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. An overview of the obtained assay validation parameters is presented in the table below.
Acceptability of theDirect Peptide Reactivity Assay (DPRA)
Cysteine reactivity assay
Lysine reactivity assay
Acceptability criteria
Results for SPCC
Acceptability criteria
Results for SPCL
Correlation coefficient (r2) standard calibration curve
>0.99
0.994
>0.99
0.997
Mean peptide concentration RC-A samples (mM)
0.50 ± 0.05
0.496 ± 0.005
0.50 ± 0.05
0.512 ± 0.007
Mean peptide concentration RC-C samples (mM)
0.50 ± 0.05
0.497 ± 0.007
0.50 ± 0.05
0.511 ± 0.010
Mean peptide concentration RC-Cisopropanolsamples (mM)
0.50 ± 0.05
0.481 ± 0.003
0.50 ± 0.05
0.522 ± 0.004
CV (%) for RC samples
B and C
<15.0
1.2
<15.0
1.6
Mean peptide depletion cinnamic aldehyde (%)
60.8-100
77.3
40.2-69.0
56.3
SD of peptide depletion cinnamic aldehyde (%)
<14.9
0.2
<11.6
0.4
SD of peptide depletion for the test item (%)
<14.9
1.1
<11.6
0.5
RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.
The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and Cisopropanol, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA.
Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed.
An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 13.1% SPCC depletion while in the lysine reactivity assay the test item showed 0.6% SPCL depletion. The mean of the SPCC and SPCL depletion was 6.9% and as a result the test item was considered to be positive in the DPRA and classified in the “lowreactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification forthe Test Item
Test item
SPCC depletion
SPCL depletion
Mean of SPCC and SPCL depletion
DPRA prediction and reactivity classification
Mean
± SD
Mean
± SD
Cysteine 1:10 / Lysine 1:50 prediction model
Test Item
13.1%
±1.1%
0.6%
±0.5%
6.9%
Positive: Low reactivity
SD = Standard Deviation.
In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. test item was positive in the DPRA and was classified in the “lowreactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed upon addition of the test item to the SPCC and SPCL peptide solutions,one cannot be sure how much test item remained in the solution to react with the peptides. Consequently,the percentages of SPCC and SPCL depletion might be underestimated.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 19 Jan 18 - 09 Feb 18
- 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:
- adopted February, 2015
- Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- activation of keratinocytes
- Details on the study design:
- VEHICLE
* The vehicle of the test item, i.e. dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).
Vehicle Control: The vehicle control is the vehicle of the test item.
* Preparation of the Vehicle Control: The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.
POSITIVE CONTROL
* Ethylene dimethacrylate glycol CAS Number 97-90-5
Batch SHBG0572V
Purity 98.3%
Storage conditions In refrigerator (2-8°C)
Stable under storage conditions until 10 March 2020
* Preparation of the Positive Control: The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.7.1, so that the final concentration of the positive control ranges from 7.8 to 250 μM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.
TEST ITEM
No correction was made for the composition/purity of the test item.
A solubility test was performed. The test item was suspended in DMSO to a final concentration of 200 mM (light yellow homogenous suspension). The stock was sonicated (Time: 41 min ; Temp.: 19 – 34 °C). The 100-fold dilution in DMEM glutamax of 200 mM and 100 mM DMSO stock formed a non-homogeneous solution and was therefore not suitable to test. The 100-fold dilution of the 50 mM DMSO stock formed a homogeneous solution (slight precipitation). This concentration was selected as highest concentration for the
main assay (limit of solubility). In the main experiments the test item was suspended in dimethyl sulfoxide (DMSO) at 50 mM (yellow suspension). The stock was sonicated (Time: 15 – 19 min; Temp.: 16 –
24 °C). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series).
The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49 and 0.24 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear
solution.
Precipitation was observed at the start and end of the incubation period at test concentrations of 125 μM and upwards in the 96-well plates.
Test item concentrations were used within 3 hours after preparation.
Blank
On each plate three blank wells were tested (no cells and no treatment).
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). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). 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 from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).
CELL CULTURE
Basic medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Maintenance medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 μg/mL).
Exposure medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 75 – 98%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.8 – 36.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Subculturing
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, and were not cultured for more than 25 passages from the frozen stock (P+25).
EXPERIMENTAL DESIGN
* Plating of Cells: For testing, cells were 80-90% confluent. 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. The passage number used was P+10 in experiment 1 and P+12 in experiment 2.
* 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 items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2. In total 2 experiments were performed.
* Luciferase Activity 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 TECAN Infinite® M200 Pro Plate Reader 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 overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
DATA INTERPRETATION
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative (Figure 1):
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the vehicle (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 nodefined MW)
4. There is an apparent overall dose-response for luciferase induction - Key result
- Run / experiment:
- run/experiment 1
- Parameter:
- IC50 [442D]
- Value:
- 0.61 µM
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- run/experiment 2
- Parameter:
- IC50 [442D]
- Value:
- 0.67 µM
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In conclusion, test item is classified as inconclusive (no biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
- Executive summary:
The objective of this study was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.
The study procedures described in this report were based on the most recent OECD guideline. Batch 17J31PR252A of test item was a slightly yellow powder. The test item was suspended in dimethyl sulfoxide at 50 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 0.24 – 500 μM (2-fold dilution series). The highest test concentration was considered to be the limit of solubility. The test item precipitated at test concentration of 125 μM and upwards. Two independent experiments were performed.
Both experiments passed the acceptance criteria:
- 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 (95 μM and 53 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.30-fold and 2.59-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (5.5% and 4.9% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.
test item showed toxicity (IC30 values of 0.42 μM and 0.44 μM and IC50 values of 0.61 μM and 0.67 μM in experiment 1 and 2, respectively). A dose-related induction of the luciferase activity (EC1.5 values of 6.4 and 5.6 in experiment 1 and 2, respectively) was measured in both experiments. However, the IC30 values are lower than the EC1.5 values in both experiments and therefore the inducations of the luciferace activity are not considered biologically relevant. The maximum luciferase activity induction (Imax) was 3.59-fold and 3.12-fold in experiment 1 and 2 respectively. Test item is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations with a cell viability of > 70% and test concentrations < 1000 μM.
In conclusion, test item is classified as inconclusive (no biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Referenceopen allclose all
Main study -results
Skin Reactions / Irritation
The very slight erythema of the ears as shown by three animals treated at 10% on Day 3 and the scaliness as noted for animals of all groups between Days 4 and 6 were considered not to have a toxicologically significant effect on the activity of the nodes.
Light yellow test item remnants were present on the dorsal surface of the ears all test item treated animals throughout the observation period, which did not hamper scoring of the skin reactions.
Systemic toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period
Macroscopic Examination of the Lymph Nodes and Surrounding Area
The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 5% and three animals treated at 10% which were considered to be enlarged.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.
Main Study: Body Weights and Skin Reactions
group | TS1 (%) | animal | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | ||||||||
bw (g)2 | erythema3 | erythema | erythema | erythema | erythema | erythema | bw (g) | |||||||||
left | right | left | right | left | right | left | right | left | right | left | right | |||||
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
1 | 0 | 1 | 22.4 | 0 | 0 | 0 | 0 | 0 | 0 | 0s | 0s | 0s | 0s | 0 | 0 | 24.1 |
|
| 2 | 19.9 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 21.5 |
|
| 3 | 19.1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0s | 0s | 0s | 0 | 0 | 20.6 |
|
| 4 | 23.5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 24.5 |
|
| 5 | 20.7 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0s | 0 | 0 | 0 | 23.5 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
2 | 2 | 6 | 20.6 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 0fs | 0 | 0 | 24.6 |
|
| 7 | 19.3 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0f | 0s | 0 | 22.1 |
|
| 8 | 18.9 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 22.3 |
|
| 9 | 19.6 | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 0fs | 0fs | 0 | 0s | 21.7 |
|
| 10 | 18.4 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 21.6 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
3 | 5 | 11 | 20.9 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 21.9 |
|
| 12 | 21.0 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0s | 19.5 |
|
| 13 | 19.0 | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 0fs | 0fs | 0f | 0 | 22.5 |
|
| 14 | 21.5 | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0f | 0fs | 0fs | 0s | 0s | 23.5 |
|
| 15 | 21.5 | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 0fs | 0fs | 0s | 0s | 24.0 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
4 | 10 | 16 | 22.9 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0f | 0fs | 0fs | 23.1 |
|
| 17 | 21.5 | 0f | 0f | 0f | 0f | 1f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 22.5 |
|
| 18 | 20.5 | 0f | 0f | 0f | 0f | 1f | 1f | 0f | 0f | 0f | 0f | 0fs | 0fs | 21.1 |
|
| 19 | 21.4 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 22.7 |
|
| 20 | 21.2 | 0f | 0f | 0f | 0f | 1f | 0f | 0f | 0f | 0f | 0f | 0fs | 0fs | 23.3 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
f. Light yellow staining of the dorsal surface of the ears by test item remnants which did not hamper the scoring
of the ears s.Scaliness
1 TS = test item (% w/w).
2 Body weight (grams).
3 Grading erythema and eschar formation(Left = dorsal surface of left ear; right = dorsal surface of right ear):
0 = No erythema
1 = Very slight erythema (barely perceptible)
Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)
group | TS1 (%) | animal | Size nodes2 | DPM3/ animal | mean DPM ± SEM4 | mean SI ± SEM | |||||
left | right | ||||||||||
|
|
|
|
|
|
|
| ||||
1 | 0 | 1 | n | n | 482 | 538 | ± | 94 | 1.0 | ± | 0.2 |
|
| 2 | n | n | 660 | ||||||
|
| 3 | n | n | 223 | ||||||
|
| 4 | n | n | 548 | ||||||
|
| 5 | n | n | 778 | ||||||
|
|
|
|
|
|
|
|
|
|
|
|
2 | 2 | 6 | n | n | 475 | 727 | ± | 76 | 1.4 | ± | 0.1 |
|
| 7 | n | n | 806 | ||||||
|
| 8 | n | n | 877 | ||||||
|
| 9 | n | n | 630 | ||||||
|
| 10 | n | n | 849 | ||||||
|
|
|
|
|
|
|
|
|
|
|
|
3 | 5 | 11 | n | n | 541 | 1166 | ± | 185 | 2.2 | ± | 0.3 |
|
| 12 | n | n | 1584 | ||||||
|
| 13 | n | n | 1142 | ||||||
|
| 14 | + | n | 1494 | ||||||
|
| 15 | n | n | 1071 | ||||||
|
|
|
|
|
|
|
|
|
|
|
|
4 | 10 | 16 | + | + | 1152 | 982 | ± | 131 | 1.8 | ± | 0.2 |
|
| 17 | n | + | 1395 | ||||||
|
| 18 | n | n | 919 | ||||||
|
| 19 | n | n | 757 | ||||||
|
| 20 | + | n | 686 | ||||||
|
|
|
|
|
|
|
| ||||
1 TS = test item (% w/w).
2 Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +, ++ or +++: degree of enlargement, n: considered to be normal).
3 DPM= Disintegrations per minute.
4 SEM = Standard Error of the Mean.
Pre-screen Test - Results
At a 50% and 25% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and therefore this concentration did not meet the selection criteria.
