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Diss Factsheets

Administrative data

Description of key information

The results from the Buehler, Keratinosens assay and DPRA analysis were negative for skin sensitization of the test material. Therefore the test material did not meet the GHS criteria to be classified as a  skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
Guinea Pig
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15th June 2018 to 7th January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Skin Sensitisation study (406) published by the Ministry of Environmental Protection of Peoples Republic of China in the year 2013
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
According to the Guidelines for the testing of chemicals "Skin Sensitisation Study" (406) published by the Ministry of Environmental Protection of People's Republic of China in the year of 2013, guinea pig is the preferred strain for the heredity characters, stability and available background data. Based on the Skin Sensitization result of positive control test item DNCB, this strain is reliable.
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
Source: Beijing Vital River Laboratory animal technology
A physical check up was carried out on all animals on arrival. Animals were acclimatised for 6 days and housed 10/12 animals in a plastic cage with corn cob bedding in Room A118. All animals were weighed and marked on the hair. Clinical observations were performed daily.
Temperature: 20-26oC
Humidity: 40-70%
Light sequence: 12 hour light / 12 hour dark
Food: Pellet rodent diet (Beijing keaoxieli Feed Co. Ltd.)
Food and water: ad libitum
Route:
epicutaneous, semiocclusive
Vehicle:
other: Ethyl alcohol
Concentration / amount:
0.3g
Day(s)/duration:
6 hours
Route:
epicutaneous, semiocclusive
Vehicle:
other: Ethyl alcohol
Concentration / amount:
0.3g
Day(s)/duration:
6 hours
No. of animals per dose:
20
Details on study design:
Animals were randomly assigned to two groups (control and treated) using Microsoft Excel 2007. There were 10 animals in the control group and 20 animals in the treated group.

0.3g of the test item was mixed with 0.3ml of the vehicle to moisten completely.

Induction phase
The left flanks of guinea pigs were clipped free of hair prior to each induction (approximately 4cm x 4cm). For the treated group, a piece of filter paper (2cm x 2cm) loaded with prepared test item was placed on the clipped area, covered with two layers of gauze and one layer of waterproof plastic film. The patch was then wrapped with non-allergenic medical adhesive tape. After 6 hours of exposure, the patch was removed and residual test item removed by cotton wool soaked in water. On days 7 and 14 after the first induction, dosing was repeated according to the above method. Animals in the control group were not given the test item and were treated according to the above method.

Challenge phase
The right flanks of the guinea pigs were clipped free of hair the day prior to challenge. (approximately 4cm x 4cm). On Day 28 after the first induction, the prepared test item was given to the control and treated animals with the above method. After 6 hours of exposure, the patch was removed and residual test item was removed using cotton wool soaked in water.

Observations
24 and 48 hours after the patch was removed in each induction the skin reactions were observed.
Inspections for moribundity/mortality were made daily.
Animals were weighed on the grouping day and at conclusion of the study.
Challenge controls:
10 animals
Positive control substance(s):
yes
Remarks:
2,4-dinitrochlorobenzene (DNCB)
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
Not specified
No. with + reactions:
17
Total no. in group:
20
Clinical observations:
Not documented
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
Not specified
No. with + reactions:
17
Total no. in group:
20
Clinical observations:
Not documented
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.3g
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.3g
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None observed
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the above results, the test item E Stage 3 intermediate was a non-sensitiser. The test result did not meet GHS criteria for skin sensitisation (BUEHLER) 
Executive summary:

The study was designed to determine the potential for E Stage 3 Intermediate to elicit a skin sensitisation reaction. The method was designed to be meet the Guidelines for the testing of chemicals 'Skin sensitisation study' (406) published by the Ministry of Environmental Protection of People's Republic of China in the year 2013. 


Thirty animals were used in the study, which included one control group (10 animals) and one treated group (20 animals). In the induction phase, for treated group on Day 0, Day 7 and Day 14, 0.3g of the test item was applied to the left flank of each animal in the treated group three times. In the challenge phase, on Day 28, 0.3g of the test item was applied to the right flank of each animal in the control and treated group. 24 and 48 hours after the patch removal in each induction, the skin reactions were observed. 24 and 48 hours after patch removal in the challenge group, the skin reactions were observed and scored. Individual animal body weights were recorded on grouping day and at conclusion of the study.


Mortality: There were no deaths or moribund during the test. 


Induction Phase: No abnormalities were found during the three times induction phase at the 24 hour observations after removing patches.


Challenge phase: No abnormalities were found in the control or treated animals observed at 24 and 48 hour observation. The score of skin reaction is 0 (0/20) for both 24 and 48 hour observation.


All animals showed expected gain in bodyweight during the study. 


Based on the above results, E Stage 3 Intermediate was a non-sensitiser. The test result did not meet GHS criteria for skin sensitisation (BUEHLER) 

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25th September 2018 to 5th October 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
no
Remarks:
The study has been conducted with a reputable lab for other international submission.
Type of study:
activation of keratinocytes
Details on the study design:
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 KeratinoSensTM cell line).
For testing, cells were defrosted when they were 60-90% confluent. On the day prior to testing cells were harvested, and seeded into 96-well plates and incubated for 24 hours in assay medium.
The test article was tested for solubility in DMSO. 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 for 24 hours.
Each definitive assay included a set of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). Each plate tested a range of 12 dosing concentrations for each test article. Each plate also included 5 wells designated for the positive control (tested over a range of 5 dosing concentrations), 6 wells designated as the DMSO solvent control, and 1 well which was left blank
The treated plates were incubated for 48 hours ± 1.

Visual Observation
After approximately 48 hours of post-treatment incubation, visual observations of the cultures were performed for the cytotoxicity plate and recorded.

Luciferase Activity Measurement
After 48 ±1 hours of exposure, each white-walled culture plate was removed from the incubator and allowed to equilibrate to room temperature for at least 30 minutes. Once at room temperature, the treatment medium was decanted from each plate. The cultures were rinsed with
250 μL of CMF-DPBS (room temperature), the CMF-DPBS rinsate was decanted from the wells, and the plates were gently blotted onto paper towels.
Fifty microliters of CMF-DPBS was added to each well followed by fifty microliters of ONE-Glo™ Reagent. The plates remained at room temperature in the dark for at least 5 minutes before being read by the luminometer. The plates were read within 45 minutes of addition of the ONE-Glo™ Reagent. The luminescence determination of each plate was performed by a Berthold Detection Systems luminometer initiated from an IBM-PC hosting the Windows-based Simplicity™ software. The light intensity in each well was measured at 565 nm in the form of relative light units (RLUs).

Cytotoxicity Assessment
After 48 ±1 hours, the clear 96-well plates designated for the MTT endpoint were decanted and gently blotted on paper towels. No rinsing was performed. Two hundred μL of 1% DMEM containing 0.59 mg/mL MTT was added to each well. The plate was incubated with a plate seal at standard culture conditions for approximately 4 hrs.
After approximately 4 hours, the MTT solution was decanted, the plate was blotted, and 200 μL of 10% SLS was added to each well. The plate was covered with a plate seal and incubated at standard culture conditions overnight.
After the overnight incubation, each plate was placed on a plate shaker and shaken for at least 20 minutes at room temperature. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader.
Positive control results:
The positive control, Cinnamic Aldehyde, was tested at 5 concentrations ranging from 4 to 64 μM.
Cinnamic aldehyde produced a statistically significant induction above 1.5 fold below 64 μM in each definitive assay.
Key result
Run / experiment:
other: 0.977 - 2000 µM
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
The mean EC1.5 and IC50 (µM) of A-1298847.0 was >2000 and 19.21 respectively. The mean Imax and mean CImax of A-1298847.0 was 0.86 and 15.6µM respectively.
Interpretation of results:
GHS criteria not met
Conclusions:
According to the current prediction model, the test article A-1298847.0 was predicted to be a non-sensitizer.
Executive summary:

The objective of this study was to evaluate the ability of A-1298847.0 to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay. The study procedures described in this report were based on the most recent OECD guideline. The test item was dissolved in dimethyl sulfoxide at 200 mM. The final 12 tested concentrations were 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μM.  


The KeratinoSens assay was accepted when the positive control (cinnamic aldehyde) caused an EC1.5 value that fell within two standard deviations of the historical mean. Additionally, the results of the three definitive trials for each plate are assessed using similar criteria outlined in the validation ring trial 4. Those acceptance criteria included: 1) variability in DMSO solvent control wells for each definitive assay was <20%; and 2) the positive control produced a statistically significant induction above 1.5 fold below 64 μM in each definitive assay.


Overall it is concluded that the test conditions were adequate and that the test system functioned properly.
The mean EC1.5 and IC50 (µM) of A-1298847.0 was >2000 and 19.21 respectively.
The mean Imax and mean CImax of A-1298847.0 was 0.86 and 15.6µM respectively.
In conclusion, according to the current prediction model, the test article A-1298847.0 was predicted to be a non-sensitizer.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 November
2018 to 26 November 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
no
Type of study:
other: Direct Peptide Reactivity Assay
Details on the study design:
Test Article Preparation


The test articles were prepared at 100 mM concentrations in an appropriate solvent.
Calculations using the molecular weight and purity of the test article were performed to
determine the appropriate amount of test article to weigh out in order to achieve approximately 3
mL of the 100 mM sample. On the day of testing, the test articles were weighed into prelabeled
glass vials and stored at room temperature until time to perform the assay.

