Benzene-1,3,5-tricarboxylic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-09 to 2017-08-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Benzene-1,3,5-tricarboxylic acid

EC Number:

209-077-7

EC Name:

Benzene-1,3,5-tricarboxylic acid

Cas Number:

554-95-0

Molecular formula:

C9H6O6

IUPAC Name:

benzene-1,3,5-tricarboxylic acid

Test material form:

other: powder

Details on test material:

benzene-1,3,5-tricarboxylic acid by Sigma Aldrich, batch no. C1511005, expire date: 2020

In vitro test system

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
42.0 mg of the test item were dissolved in 1 mL dimethyl sulfoxide (DMSO) to a concentration of 200 mM. 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 will then be 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).

Results and discussion

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.

In vitro / in chemico

Results

Results 1

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

Results 2

Run / experiment:

other: Luciferase determinations

Parameter:

other: I max

Remarks:

maximal fold-induction over solvent control (Imax)

Value:

1.18

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

Results 3

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.

Results 4

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 and IC30 value was > 2000 µM for 50% and 30% reduction of cellular viability, respectively

Results 5

Run / experiment:

other: Cytotoxicity determinations

Parameter:

other: IC 30 [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 and IC30 value was > 2000 µM for 50% and 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:
4) 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);
5) 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);
6) the EC1.5 value is less than (<) 1000 µM;
7) 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.

Any other information on results incl. tables

Numerical results for the test item

	Luciferase determinations		Cytotoxicity determinations
Parameter	Imax	EC1.5[µM]	IC50[µM]	IC30[µM]
Test item	1.18 ± 0.01	-	>2000	>2000

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	1.23	1.28	1.36	2.00*	2.53*	19.46	TRUE	TRUE
Replicate 2	0.99	1.08	1.47	1.91*	3.40*	17.05	TRUE	TRUE
Average	1.11	1.18	1.42	1.95	2.96	18.25	TRUE	TRUE

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

Test item 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 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.

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 was tested at 12 concentrations in the range from 0.98 to 2000 µM. 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            1.18 ± 0.01        -            >2000   >2000

- = 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 1.18 ± 0.01 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 and IC30 value was > 2000 µM for 50% and 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 solvent control and the positive control cinnamic aldehyde were run in both repetitions. All quality criteria for luciferase induction and variability of the solvent control and positive control required were fulfilled.

 

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