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Diss Factsheets

Administrative data

Description of key information

One In Vitro and one In Chemico Skin Sensitisation have been performed: Keratinosens and DPRA in a stepwise approach

The In Vitro Skin Sensitisation Human Cell Line Activation Test (h-CLAT) could not have been performed because of the logKow value of the test item (>3.5).

So, QSAR data have been provided too.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2018 - january 2019
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)
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The objective of the KeratinoSens assay is to evaluate the potential of the test item to activate the Nrf2 transcription factor. This test is part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non sensitizers in the context of an integrated approach to testing and assessment. It is an alternative method in order to avoid animal study
Specific details on test material used for the study:
At room temperature (on 29 October 2018, the test item was found at +4°C, whereas it should have been at room temperature. After investigations, it was deducted that the test item was mistakenly stored at +4°C for up to 12 days. This was considered not to have any impact on test item integrity, as stated by the Sponsor in an email dated 29 October 2019

. Protected from light

. Protected from humidity
Details on the study design:
The study design was based on the OECD Guideline 442D: In Vitro Skin Sensitisation assays addressing the AOP key event on keratinocyte activation, adopted on June 2018.
With the following exception:
. the OECD guideline 442D mentions that test items prepared in sterile water or culture medium have to be filtered after preparation for sterilization prior treatment. Filtration of such preparations can be possible when a test item formulation is a solution. However, in case the test item formulation is a suspension, filtration process can retain test item particles in the filter and considerably decrease test item concentration in the filtered formulation. Therefore, in view of the above and considering that pre-treatment filtration procedure is not of common practice in other in vitro cell-based assays, and that water and culture media used in the assay are already sterile, test item formulations were not filtered prior treatment.
The test item was tested in three independent runs using cells from a different passage number. A third run was performed since non concordant results were obtained between the two first runs.
Positive control results:
Gene induction values, Imax and EC1.5 values obtained with the positive control (cinnamic aldehyde)
Imax mean = 4.24
EC1.5 geometric mean = 13.08 µM

Evaluation of the viability (%) of cultures treated with the positive control for each run:
cinnamic aldehyde Concentrations (μM) : 4 ; 8 ; 16 ; 32 ; 64
Viability (%) in Run 1 106 ; 113 ; 116 ; 117 ; 97
Viability (%) in Run 2 111 ; 109 ; 128 ; 126 ; 129
Viability (%) in Run 3 107 ; 108 ; 112 ; 116 ; 106
Mean viability (%) 108 ; 110 ; 119 ; 120 ; 111
Geometric Mean (%) 108 ; 110 ; 119 ; 120 ; 110
SD 3 ; 3 ; 8 ; 5 ; 17
Key result
Run / experiment:
other: 1
Parameter:
other: IC30
Remarks:
(µM)
Value:
3.69
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: IC50
Remarks:
(µM)
Value:
18.91
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
1.5
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: IC30
Remarks:
(µM)
Value:
25.37
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: IC50
Value:
29.65
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: Imax
Value:
1.67
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5
Remarks:
calculated (µM)
Value:
1.87
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: IC30
Value:
15.23
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: IC50
Value:
19.29
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: Imax
Value:
1.5
Vehicle controls validity:
other: same as negative control
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
First run
All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid.
Second run
All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.
Third run
Since non-concordant results were obtained throughout the two first runs, a third run was performed. In the third run, all acceptance criteria were met for the positive and negative controls, with one exception regarding positive control EC1.5 slightly higher than historical data (i.e. 15.6 μM instead of ranging from 2.8 to 13.9 μM) which was considered not to have any impact on the validity of the results, the run was therefore considered to be valid.

IC30 and IC50: Concentrations effecting a reduction of cellular viability by 50% and 30%

Imax : Maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item, 80ACQ, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.
Executive summary:

test item was found soluble in DMSO at 200 mM and 100 mM. Precipitates were observed once these formulations were 100-fold diluted in culture medium.

The three runs were performed using the following concentrations in culture medium containing 1% DMSO:

. first run: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM,

. second run: 0.71, 1.01, 1.42, 2, 2.82, 3.98, 5.61, 7.91, 11.15, 15.72, 22.16 and 31.25 μM,

. third run: 0.02, 0.03, 0.06, 0.12, 0.24, 0.49, 0.98, 1.95, 3.91, 7.81, 15.63 and 31.25 μM.