At a 10% and 5% test item concentration, no signs of systemic toxicity were noted and no significant irritation was observed and therefore the 10% concentration was selected as highest concentration for the main study
| Table 1 Pre-Screen Test: Body Weights and Skin Reactions | ||||||||||||||||
| TS1 | (%) | animal | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | ||||||||
| bw | erythema3 | erythema | erythema | erythema | erythema | erythema | bw | |||||||||
| (g)2 | left | right | left | right | left | right | left | right | left | right | left | right | g) | |||
| 504 | 3 | 23,6 | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 0 | 0 | 0s | 0s | 25,1 | |
| 4 | 22,2 | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 0 | 0 | 0s | 0 | 22,4 | ||
| 25 | 1 | 24,7 | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 0 | 0 | 0s | 0s | 25,8 | |
| 2 | 22,8 | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0 | 0 | 0 | 0 | 0s | 23,6 | ||
| 10 | 7 | 21 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0s | 0s | 21,6 | |
| 8 | 21,3 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0fs | 0s | 22,2 | ||
| 5 | 5 | 21 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0 | 0f | 0f | 20,8 | ||
| 6 | 18,4 | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0f | 0s | 0s | 19,8 | ||
| f.White staining of the dorsal surface of the ears by test item remnants which did not hamper the scoring of the ears | ||||||||||||||||
| s.Scaliness | ||||||||||||||||
| 1TS = test item (% w/w). | ||||||||||||||||
| 2Body weight (grams). | ||||||||||||||||
| 3Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear): 0 = No erythema | ||||||||||||||||
| 4Applied using spatula instead of using a pipette. | ||||||||||||||||
| Table 2 Pre-Screen Test: Ear Thickness Measurements | ||||||||||||
| TS1 | (%) | animal | Day 1 | Day 3 | Day 6 | |||||||
| left | right | left | right | right | left | right | left | right | ||||
| (mm) | (mm) | (mm) | %2 | (mm) | %2 | (mm) | %2 | (mm) | %2 | |||
| 50 | 3 | 0,22 | 0,215 | 0,28 | 27 | 0,285 | 33 | 0,23 | 5 | 0,225 | 5 | |
| 4 | 0,22 | 0,225 | 0,285 | 30 | 0,29 | 29 | 0,225 | 2 | 0,225 | 0 | ||
| 25 | 1 | 0,22 | 0,225 | 0,29 | 32 | 0,3 | 33 | 0,22 | 0 | 0,225 | 0 | |
| 2 | 0,225 | 0,225 | 0,29 | 29 | 0,295 | 31 | 0,225 | 0 | 0,23 | 2 | ||
| 10 | 7 | 0,235 | 0,23 | 0,24 | 2 | 0,24 | 4 | 0,23 | -2 | 0,225 | -2 | |
| 8 | 0,225 | 0,23 | 0,24 | 7 | 0,24 | 4 | 0,22 | -2 | 0,225 | -2 | ||
| 5 | 5 | 0,225 | 0,23 | 0,24 | 7 | 0,235 | 2 | 0,22 | -2 | 0,225 | -2 | |
| 6 | 0,225 | 0,225 | 0,24 | 7 | 0,23 | 2 | 0,22 | -2 | 0,23 | -2 | ||
| Left (mm) = thickness of left ear in millimeters; right (mm) = thickness of right ear in millimeters. | ||||||||||||
| 1TS = test item (% w/w). | ||||||||||||
| 2Percent increase compared to Day 1 pre-dose value. A 25% value is used as the threshold for selection for use in the main study. | ||||||||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
Weight of Evidence for Skin Sensitization :
DEREK NEXUS version 6.0.1 yielded an alert for our product for skin sensitization based on the presence of the para hydroxyl-aniline group.
Our product is predicted to be sensitizing to the skin (plausible). DEREK NEXUS predicted an LLNA EC3 based on data of closest structurallyrelated substances which resulted in an EC3 of 1.0% (moderate sensitizer).
A valid DPRA test was performed according to OECD 442C and GLP. For the DPRA assay our product was dissolved in Isopropanol at 100 mM. Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed.
In the cysteine reactivity assay the test item showed 13.1% SPCC depletion while in the lysine reactivity assay the test item showed 0.6% SPCL depletion. The mean of the SPCC and SPCL depletion was 6.9% and as a result the test item was considered to be positive in the DPRA and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed upon addition of the test item to the SPCC and SPCL peptide solutions, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the percentages of SPCC and SPCL depletion might be underestimated.
