Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2017 - 29 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in chemico skin sensitization tests is the Direct Peptide Reactivity assay (DPRA), which is recommended in international guidelines (e.g. OECD).

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)

Justification for non-LLNA method:

The purpose of this study was to evaluate the reactivity of the test substance towards model synthetic peptides containing either cysteine or lysine, and to categorize the test item in one of four classes of reactivity for supporting the discrimination between skin sensitizers and nonsensitizers

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light container flushed with nitrogen

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

Test system: 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.
Stock solutions of SPCC and SPCL were prepared by dissolving 10 mg of the peptide in 19.96 mL phosphate buffer or 19.31 mL ammonium acetate buffer, respectively. The reference control solutions (0.5 mM) were prepared by diluting the stock solutions with acetonitrile. The samples for the calibration curves for both peptides were prepared at concentrations 0.017, 0.033, 0.067, 0.133, 0.267 and 0.533 mM.
Cinnamic aldehyde at concentration 100 mM in acetonitrile was used as a positive control.
Test item solution was prepared in methanol at concentration 100 mM based on the results of preliminary solubility tests.

Sample incubations:
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in an incubator at 25 ± 2.5 °C. After incubation, the samples were transferred to the autosampler. The incubation time between placement of the samples in the incubator and analysis of the first RCcysB- or RClysB- sample was 24.5 hours. The time between the first RCcysB- or ClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.

Analysis:
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA at 220 nM. 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)] x 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%
Acceptability criteria:
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.

The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50 ± 0.05 mM.

Data interpretation:
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see table in the section "Any other information on materials and methods incl. tables"), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

Positive control results:

The mean Percent SPCC Depletion and mean Percent SPCL Depletion for the positive control cinnamic aldehyde were 74.9% ± 1.8% and 57.3% ± 2.9%, respecitvely. These values were within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%) for SPCC and of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%) for SPCL.

Key result

Run / experiment:

other: 1

Parameter:

other: % SPCC depletion

Value:

1.9

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: % SPCC depletion

Value:

1.2

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 3

Parameter:

other: % SPCC depletion

Value:

2.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: % SPCL depletion

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: % SPCL depletion

Value:

0.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 3

Parameter:

other: % SPCL depletion

Value:

5.9

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:

OTHER EFFECTS:
- Visible damage on test system:

DEMONSTRATION OF TECHNICAL PROFICIENCY: the suitability of the HPLC system was demonstrated by measuring the reference control samples for SPCC and SPCL. The mean peptide concentrations of reference control sames were all within the acceptance criteria of 0.50 ± 0.05 mM. The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls was 1.0% for SPCC and 1.9% for SPCL. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 17.81 for SPCC and 16.21 for SPCL. The mean A220/A258 ratio ± 10% range were 16.03-19.59 and 14.59-17.83, respectively. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes, the mean Percent SPCC Depletion and mean Percent SPCL Depletion for the positive control cinnamic aldehyde were 74.9% ± 1.8% and 57.3% ± 2.9%, respecitvely. These values were within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%) for SPCC and of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%) for SPCL.
- Acceptance criteria met for variability between replicate measurements: yes, the standard deviations were 0.6% and 3.2%, which is below the acceptability criteria of 14.9% and 11.6% for SPCC and SPCL depletion, respectively.

Interpretation of results:

study cannot be used for classification

Remarks:

This study is part of a weight of evidence approach on which the classification is based.

Conclusions:

Based on the outcome of a Direct Peptide Reactivity Assay (DPRA) performed according to OECD guideline and GLP principles, Polyambrol is classified as negative (no depletion of synthetic peptides containing either cysteine or lysine) under the experimental conditions described in this report.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 September 2017 - 06 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise 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).

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 3 November 2015

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The purpose of this study was to evaluate the ability of Polyambrol to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. Activation of this pathway can lead to skin sensitisation.

Specific details on test material used for the study:

Specific gravity/density: 0.946 (20ºC); 0.943 (25ºC)

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

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

CONTROLS:
Positive control: ethylene dimethacrylate glycol (purity: 98.3%), 7.8-250 µM, tested in triplicate, DMSO was used as a vehicle;
Negative control: DMSO (1% in exposure medium);
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values.

TEST SYSTEM:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (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) 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. The passage number used was 22 in experiment 1 and 24 in experiment 2.

TEST ITEM PREPARATION:
The test item was suspended in DMSO to a final concentration of 40 mg/mL (clear and colourless). 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 400, 200, 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39 and 0.20 μg/mL (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates.

TEST CONCENTRATIONS:
- Both experiments: 400, 200, 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39 and 0.20 μg/mL

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 35 – 99 %) at 37.0 ± 1.0°C (actual range 35.4 – 44.6°C), in the presence of 5% ± 0.5% CO2.

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 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 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.

Positive control results:

The EC1.5 of the positive control was between 5 and 125 μM (59 μM and 78 μM in experiment 1 and 2, respectively). A dose related response was observed and the induction at 250 μM was higher than 2-fold (2.27-fold and 2.18-fold in experiment 1 and 2, respectively).

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

1.13

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: No unit, 1.13-fold induction

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

1.18

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: No unit, 1.18-fold induction

Other effects / acceptance of results:

- In the first experiment, the test item showed toxicity (IC30 value of 34 μg/mL, IC50 value of 38 μg/mL). No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.13 and therefore no EC1.5 could be calculated.
- In the second experiment, the test item showed toxicity (IC30 value of 65 μM, IC50 value of 75 μM). No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.18 and therefore no EC1.5 could be calculated.

- In both experiments, no precipitation was observed at the start and end of the incubation period in the 96-well plates.

All three 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 (59 μM and 78 μM in experiment 1 and 2, respectively). A dose related response was observed and the induction at 250 μM was higher than 2-fold (2.27-fold and 2.18-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (18.8% and 6.8% in experiment 1 and 2, respectively).

Table1          Overview Luminescence Induction and Cell Viability of POLYAMBROL in Experiment 1 and 2

Concentration (µg/mL)	0.20	0.39	0.78	1.6	3.1	6.3	13	25	50	100	200	400
Exp 1 luminescence	0.61	0.69	0.63	0.70	0.79	0.88	0.92	1.13	0.09	0.17	0.00	0.00
Exp 1 viability (%)	86.9	89.9	98.3	99.3	90.7	99.4	103.4	104.8	3.2	0.2	0.0	-0.2
Exp 2 luminescence	1.06	1.08	1.11	1.06	1.05	1.08	1.07	1.18	1.16	0.00	0.00	0.00
Exp 2 viability (%)	105.4	100.3	98.8	99.6	98.5	101.5	98.9	103.3	99.2	0.1	0.0	0.1

 

Table2          Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	0.90	1.07	1.25	1.53***	1.94***	2.27***
Exp 1 viability (%)	105.5	108.3	116.6	111.6	108.1	112.5
Exp 2 luminescence	1.10	1.26	1.22	1.46	1.64***	2.18***
Exp 2 viability (%)	96.3	99.7	102.9	106.6	111.5	110.2

^***p<0.001 Student’s t test

 

Table3          Overview EC_1.5, I_max, IC₃₀ and IC₅₀Values

	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	NA	1.13	34	38
Test item Experiment 2	NA	1.18	65	75
Pos Control Experiment 1	59	2.27	NA	NA
Pos Control Experiment 2	78	2.18	NA	NA

NA = Not applicable

 

 

Interpretation of results:

study cannot be used for classification

Remarks:

This study is part of a weight of evidence approach on which the classification is based.

Conclusions:

Based on the outcome of a KeratinoSens™ assay performed according to OECD guideline and GLP principles, Polyambrol is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Polyambrol has been assessed for its potential to induce skin sensitization in two Assays.

The in chemico DPRA assay is negative and the in vitroKeratinoSens is also negative.

In conclusion, this substance has no potential to induce skin sensitization in humans.