At these tested concentrations:

. slight to strong precipitates were observed in treated wells at concentrations ≥ 500 μM,

. a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 3.91 μM in the first run, ≥ 31.25 μM in the second run and ≥ 0.98 μM in the third run,

. the corresponding geometric means IC30 and IC50 of the three validated runs were calculated to be 11.26 and 22.11 μM, respectively,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to

the negative control in non-cytotoxic and non-precipitating concentrations for both the first and third runs. In the second run, statistically gene-fold inductions above the threshold of 1.5 were noted at

2 μM and at concentrations between 3.98 to 11.15 μM with an apparent dose-response until 11.15 μM.

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

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 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:
04 February 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.
It is an alternative method in order to avoid in vivo study.
Specific details on test material used for the study:
Storage conditions:
. At room temperature
. Protected from light
. Protected from humidity
Details on the study design:
Methods
The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.
Peptide reactivity was reported as percentage depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate vehicle).

Based on solubility results, the retained vehicle was acetone.
Positive control results:
The positive control was dissolved in acetonitrile at 100 mM. The obtained formulation was a colorless limpid solution. The formulation was used just after its preparation.
Mean depletion rate (%) of Cinnamaldehyde : 67.34 / depletion clasiification : high reactivity (42.47% < Mean % depletion ≤ 100%)
Key result
Run / experiment:
other: 1
Parameter:
other: %depletion
Remarks:
cysteine peptide
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Value set to 0 due to negative depletion
Key result
Run / experiment:
other: 1
Parameter:
other: %depletion
Remarks:
lysine peptide
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Value set to 0 due to negative depletion
Key result
Run / experiment:
other: 2
Parameter:
other: %depletion
Remarks:
cysteine
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Value set to 0 due to negative depletion
Key result
Run / experiment:
other: 2
Parameter:
other: %depletion
Remarks:
lysine
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Value set to 0 due to negative depletion
Key result
Run / experiment:
other: 3
Parameter:
other: %depletion
Remarks:
cysteine
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Value set to 0 due to negative depletion
Key result
Run / experiment:
other: 3
Parameter:
other: %depletion
Remarks:
lysine
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Value set to 0 due to negative depletion
Other effects / acceptance of results:
ACCEPTANCE CRITERIA
The run was considered valid if the following criteria were fully met:
. the calibration curves should have a coefficient of determination (r2) ≥ 0.99,
. the mean peptide concentrations of the reference control A samples should be within ± 10% of the nominal concentration,
. the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
− for the cysteine peptide, the mean percentage depletion value should be between 60.8 and 100% with a SD < 14.9%,
− for the lysine peptide, the mean percentage depletion value should be between 40.2 and 69.0% with a SD < 11.6%,
. the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0%.
The test item’s results were considered valid if the following criteria were fully met:
. the mean peptide concentrations of the reference control C samples prepared in the appropriate vehicle should be within ± 10% of the nominal concentration,
. the maximum SD for the test item replicates should be < 14.9% for the percentage cysteine depletion value and < 11.6% for the percentage lysine depletion value.

Lysine peptide: due to a mistaken quantity of ammonium acetate weighed during the preparation of the ammonium acetate buffer solution, its concentration was 60 mM instead of the 100 mM, as specified in the internal analytical method. This buffer solution is then used for the preparation of lysine peptide. However, since all acceptance criteria were fulfilled with this peptide, this deviation was considered not to have compromised the validity or integrity of the study.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item 80ACQ, was considered to have no or minimal peptide reactivity, though with limitations due to its precipitation with the peptides.
Executive summary:

Results

The test item was dissolved at 100 mM in acetone.

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

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percentage depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:

. for the cysteine peptide, the mean depletion value was set to 0%,

. for the lysine peptide, the mean depletion value was set to 0%.

The mean of the percentage cysteine and percentage lysine depletions was set to 0%. Accordingly, the test item was considered to have no or minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization.

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

Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
other information
Study period:
May 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Justification for type of information:
The In Vitro Skin Sensitisation Human Cell Line Activation Test (h-CLAT) could not have been performed because of the logKow value of the test item (>3.5).
QSAR data have been provided too.
Interpretation of results:
other: QSAR
Remarks:
predicted value: negative
Conclusions:
According to the QSAR prediction Skin Sensitization (Danish EPA), 80ACQ is predicted to be not sensitising
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification