Tetraethylammonium benzoate

Endpoint summary

Description of key information

The test item revealed no sensitising properties in the in vitro ARE-Nrf2 Luciferase test method according to OECD 442D (LPT, 2018). Additionally, in the Direct Peptide Reactivity Assay (DPRA) according to OECD 442C the test item revealed a mean cysteine and lysine peptide depletion of 0.316% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) (LPT, 2017).

Based on these clear negative results in the two in vitro/in chemico studies no further study regarding skin sensitisation was performed for the test item. A data waiver was claimed.

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:

2017-08-22 to 2017-09-01

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:

adopted 4 February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Preparation of the cysteine or lysine-containing peptides
- Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 95% were freshly prepared just before their incubation with the test item.
- The final concentration of the cysteine peptide was 0.666 mM in pH 7.5 phosphate buffer, whereas the final concentration of the lysine peptide was 0.666 mM in pH 10.2 ammonium acetate buffer.

Preparation of the test item
- Solubility of the test item in an appropriate solvent was assessed before performing the assay.
- 151.10 mg test item were dissolved in 3 mL highly purified water immediately before testing to prepare a 100 mM solution.
- The test item solution was then tested as such without any further dilution by incubating at 1:10 or 1:50 ratio with the cysteine peptides and lysine peptides, respectively

Positive control, reference controls and coelution control
- Cinnamic aldehyde (CAS no. 14371-10-9) was used as positive control (PC) at a concentration of 100 mM in acetonitrile.
- In addition reference controls (i.e. samples containing only the peptide and added acetonitrile) were also included in the HPLC run sequence and these were used to verify the HPLC system suitability prior to the analysis (reference controls A), the stability of the reference controls over time (reference control B) and to verify that the solvent used to dissolve the test item does not impact the percent peptide depletion (reference control C).
- The appropriate reference control for the test item was used to calculate the percent peptide depletion for the test item.
- In addition a coelution control constituted by the test item alone for the test item analysed was included in the run sequence to detect possible coelution of the test item with either the lysine or the cysteine peptide.

Incubation of the test item with the cysteine and lysine peptide solutions
- Cysteine and lysine peptide solutions were incubated in glass autosampler vials with the test item at 1:10 and 1:50 ratio, respectively.
- The reaction solution was left in the dark at 25 ± 2.5°C for 24 ± 2 hours before running the HPLC analysis.
- The test item assay was analyzed in triplicate for both peptides.
- Samples were visually inspected prior to HPLC analysis.
- If a precipitate would be observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item, in this case one cannot be sure how much test item remained in the solution to react with the peptide.
- Therefore, in such a case, a positive result could still be used, but a negative result is uncertain and would be interpreted with due care.
- No precipitate or phase separation was observed.

Preparation of the HPLC standard calibration curve
- A standard calibration curve was generated for both the cysteine and the lysine peptides.
- Peptide standards were prepared in a solution of 20% acetonitrile : buffer using 100 mM sodium phosphate buffer (pH 7.5) for the cysteine peptide and 100 mM ammonium acetate buffer (pH 10.2) for the lysine peptide.
- Using serial dilution standards of the peptide stock solution (0.666 mM of cysteine peptide in sodium phosphate or 0.666 mM lysine peptide in ammonium acetate), 6 calibration standards were prepared to cover the range from 0.534 to 0.0167 mM.
- A blank of the dilution buffer was also included in the standard calibration curve.
- Suitable calibration curves should have an r2 > 0.99.
- If a test item promotes the oxidation of the cysteine peptide, the peak of the dimerised cysteine peptide would have been visually monitored.
- If dimerisation appears to have occurred, this would be noted as percent peptide depletion would be over-estimated leading to false positive predictions and/or assignment to a higher reactivity class.
- HPLC analysis for the cysteine and lysine peptides were performed on one day.
- All test item solutions were freshly prepared for both assays on one day.
- The analysis was timed to assure that the injection of the first sample (reference control C) starts 22 to 26 hours after the test item was mixed with the peptide solution.
- The HPLC run sequences were set up in order to keep the HPLC analysis time to less than 30 hours.

Positive control results:

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 70.11% for cysteine and 50.98% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

Key result

Run / experiment:

other: incubating at 1:10 and 1:50 ratio with the cysteine peptides

Parameter:

other: cysteine peptide depletion

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: incubating at 1:10 and 1:50 ratio with the lysine peptides

Parameter:

other: lysine peptide depletion

Value:

0.63

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

No precipitate in the reaction mixture at the end of the incubation time and no coelution were observed.

Data evaluation
The concentrations of cysteine or lysine peptide were photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks 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 control C according to the formula described below.

% peptide depletion= 1-[( Peptide peak area in replicate injection / Mean peptide peak area in reference controls C)] x 100


Acceptance criteria
The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
c) The mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.

The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should 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 should be 0.50 ± 0.05 mM.
If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.


Prediction model see table "any other information on results"

Prediction model

The mean percent cysteine and percent lysine depletion value was calculated for each test item. Negative depletion was considered as “0” when calculating the mean. By using the cysteine 1:10/lysine 1:50 prediction model shown in Table below, the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an Integrated Approach to Testing and Assessment(IATA). Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate and high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA.

Cysteine 1:10/lysine 1:50 prediction model

Mean of cysteine and lysine % depletion	Reactivity Class	DPRA Prediction
0% <mean % depletion≤6.38%	No or minimal reactivity	Negative
6.38%<mean % depletion≤22.62%	Low reactivity	Positive
22.62%<mean % depletion≤42.47%	Moderate reactivity
42.47%<mean % depletion≤100%	High reactivity

There was an overlap in retention time between test item dissolved in water and the cysteine peptide (co-elution). As the overlap between the test item and cysteine peptide was incomplete, the percent peptide depletion values could be estimated and used in the cysteine 1:10/lysine 1:50 prediction model. In this case the co-elution peak could exactly be integrated and subtracted from the test item solution peak.

A single HPLC analysis for both the cysteine and the lysine peptide would be sufficient for a test item when the result is unequivocal. However, in cases of results close to the threshold used to discriminate between positive and negative results (i.e. borderline results), additional testing may be necessary. If situations where the mean percent depletion falls in the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model or the cysteine percent depletion falls in the range of 9% to 17% for the cysteine 1:10 prediction model, a second run would be considered, as well as a third one in case of discordant results between the first two runs.

 

Conclusions:

The test item revealed a mean cysteine and lysine peptide depletion of 0.316% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Executive summary:

The purpose of this study was to determine the sensitising potential of test item in a Direct Peptide Reactivity Assay (DPRA). The study was performed according to OECD guideline 442C. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values are then 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 sensitisers and non-sensitisers.

The test item was dissolved at a concentration of 100 mM in highly purified water.

Two reference controls containing only 0.5 mM cysteine peptide solution or 0.5 mM lysine peptide solution and acetonitrile were also included in the HPLC run sequence and were used to verify the HPLC system suitability prior to analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding highly purified water (vehicle) to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. Each sample was tested in triplicate.

There was an overlap in retention time between test item dissolved in water and the cysteine peptide (co-elution). As the overlap between the test item and cysteine peptide was incomplete and the co-elution peak from the co-elution control could exactly be integrated and subtracted from the test item solution peak, the percent peptide depletion values could be estimated and used in the cysteine 1:10/lysine 1:50 prediction model.

Test item samples revealed a corrected cysteine peptide depletion of 0.0% and lysine peptide depletion of 0.63% (mean peptide depletion of 0.316%) and, hence, well below 6.38% Test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile.Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 70.11% for cysteine and 50.98% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.Therefore, the study can be regarded as valid.

The acceptance criteria of validity were fulfilled in this test.

 

Conclusion

The test item revealed a mean cysteine and lysine peptide depletion of 0.316% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-22 to 2017-09-29

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

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Preparation of the keratinocyte cultures
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element was used (KeratinoSens™ cell line ). Cells were propagated and stored frozen as a homogeneous stock. Cells from this original stock were propagated up to a maximum passage number of 25 and were employed for routine testing using the appropriate maintenance medium.
For testing, cells were 80-90% confluent, and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested and distributed into 96-well plates (10 000 cells/well). Attention was paid to avoid sedimentation of the cells during seeding to ensure homogeneous cell number distribution across wells. For each repetition, three technical replicates were used for the luciferase activity measurements, and three parallel technical replicates used for the cell viability assay.

