Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Endpoint summary

Description of key information

Two in vitro skin sensitisation tests (ARE-Nrf2 Luciferase Test and human Cell Line Activation Test) were performed. The results indicate that benzoyl-m-toluoylmethylresorcinol has skin sensitising properties. Further test work is ungoing to determine the skin sensitisation potency of BTMR.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-June-2021 to 01-July-2021 (experimental dates)

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes

Type of study:

human Cell Line Activation Test (h-CLAT)

Specific details on test material used for the study:

Description: Light brown powder
Storage conditions: Stored at 15-25°C, protected from light.

Details of test system:

THP-1 cell line [442E]

Details on the study design:

Preliminary solubility data indicated that BTMR was not directly soluble in culture medium (RPMI-1640) or saline. Solubility was however achieved in DMSO at a concentration of 110 mg/mL.

A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The test article was formulated at 110 mg/mL in DMSO and then diluted a series of times with the corresponding solvent and in culture medium. The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate to obtain a final range of concentrations in the plate of 1.72 220 µg/mL. As there was no effect on viability in the dose finding assay and no CV75 was obtained, eight stock solutions of test article were prepared by 1.2-fold serial dilutions to give eight concentrations, and the stock solutions were then further diluted 250-fold into the culture medium (working solutions). The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate. Final test concentrations in a range from 61.4 to 220 µg/mL (after dilution in medium) were used for the expression measurements.

THP-1 cells were cultured in a humidified incubator set to 37ºC, 5% CO2, in RPMI 1640 medium supplemented with 10% heat inactivated (HI) foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 µg/mL streptomycin. Cell density did not exceed 1 x 106 cells/mL, and cells did not exceed 30 passages.

Reactivity check was performed using DNCB, nickel sulphate and lactic acid two weeks after thawing. Only cells which passed the reactivity check were used for the assay.

The test article working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24±0.5 hours (incubator set to 37ºC, 5% CO2). After the 24-hour incubation period, all cells from the 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 µL of phosphate buffered saline containing 0.1% bovine serum albumin (FACS buffer) and re-suspended in 190 µL of FACS buffer. 10 µL of propidium iodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 µg/mL). PI uptake was analyzed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired.
Cell viability and the CV75 value were calculated.

Two independent runs (experiments) were needed to drive a prediction. Each independent run was performed on a different day. On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium and distributed into a 24-well plate. The test article working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24 hours (incubator set to 37ºC, 5% CO2).
After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) on ice for 15 minutes. After blocking, the cells were centrifuged and stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes. The stained cells were washed three times with an excess of FACS buffer, resuspended in FACS buffer and 12.5 µg/mL PI solution was added (to give a final PI concentration of 0.625 µg/mL).
The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

Vehicle / solvent control:

DMSO

Positive control:

other: 2,4-dinitrochlorobenzene (DNCB)

Positive control results:

RFI values were 150% for CD86 and 200% for CD54, and cell viability was >50% in each independent run.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC150, CD86 [442E]

Value:

140.49 µg/mL

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

In Experiment 1, the RFI values for CD86 were >150% (with cell viability >50%) at multiple concentrations and the RFI values for CD54 were <200% at all concentrations. In Experiment 2, the RFI values for CD86 were >150% and the RFI values for CD54 were >200% at one or more concentrations (with cell viability >50%). The test article therefore gave a positive prediction in the assay.
The EC150 value for CD86 calculated by linear regression of endpoint assay data was 140.49 µg/mL. No EC200 value was calculated for CD54 as this marker was negative in Experiment 1.

All assay acceptance criteria were met.
- The cell viabilities of medium and solvent/vehicle control were higher than 90% in each independent run.
- In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI =150% and CD54 RFI =200%).
- For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
- For the positive control, RFI values were =150% for CD86 and =200% for CD54, and cell viability was >50% in each independent run.
- For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.

Table 1 - BTMR relative fluorescence intensity (RFI) 

Concentration (µg/mL)	RFI (CD86)	RFI (CD86)	RFI (CD54)	RFI (CD54)
	Exp 1	Exp 2	Exp 1	Exp 2
61.40	112	97	79	93
73.68	196	113	164	108
88.41	167	115	94	103
106.10	189	142	121	157
127.31	169	127	92	116
152.78	134	171	96	144
183.33	146	153	107	240
220.00	130	111	121	142
Solvent/vehicle control (DMSO)	100	147	89	116
Positive control (DNCB)	534	431	1070	879

Interpretation of results:

study cannot be used for classification

Remarks:

The data can only be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitizers and non-sensitizers for the purpose of hazard classification and labelling.

Conclusions:

In a human Cell Line Activation Test (h-CLAT), performed according to OECD TG 442E, BTMR was considered to be positive.

Executive summary:

A human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E.

For the dose finding assay, the test article was dissolved in dimethyl sulfoxide (DMSO) at a maximum attained of concentration of 110 mg/mL giving a maximum test concentration of 220 µg/mL. No reduction in viability was observed.

For the expression measurements, test concentrations in a range from 61.4 to 220 μg/mL (after dilution in medium) were used. Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 10E6 cells per well. After blocking, the cells were stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes. The stained cells were washed, re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

In Experiment 1, the RFI values for CD86 were >150% (with cell viability >50%) at multiple concentrations and the RFI values for CD54 were <200% at all concentrations. In Experiment 2, the RFI values for CD86 were >150% and the RFI values for CD54 were >200% at one or more concentrations (with cell viability >50%). The test article therefore gave a positive prediction in the assay.