A valid KeratinoSensTM assay was performed according to OECD 442D and GLP. For the KeratinoSensTM assay: our product was dissolved in DMSO at 50 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 0.24 – 500 μM (2-fold dilution series). The highest test concentration was considered to be the limit of solubility. The test item precipitated at test concentration of 125 μM and upwards. Two independent experiments were performed. Both experiments passed the acceptance criteria. SOur product showed toxicity (IC30 values of 0.42 μM and 0.44 μM and IC50 values of 0.61 μM and 0.67 μM in experiment 1 and 2, respectively). A dose-related induction of the luciferase activity (EC1.5 values of 6.4 and 5.6 in experiment 1 and 2, respectively) was measured in both experiments. However, the IC30 values are lower than the EC1.5 values in both experiments and therefore the inductions of the luciferase activity are not considered biologically relevant. The maximum luciferase activity induction (Imax) was 3.59-fold and 3.12-fold in experiment 1 and 2, respectively. Our product is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations with a cell viability of > 70% and test concentrations < 1000 μM.
DEREK predicted our product to be a moderate skin sensitizer with an EC value of 1.0%. The DPRA assay gave a positive result, whereas the KeratinoSensTM assay resulted inconclusive (due to induction of non-biologically relevant luciferase activity).
Based on these results, it can be concluded that our product has skin sensitizing properties and is predicted to be a moderate sensitizer based on DEREK EC3 prediction of 1.0%.
Performance of an additional in vitro assay, U-Sens assay, addressing the activation of dendritic cells would not yield additional information if it was negative or positive.
Therefore, further in vitro testing is considered not scientifically required. Based on the EC3 prediction related to 10 structurally similar compounds, classification of our compound is possible, and no further in vivo testing is needed. However, as the analogues used for the calculation of EC3 had a similarity of 46-22%, with skin sensitizing properties varying from non-sensitizing to extreme sensitizer, the predicted EC3 should be considered with caution.
Conclusion
Based on a positive DPRA assay and an inconclusive KeratinoSensTM, it can be concluded that our product has skin sensitizing properties and is predicted to be a moderate sensitizer based on DEREK EC3 prediction of 1.0%. Performance of an additional in vitro assay, U-SensTM assay, addressing the activation of dendritic cells, would not yield additional information if it was negative or positive.
Therefore, further in vitro testing is considered not scientifically required. Based on the EC3 prediction by DEREK related to 10 structurally similar compounds, classification of our product is possible, and no further in vivo testing is needed. However, as the analogues used for the calculation of EC3 had a similarity of 46-22%, with skin sensitizing properties varied from non-sensitizing to extreme sensitizer, the predicted EC3 should be considered with caution. In the absence of further reliable in vitro tests for skin sensitizing potency/potential, a LLNA test will provide more reliable information on potential skin sensitizing properties.
Justification for classification or non-classification
The objective of this study was to reach an overall conclusion on the endpoint skin sensitization based on all available relevant information, including alternative testing data.
A DEREK assessment, DPRA assay and KeratinoSensTM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA.
Guidance on information requirements and chemical safety assessment Chapter R.7a. DEREK predicted Test item to be a moderate skin sensitizer with an EC value of 1.0%. The DPRA assay gave a positive result, whereas the KeratinoSensTM assay resulted inconclusive (due to induction of non-biologically relevant luciferase activity). Based on these results, it can be concluded that test itm has skin sensitizing properties, and is predicted to be a moderate sensitizer based on DEREK EC3 prediction of 1.0%.
Performance of an additional in vitro assay, U-Sens assay, addressing the activation of dendritic cells would not yield additional information if it was negative or positive. Therefore, further in vitro testing is considered not scientifically required. Based on the EC3 prediction related to 10 structurally similar compounds, classification of our product is possible, and no further in vivo testing is needed. However, as the analogues used for the calculation of EC3 had a similarity of 46-22%, with skin sensitizing properties varied from non-sensitizing to extreme sensitizer, the predicted EC3 should be considered with caution. In the absence of further reliable in vitro tests for skin sensitizing potency/potential, a LLNA test will provide more reliable information on potential skin sensitizing properties. The LLNA test has been performed and according to this test , since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, test item was not considered to be a skin sensitizer.
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified as skin sensitising.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