Preparation of the test and control items
100.54 mg of the test item were dissolved in 1 mL dimethyl sulfoxide (DMSO) to a concentration of 200 mM N,N,N-triethylethanaminium benzoate. A correction factor of 2 was used as the content of N,N,N-triethylethanaminium benzoate was 49.9% in water. All concentrations refer to N,N,N-triethylethanaminium benzoate. Fresh preparations of the test and control items were used for the treatment. The final concentration of the vehicle in the culture system did not affect cell viability or growth rate.
Based on the stock solution of the test item, serial dilutions were made using solvent to obtain 12 master concentrations to be tested (from 0.098 to 200 mM). The master concentrations were then further diluted in treatment culture medium containing 1% serum , so that the final concentrations of the test item range from 0.98 to 2000 µM. The solvent DMSO was used as the negative control. Six wells per plate were prepared. It was diluted following the same dilution scheme as described for the master concentrations, so that the final negative control concentration is 1%, which is known to not affect cell viability and corresponds to the same concentration of DMSO found in the test item and in the positive control.
Cinnamic aldehyde was used as the positive control. A series of 5 master concentrations ranging from 0.4 to 6.4 mM was prepared in DMSO and diluted as described for the master concentrations, so that the final concentration of the positive control range from 4 to 64 µM.

Application of the test and control items
For each test chemical and positive control item, one experiment is needed to derive a prediction (positive or negative), consisting of at least two independent repetitions each containing three replicates (i.e. n=6). Each independent repetition was performed on a different day with fresh stock solution of test chemicals and independently harvested cells. Cells may come from the same passage however.
After seeding, cells were grown for 24 hours in the 96-well microtiter plates. The medium was then removed and replaced with fresh culture medium (150 µL culture medium containing 1% serum but without Geneticin to which 50 µL of the diluted test and control items were added. Three wells per plate were carried out containing no cells to assess background values.
The treated plates were then incubated for about 48 hours at 37 ± 1°C in the presence of 5% CO2. Evaporation of volatile test chemicals and cross-contamination between wells by test items were avoided by covering the plates with a foil prior to the incubation with the test items.

Luciferase activity measurements
After the 48 hour exposure time with the test and control items, cells were washed with a phosphate buffered saline, and the relevant lysis buffer (One GlowTM Luciferase Assay System) for luminescence readings added to each well for an adequate time at room temperature. Plates with the cell lysate will then be placed in the luminometer (Tecan Infinite 200Pro) for reading.

Cytotoxicity assessment
For the KeratinoSensTM cell viability assay, the 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) and cells incubated for 4 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed by adding 10% aqueous SDS solution to each well overnight or for up to 3 days at 37 °C. After shaking, the absorption was measured at i.e. 620 nm with a photometer (TecanSunrise Magellan Version 7.2).

Positive control results:

Cinnamic aldehyde tested at five concentrations from 4 – 64 µM was used as the positive control.
The positive control cinnamic aldehyde were run in both repetitions. All quality criteria for luciferase induction and variability of the positive control required were fulfilled.

Key result

Run / experiment:

other: Luciferase induction

Parameter:

other: KeratinoSens

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Solvent control

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Luciferase determinations

Parameter:

other: I max

Remarks:

maximal average fold induction of the luciferase activity (Imax) value

Value:

1.39

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Solvent control

Positive controls validity:

valid

Remarks:

see table below "any other information"

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Luciferase determinations

Parameter:

other: EC1.5 [µM]

Remarks:

the concentration needed to reach an 1.5 fold induction (EC1.5)

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Solvent control

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

no EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) could be calculated.

Run / experiment:

other: Cytotoxicity determinations

Parameter:

other: IC 50 [µM]

Value:

2 000

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Solvent control

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The calculated IC50 value was > 2000 µM for 50% reduction of cellular viability, respectively

Run / experiment:

other: Cytotoxicity determinations

Parameter:

other: IC 30 [µM]

Value:

1 348.38

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Solvent control

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The calculated IC30 value was 1348.38 µM for 30% reduction of cellular viability, respectively

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Acceptance criteria
The following acceptance criteria should be met.
1) the luciferase activity induction obtained with the positive control (cinnamic aldehyde), should be statistically significant above the threshold of 1.5 compared to the negative (solvent) control (e.g. using a t-test) in at least one of the tested concentrations (from 4 to 64 µM).
2) the EC1.5 value should be within two standard deviations of the historical mean. In addition, the average induction in the three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of the positive control (cinnamic aldehyde) should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
3) the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 6 wells tested in triplicate. If the variability is higher, results are discarded.

Interpretation of results and prediction model
A prediction is considered positive if the following 4 conditions are all met in 2 of 2 or, if necessary in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1) the Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test);
2) the cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration);
3) the EC1.5 value is less than (<) 1000 µM;
4) there is an apparent overall dose-response for luciferase induction. The Spearman's rank correlation coefficient was employed for investigation of a possible dose-relationship.

If in a given repetition, all of the first three conditions are met but a clear dose-response for the luciferase induction cannot be observed, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM should also be considered as inconclusive.
In rare cases, test items which induce the luciferase activity very close to the cytotoxic levels can be positive in some repetitions at non-cytotoxic levels (i.e. EC1.5 determining concentration below (<) the IC30), and in other repetitions only at cytotoxic levels (i.e. EC1.5 determining concentration above (>) the IC30). Such test items would have been retested with more narrow dose-response analysis using a lower dilution factor (e.g. 1.33 or √2 (=1.41) fold dilution between wells), to determine if induction has occurred at cytotoxic levels or not.

Numerical results for the test item

	Luciferase determinations		Cytotoxicity determinations
Parameter	Imax	EC1.5[µM]	IC50[µM]	IC30[µM]
Test item	1.39 ± 0.09	-	>2000	1348.38

- = no concentration with calculated ≥ 1.5 fold luciferase induction

Numerical results for the positive control (cinnamic aldehyde)

Positive control: Induction values Reference							Criteria#
cinnamic aldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Induction 64 µM	EC1.5
Replicate 1	1.17	1.25	1.50	1.99*	3.28*	15.98	TRUE	TRUE
Replicate 2	1.11	1.06	1.29	1.57*	2.63*	27.97	TRUE	TRUE
Average	1.14	1.15	1.39	1.78	2.96	21.14	TRUE	TRUE

#  the induction in the two replicates 64 µM should be between 2 and 8, the EC_1.5value should be between 7 µM and 30 µM.

 

Conclusions:

In conclusion, test item revealed no sensitising properties in the ARE-Nrf2 Luciferase test method.

Executive summary:

Test item 50% was examined for sensitising properties in the ARE-Nrf2 luciferase test method. The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.

Two endpoints were measured: luciferase induction after a 48 hour treatment with test item 50% and cytotoxicity determined with the MTT assay with the same cell batch and employing the same dilutions of the test item. DMSO was used as solvent control. A correction factor of 2 was used as the content of test item was 49.9% in water. All concentrations refer to the test item benzoate.

For Luciferase induction the maximal fold-induction over solvent control (Imax) and the concentration needed to reach an 1.5 fold induction (EC1.5) were calculated. For cytotoxicity the IC50 and IC30 values were interpolated.

Test item 50% was tested at 12 concentrations in the range from 0.98 to 2000 µM test item. Cinnamic aldehyde tested at five concentrations from 4 – 64 µM was used as the positive control. Two independent repetitions with three parallel technical replicates were run with this same set-up, and one parallel plate was prepared for cytotoxicity determination.

For the MTT data the % viability was then calculated for each well in the test plate in relation to average of the six solvent control wells.

For the luciferase data the average value of the six solvent control wells was set to 1, and for each well in the test plate the fold induction was calculated in relation to this value.

 

The following parameters of luciferase induction and cytotoxicity determinations were calculated for the test item-treated cells:

Luciferase determinations          Cytotoxicity determinations

Parameter        Imax     EC1.5 [µM]               IC50 [µM]          IC30 [µM]

Test item            1.39 ± 0.09        -                   >2000   1348.38

- = no concentration with calculated≥1.5 fold luciferase induction

 

The maximal average fold induction of the luciferase activity (Imax) value observed at any concentration of the test item was 1.39 ± 0.09 and no EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) could be calculated.

The calculated IC50 value was > 2000 µM test item for 50% and IC30 and 1348.38 µM for 30% reduction of cellular viability, respectively.

The KeratinoSensTM prediction of the test item is considered negative as the luciferase induction value was < 1.5 compared to the solvent control.

The positive control cinnamic aldehyde was run in both repetitions. Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This was the case in both repetitions. The induction at 64 µM and the EC1.5 for cinnamic aldehyde were also calculated. The targets are:

•            the average induction in the two replicates for cinnamic aldehyde at 64 µM should be between 2 and 8,

•            the EC1.5 value should be between 7 µM and 30 µM or within two standard deviations of the historical mean value.

At least one of these two numerical criteria must be met in order to accept a repetition. In the experiments performed both criteria were fulfilled in both repetitions. Thus both repetitions were valid for the positive control.

In addition, the EC1.5 value of the positive control of 21.14 µM is within two standard deviations of the historical mean.

As further performance criterion the variability of the solvent control must be below 20%. The average coefficients of variation (CV) for the solvent control were 6.22% or 7.43%.

All quality criteria required (see section 6.2) were fulfilled.

In conclusion, test item 50% revealed no sensitising properties in the ARE-Nrf2 Luciferase test method.

Reference 3

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the clear negative results of the two in vitro skin sensitization studies the test item is not classified according to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008.