The EC150 value for CD86 was calculated to be 140.49 μg/mL.

All acceptance criteria of the h-CLAT assay parameters were met in each experiment.

BTMR was considered to be positive in the human Cell Line Activation Test.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01-June-2021 to 11-June-2021 (experimental dates)

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Specific details on test material used for the study:

Description: Light brown powder
Storage conditions: Stored at 15-25°C, protected from light.

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

Preliminary solubility data indicated that BTMR was soluble in dimethyl sulfoxide (DMSO) up to at least 72.42 mg/mL, which was in excess of the target concentration 200 mM (equivalent to 69.20 mg/mL).

The test article was dissolved in dimethyl sulfoxide (DMSO) to the final stock concentration (200 mM). Serial dilutions were then made using DMSO to obtain 12 master concentrations of the test article in the range 0.098 to 200 mM, producing a visually clear solution. The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations range from 0.98 to 2000 µM. Formulations were prepared shortly before testing. The positive control was prepared at a concentration of 6.4 mM in DMSO. Five master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 6.4 mM stock solution). The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations range from 4 to 64 µM.

Aliquots of 50 µL of each of the final concentrations were transferred to give three luciferase replicates on a white-walled plate and a single viability replicate on a clear-walled plate. Treatment plates were prepared using cells (passages 12 or 13) that were 80 - 90% confluent. On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated for 24±1 hours an incubator set to 37°C, 5% CO2. For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay. After addition of the test item or control samples, each plate was sealed using a plate sealer and then incubated for 48±1 hours in a humidified incubator set to 37°C, 5% CO2.
After the 48 hour exposure period, the medium in the viability plate was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The plate was sealed and incubated for 4 hours in an incubator set to 37°C, 5% CO2.

The MTT medium was then removed and 200 µL SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator set to 37°C, 5% CO2, and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm.
After the 48 hour exposure period, the cells in the luciferase plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plate was then incubated for 20 minutes in an incubator set to 25°C, loaded into the luminescence plate reader and read.

The following parameters were calculated:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test article and positive control;
• The 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) was obtained; and
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Vehicle / solvent control:

DMSO

Positive control:

cinnamic aldehyde [442D]

Positive control results:

Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 8 to 64 µM in Experiment 1 and at concentrations of 16 to 64 µM in Experiment 2.
The EC1.5 value for the positive control was 7.61 and 8.60 µM in Experiments 1 and 2, respectively. The average induction in the three replicates for the positive control at 64 µM was 4.87 and 12.75 in Experiments 1 and 2, respectively. The average induction for the positive control at 64 µM in Experiment 2 was outside the range specified in the protocol. As however a clear positive dose response was observed for luciferase induction, these data were considered to be acceptable.

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.88

Cell viability:

125.12%

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

3.35 µM

Cell viability:

125.12%

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

2.38

Cell viability:

145.39%

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

2.35 µM

Cell viability:

145.39%

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 9.48% and 17.08% in Experiments 1 and 2, respectively.

Detailed results are included as attachment.
A clear biphasic dose response was observed for luciferase induction (1.5 threshold was crossed twice). This response was therefore considered to be specific to the test chemical and not an experimental artefact.
No significant cytotoxic effects occurred at the lowest concentration leading to >1.5-fold luciferase induction as cell viability at the EC1.5 determining concentrations was 125.12% and 145.39% in Experiments 1 and 2, respectively.
The cell viability was above 70% at concentrations of 0.98 to 500 μM in Experiment 1 and at all concentrations in Experiment 2. The IC50 and IC30 values in experiment 1 were 1187.14 μM and 1351.98 μM, respectively. As all viability results for Experiment 2 were above 70%, the IC50 and IC30 values for this experiment were >2000 μM and could not be calculated.

Interpretation of results:

study cannot be used for classification

Remarks:

The data can only be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitizers and non-sensitizers for the purpose of hazard classification and labelling.

Conclusions:

Based on the results of an ARE-Nrf2 Luciferase Test, performed according to OECD guideline 442D and according to GLP principles, BTMR was predicted to be positive.

Executive summary:

An ARE-Nrf2 Luciferase Test was performed according to OECD guideline 442D and according to GLP principles.

The test article was dissolved in dimethyl sulfoxide (DMSO) to the final stock concentration (200 mM). The final test concentrations ranged from 0.98 to 2000 μM. Aliquots of 50 μL of each of the final concentrations were transferred to give three luciferase replicates. Luminescence was measured.

A dose response was observed in both experiments.  The Imax was calculated to be 1.88 and 2.38 in Experiment 1 and Experiment 2 respectively. Cell Viability of 125.12% and 145.39% were observed in Experiment 1 and Experiment 2, respectively. The EC1.5 was calculated to be 3.35 μM and 2.35 μM for Experiment 1 and Experiment 2, respectively.

The results for the negative control (DMSO) confirmed the validity of the assay. 

All four conditions required for a positive prediction were met in both experiments, therefore BTMR was considered to be positive in the ARE-Nrf2 Luciferase Test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Based on the results of an ARE-Nrf2 Luciferase Test, performed according to OECD guideline 442D and according to GLP principles, BTMR was predicted to be positive. In a human Cell Line Activation Test (h-CLAT), performed according to OECD TG 442E, BTMR was considered to be positive. 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Justification for classification or non-classification

The results of in vitro skin sensitisation tests indicate that benzoyl-m-toluoylmethylresorcinol has skin sensitising properties (cat. 1). As no conclusion can be drawn on the potency based on the currently available data-set, an animal study will be initiated (as a last resort) to determine its potency.