Test Article Solubility Test


A solubility test was performed for the test articles in order to determine an appropriate
solvent that completely dissolved each test article at a 100 mM concentration. The test articles,
A-1298847.0, A-509472.0, A-873806.0, A-560815.0, A-641294.0, A-1278823.5, A-1578921.0,
and A-1371022.0, were found to be soluble in acetonitrile with brief vortexing. The test article,
A-1354221.0, was found to be soluble in isopropanol with brief vortexing. The test articles, A1298740.0, A-1325053.0, and A-1298540.0, were not found to be soluble in any of the
solvents typically used in the DPRA with vortexing, heating and sonication.
The test articles that were not soluble were not tested in the DPRA, since no firm conclusion can be made from negative results for these test articles.

Peptide Preparation

Custom synthetic peptides containing cysteine or lysine as the reactive centers (with
phenylalanine to aid in detection) were used in this assay. The purity of each peptide was at least
90%. Peptide samples were newly prepared for each sample set, and a single preparation of the
peptide was used throughout the sample set. The cysteine peptide was prepared by weighing an
appropriate amount of the peptide to achieve a 0.667 mM concentration in pH 7.5±0.1 phosphate
buffer. The actual pH of the phosphate buffer on the day of testing was 7.5. The lysine peptide
was prepared by weighing an appropriate amount of the peptide to achieve a 0.667 mM
concentration in pH 10.2±0.1 acetate buffer. The actual pH of the acetate buffer on the day of
testing was 10.1. The peptide solutions were gently mixed on the shaker.

Peptide Standards


A set of serially diluted standards were prepared for each peptide. The top stock of the
standards (0.534 mM) was prepared from the 0.667 mM peptide solution in acetonitrile. The
remaining standards were prepared by serial dilution in dilution buffer (20% acetonitrile in
phosphate buffer for the cysteine peptide or acetate buffer for the lysine peptide). Six standards
were prepared at concentrations of 0.534- 0.017 mM. A seventh standard was prepared
containing only dilution buffer. Approximately 1 mL of each standard was pipetted into the
appropriate prelabeled autosampler vials.

Controls


The positive control used in this assay was cinnamic aldehyde prepared at a concentration
of 100 mM. The positive control was reacted with the peptides in the same fashion as the test
articles. There were three sets of reference controls of acetonitrile run at different points throughout the assay (reference controls A-C). Triplicate reference controls were also prepared
for each solvent used in the assay. These controls consist of the solvent (acetonitrile or
isopropanol) reacted with the peptide in the absence of test article. A coelution control was also
prepared for each test article. The coelution control consisted of the test article without the
peptide. The purpose of the coelution control was to determine if the test article elution from theHPLC column overlapped with the peptide elution.

Reaction Mixture Preparation


Prior to testing, the test articles were diluted in the appropriate solvent to yield a 100 mM
test article concentration. The test article dilutions were mixed as determined during the
solubility test (vortexing). The final dosing solutions were prepared for each test article, positive
control, and reference control in the prelabeled autosampler vials. Table 1 shows the make-up ofthe reaction mixtures for each peptide. Triplicate samples were prepared for the test articles and
controls. A single sample was prepared for each coelution control.

HPLC Set-up and Operation


The separations module used in this assay was a Waters 2695 HPLC system. This system
consisted of a solvent management system for the mobile phases and a sample management
system for the test articles and controls. The HPLC system was coupled to a photodiode array
detector set at 220 nm. The column used was a Zorbax SB-C18 column (Agilent) with
dimensions of 2.1 mm x 100 mm x 3.5 micron. The column was primed for at least two hours
before the start of the assay. To prime the column, equal parts of mobile phase A (0.1%
trifluoroacetic acid in HPLC grade water) and mobile phase B (0.08% trifluoroacetic acid in
HPLC grade acetonitrile) were passed through the column.


Once the column was primed for at least two hours, and the samples were prepared, the
autosampler vials were placed into the designated locations of the separations module carousels.
The samples were incubated in the dark at room temperature for 24±2 hours.


A gradient elution was used in this assay. The mobile phase changed from 10-25%
acetonitrile over a 10 minute period to allow for sample separation and gradually elute most of
the sample from the column. This was followed by a rapid increase to 90% acetonitrile to
remove anything remaining on the column. The column was allowed to equilibrate back to
initial specs for 7 minutes between injections.


The Empower 3 software was used to convert the absorbance data from the UV detector
into chromatograms of intensity versus retention time for the samples and controls. At the end of
the run, each chromatogram was integrated in order for the software to calculate the area under
the peptide peak. Cysteine and lysine elute from the column at known times, so it was possible
to determine which peaks in the chromatograms represented the peptides and use the areas under
those peaks for the subsequent calculations.

Parameter:
other: % Mean Peptide Depletion of Cysteine and Lysine
Value:
1.15
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: % Mean Peptide Depletion of Lysine
Value:
0.94
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: % Mean Peptide Depletion of Cysteine
Value:
1.35
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
According to the results from the direct peptide reactivity assay, the test material was considered to be a non-sensitizer and therefore, did not meet the GHS criteria for classification.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification