Benzoic acid, 2-([1,1'-biphenyl]-4-ylcarbonyl)-

Endpoint summary

Description of key information

Based on the available weight of evidence from in vitro and in vivo studies, the test substance is not considered to be sensitising to the skin.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

yes

Remarks:

Precipitates of the test substance were observed at the end of the incubation with the peptides, therefore the peptide depletion may be underestimated.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Reactivity (expressed as percent of peptide depletion) is determined following 24-h contact between the test substance and peptide in acetonitrile at the ratios 1:10 cysteine: test substance and 1:50 lysine: test substance by liquid chromatography with UV-Visible spectra detection.

The reactivity of the test substance was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test substance and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test substance 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 percent 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 solvent).

% depletion = 1- (Peptide peak area in replicate injection/Mean peptide peak area in relevant reference control C samples) x 100

The test substance was dissolved at 100 mM in a 1:9 mixture of DMSO : acetonitrile. 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 substance did not co-elute with either the lysine or the cysteine peptides.

Analytical analysis:
- High Performance Liquid Chromatography (HPLC) Systems with a UV detector (220 nm),
- analytical chromatographic columns (Zorbax SB C18, 100 x 2.1 mm; 3.5 μm HPLC), in-line filter C18, 4.0 x 2.0 mm (Phenomenex)
Mobile phases run in gradient:
A: acetonitrile + 0.085 % TFA
B: milli-Q water + 0.1% TFA
Flow: 350 μL/minute
Oven temperature: 30.0 °C
Autosampler temperature: nominal temperature: +25 °C
Injection volume: 5 μL
Total analysis time: 20 minutes

Co-elution control samples preparation:
For the co-elution control with cysteine peptide: 50 μL of test substance formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.
For the co-elution control with lysine peptide: in parallel, 250 μL of test substance formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).

Reference control samples preparation:
Reference control A and B samples: In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.
Reference control C samples:
Reference control C samples were prepared for each solvent used to dissolve the test and positive control substances.
For the reference control C prepared with cysteine peptide:
50 μL of each vehicle (a 1:9 mixture of DMSO:acetonitrile or acetonitrile) was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reference control C prepared with lysine peptide:
In parallel, 250 μL of each vehicle (a 1:9 mixture of DMSO:acetonitrile or acetonitrile) was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

Cinnamaldehyde (positive control) depletion control samples preparation:
For the reactivity of cinnamaldehyde with cysteine peptide:
50 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of cinnamaldehyde with lysine peptide:
In parallel, 250 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

Test substance samples preparation:
For the reactivity of test substance with cysteine peptide:
50 μL of test substance formulation was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of substance with lysine peptide:
In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

Preparation of the calibration curve samples:
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20 % acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve.


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

The positive control was cinnamaldehyde (purity: 98.9%).
As several test items were assayed concurrently, the results of the positive control were shared.
The positive control was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in acetonitrile at 100 mM. The physical aspect of the formulation was clear colorless solution. The formulation was used just after its preparation.

Key result

Parameter:

other: mean of the percent cysteine and percent lysine depletion

Value:

0.97

Vehicle controls validity:

valid

Remarks:

1:9 mixture of DMSO:acetonitrile

Negative controls validity:

valid

Remarks:

co-elution control samples

Positive controls validity:

valid

Remarks:

cinnamaldehyde

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

DATA ANALYSIS

Calculation of the percent peptide depletion
Each appropriate peak was integrated and the peak area for calibration standards, control and test substance samples were determined. Based on the concentration of standards and their peak area, a linear calibration curve was generated. Then, the concentration of peptide was determined in each sample from absorbance at 220 nm, measuring the peak area of the appropriate peaks and calculating the concentration of peptide using the linear calibration curves. Then, for each positive control and test substance replicate, the percent depletion of peptide was determined from 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 solvent) by using the following formula:

% depletion = 1- (Peptide peak area in replicate injection/Mean peptide peak area in relevant reference control C samples) x 100

Then, the mean percent depletion of the three replicates was calculated for each peptide as well as the mean of the percent cysteine and percent lysine depletions. Negative depletion values were considered as "Zero" for the calculation of the mean %depletion.

Evaluation of the possible co-elution of the test substance with the lysine or cysteine peptides:
In order to detect possible co-elution of the test substance with a peptide, chromatograms of the co-elution control samples were analyzed and compared with those of the reference control C samples.

ACCEPTANCE CRITERIA

The run was considered valid if the following criteria were fully met:
. the calibration curve 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 percent depletion value should be between 60.8 and 100 % with a SD < 14.9%,
− for the lysine peptide, the mean percent 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 substance 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 solvent should be within ± 10% of the nominal concentration,
. the maximum SD for the test substance replicates should be < 14.9% for the percent cysteine depletion value and < 11.6% for the percent lysine depletion value.

The cysteine peptide depletion value was 1.21%,

The lysine peptide depletion value was 0.72%.

The mean of the percent cysteine and percent lysine depletions was equal to 0.97%. However, since precipitates were observed at the end of the incubation with the peptides, the peptide depletion may be underestimated.

Since the mean of the percent cysteine and percent lysine depletions was < 6.38%, the test substance was considered to have minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test substance is likely not to have any potential to cause skin sensitization, though with limitations due to test substance precipitation with the peptides.

Interpretation of results:

other: Negative

Conclusions:

Under the study conditions, the test substance was considered to have no/minimal peptide reactivity, although with limitations due to test substance precipitation with the peptides. Therefore, the test substance is considered to produce negative results in the DPRA assay.

Executive summary:

A study was conducted to determine the reactivity potential of the test substance PBBA, with synthetic cysteine and lysine peptides according to OECD Guideline 442 C (direct peptide reactivity assay (DPRA)), in compliance with GLP. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test substance for 24 h at the ratios 1:10 cysteine: test substance and 1:50 lysine: test substance. The test substance was dissolved at 100 mM in a 1:9 mixture of DMSO : acetonitrile. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with UV detection at 220 nm.Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide area in the three relevant reference control C samples. Results of the test substance reactivity indicated for the cysteine peptide, the mean depletion value of 1.21% and for the lysine peptide, the mean depletion value of 0.72%. The mean of the percent cysteine and percent lysine depletions was equal to 0.97%. However, since precipitates were observed at the end of the incubation with the peptides, depletion may be underestimated. Since the mean of the percent cysteine and percent lysine depletions was <6.38%, the test substance was considered to have no/minimal peptide reactivity. The acceptance study criteria were satisfied, the study was therefore considered to be valid. Under the study conditions, the test substance was considered to have no/minimal peptide reactivity, although with limitations due to test substance precipitation with the peptides. Therefore, the test substance was considered to produce negative results in the DPRA assay (Chevallier, 2017).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

The KeratinoSens cells were first plated on 96-well plates and grown for 24 h at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test substance and of positive controls. The treated plates were then incubated for 48 h at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed.

For each run, the test substance was solubilised in DMSO at 200 μM.

The same concentrations were used in first and second run:
This run was performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO.

Vehicle and negative control: DMSO
This vehicle was used as the negative control, and was applied to cells at a concentration of 1% in culture medium. Since several test substances were assayed concurrently, the results of the negative control were shared.

Positive control: Cinnamic Aldehyde
Since several test substances were assayed concurrently, the results of the positive control were shared.
For each run, the positive control substance was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of two, to obtain a total of five concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 μM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.

Test substance formulations:
On the basis of solubility results, the test substance was dissolved in DMSO at 200 mM.
One formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2 for the two runs, to obtain a total of 12 concentrations in a 96-well plate; this 96-well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level.
All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

TEST SYSTEM:

KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test substance of the Nrf2 transcription factor in this test.
. Supplier: this cell line was provided by Givaudan.
. Batch: C1.
. Storage conditions: In the freezer set at -80°C.
. Mycoplasm: absence of mycoplasma was confirmed.

Solubility assay:
A solubility assay was performed prior the first treatment in order to select the vehicle (among DMSO, water or treatment culture medium).
Since the test substance was found soluble in DMSO at 200 mM, this stock formulation was diluted in treatment culture medium to the final concentration of 2000 μM. Then, a visual inspection of the sample was performed to evaluate the absence of precipitate/emulsion.

METHOD FOR A RUN OF KERATINOSENS ASSAY:

Cell seeding for testing:
. Cells were grown using general culture procedures up to 80-90% confluence,
. the day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 104 cells/mL,
. cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 μL (representing 1 x 104 cells) per well taking care to avoid sedimentation of the cells during seeding,
. after seeding, the cells were grown for 24 (± 1) h in the 96-well microtiter plates prior to test item addition.

Treatment:
. After the 24-h growing period, the medium was removed by aspiration and replaced by 150 μL of treatment medium,
. from the Master plate 4x, a volume of 50 μL was added to each well of the three white assay plates and 50 μL to the transparent plate for the cytotoxicity evaluation,
. all plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells,
. the plates were then incubated for 48 (± 2) h at 37°C, 5% CO2, 90% humidity.

Endpoint measurements:
Microscopic observation to evaluate the presence or absence of precipitate - transparent plate. After the 48 (± 2) h incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.

Luminescence flash signal to evaluate induction signal - white plates:
. After incubation, the supernatants from the white assay plates were discarded,
. the cells were washed once with D-PBS,
. a volume of 20 μL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) min at room temperature and under orbital shaking,
. the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
- 50 μL of the luciferase substrate was added to each well,
- 1 second after this addition, the luciferase signal was integrated for 2 sec.
Thus the cycle time to read one plate was approximately 10 min.

Absorbance signal to evaluate the cytotoxicity - transparent plate:
. For the cell viability assay plate, the medium was replaced by 200 μL of treatment medium,
. a volume of 27 μL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate,
. the plates were covered with a sealing membrane and returned at 37°C in the incubator in humidified atmosphere for 4 h (± 10 min),
. at the end of the incubation period, the medium was removed and a volume of 200 μL of a 10% SDS solution was added to each well,
. the plates were covered with a sealing membrane and placed at 37°C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells,
. after the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.

Positive control: Cinnamic Aldehyde.
For each run, the positive control was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of two, to obtain a total of five concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 μM. All these formulations were prepared within 4 h before use, then kept at room temperature and protected from light until use.

Key result

Run / experiment:

other: first run

Parameter:

other: viability at ≥500 μM

Value:

70

Vehicle controls validity:

valid

Remarks:

DMSO

Negative controls validity:

valid

Remarks:

DMSO

Positive controls validity:

valid

Remarks:

Cinnamic Aldehyde

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: second run

Parameter:

other: cell viability at ≥ 250 μM

Value:

70

Vehicle controls validity:

valid

Remarks:

DMSO

Negative controls validity:

valid

Remarks:

DMSO

Positive controls validity:

valid

Remarks:

Cinnamic Aldehyde

Remarks on result:

positive indication of skin sensitisation

Solubility test:

In the solubility test, the test substance was found soluble in DMSO at 200 μM. Therefore, this vehicle was selected for the preparation of the test substance stock formulations. No precipitate or emulsion was observed once the test item stock formulation was diluted in the treatment culture medium to a final concentration of 2000 μM.

Results from the first run:

. no precipitate/emulsion were observed in any wells at the end of the 48-h treatment period at any tested concentrations,

. a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 500 μM,

. the corresponding IC30 and IC50 were calculated to be 302.30 and 414.78 μM, respectively,

. statistically significant gene inductions above the threshold of 1.5 were noted in comparison to the negative control at all tested concentrations (except at the too cytotoxic dose-levels) with an apparent overall dose-response for luciferase induction up to the cytotoxic concentrations,

. the Imax was 9.13 and the calculated EC1.5 was lower than 0.98 μM.

Thus, the criteria for a positive response were met.

Results from the second run:

. no precipitate/emulsion were observed in any wells at the end of the 48-h treatment period at any tested concentrations,

. a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 250 μM,

. the corresponding IC30 and IC50 were calculated to be 237.24 and 388.36 μM, respectively,

. statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control at concentrations ≥ 1.95 μM with an apparent overall dose-response for luciferase induction up to the cytotoxic concentrations,

. the Imax was 20.43 and the calculated EC1.5 was 1.93 μM.

Thus, the criteria for a positive response were met.

Global analysis from both runs

The geometric means IC30 and IC50 of the two validated runs were calculated to be 267.80 and 401.35 μM, respectively.

The evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive. This positive 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, although It cannot be used on its own to conclude on the skin sensitisation potential of the test substance.

Interpretation of results:

other: Positive

Conclusions:

Under the study conditions, the test substance showed positive results in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.

Executive summary:

A study was conducted to determine the potential of the test substance PBBA, to activate the Nrf2 transcription factor according to OECD Guideline 442 D (in vitro skin sensitisation: ARE-Nrf2 Luciferase), in compliance with GLP. In this study, the KeratinoSens cell line and an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line was used. The KeratinoSens cells were first plated on 96-well plates and grown for 24 h at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test substance and of positive controls. The treated plates were then incubated for 48 h at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed. The following concentrations of the test substance were investigated in Experiments I and II (without analytical verification): 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO. At these tested concentrations: no precipitate/emulsion were observed in any wells at the end of the 48 -h treatment period. In the first run a decrease in cell viability to less than <70% was noted at concentrations ≥500 μM. The corresponding IC30 and IC50 were calculated to be 302.30 and 414.78 μM, respectively. Statistically significant gene inductions above the threshold of 1.5 were noted in comparison to the negative control at all tested concentrations (except at the too cytotoxic dose-levels) with an apparent overall dose-response for luciferase induction up to the cytotoxic concentrations. In the second run a decrease in cell viability to less than < 70% was noted at concentrations ≥250 μM. The corresponding IC30 and IC50 were calculated to be 237.24 and 388.36 μM, respectively. Statistically significant gene inductions above the threshold of 1.5 were noted in comparison to the negative control at concentrations ≥1.95 μM with an apparent overall dose-response for luciferase induction up to the cytotoxic concentrations. The geometric means IC30 and IC50 of the two validated runs were calculated to be 267.80 and 401.35 μM, respectively. The evaluation criteria for a positive response were met in both runs, the final outcome was therefore positive. Under the study conditions, the test substance showed positive results in the KeratinoSens assay and was therefore considered to activate the Nrf2 transcription factor (Chrystel, 2017).

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 22, 2017 to May 3, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

see 'Any other information on materials and methods'

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

see 'Any other information on materials and methods'

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): DA

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

EXPERIMENTAL ANIMALS

Species and strain: CBA/CaOlaHsd mice
Source: Envigo, Italy
Hygienic level: SPF at arrival; standard housing conditions during the study
Number of animals: 4 animals/group
Sex: Female, nulliparous, non-pregnant
Age of animals at starting: 9 weeks old (age-matched, within one week)
Body weight range at starting: 19.1 – 19.9 g, (the weight variation in animals in the study did not exceed 20% of the mean weight)
Acclimatization time: 14 d
Preliminary Experiment:12 weeks of age, 20.6 - 23.0 g

HUSBANDARY

Animal health: only healthy animals were used for the study, health status was certified by the veterinarian
Housing / Enrichment: group caging / mice were provided with glass tunnel-tubes
Cage type: type II. polypropylene / polycarbonate
Bedding: bedding of certified wood chips (J. Rettenmaier & Söhne GmbH), certified nest building material (ARBOCEL)
Light: 12 h daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 18.5 – 24.8°C
Relative humidity: 23 – 80%
Ventilation: 15 – 20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases
Food and feeding: Animals received ssniff SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable “Complete feed for Rats and Mice – Breeding and Maintenance"
Water supply: animals received tap water from the municipal supply from 500 mL bottles, ad libitum

Vehicle:

dimethyl sulphoxide

Concentration:

50% and 25% (w/v) in DMSO

No. of animals per dose:

preliminary irritation/toxicity test: 2 animals/dose

Details on study design:

PRELIMINARY IRRITATION/TOXICITY

Two animals per dose were treated with the test substance at concentrations of 50% and 25% (w/v) in DMSO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed. In the preliminary irritation/toxicity test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 h after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.

PROLIFERATION ASSAY

Topical application
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Twenty-four female CBA/CaOlaHsd mice were allocated to six groups of four animals each:
- four groups received 2-(4-Phenylbenzoyl)benzoic acid (formulated in DMSO) at 50, 25, 10 and 2% (w/v) respectively,
- the negative control group received the vehicle (DMSO) only,
- the positive control group received 25% (w/v) HCA (dissolved in DMSO).

Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µL Ci of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 h (± 30 min).

Removal and preparation of draining auricular lymph nodes
Five hours after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 min at 4°C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight (approximately 18 h) incubation at 2-8°C, precipitates were centrifuged (approximately 190 x g for 10 min at 4°C), and supernatants were removed. Pellets were resuspended in 1 mL of 5% (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-min measurement). The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by a scintillation vial filled with 10 mL of scintillation liquid.

OBSERVATIONS

Clinical Observations: during the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.

Measurement of Body Weight: individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

50%

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

2%

Cellular proliferation data / Observations:

CLINICAL OBSERVATION

No mortality or signs of systemic toxicity were observed during the study. Rigid ears or slightly rigid ears were observed on Day 2 and Day 3 in all of the 50 and 25% (w/v) dose group animals. Minimal amount of test substance precipitate were present up to Day 5 in the 50 and 25% (w/v) dose groups, and up to Day 3 in the 10% (w/v) dose group. The results of the observations are summarized in Appendix 3.

BODY WEIGHT MEASUREMENT

No marked body weight losses (≥ 5%) was observed on the mean body weight changes; although the body weight loss was ≥5 % for 1/4 animals in the 10% (w/v) dose group. This change was considered incidental.

AR THICKNESS VALUES AND EAR PUNCH WEIGHTS

Increased ear thickness values (≥ 25%) were detected for one animal (only left ear) in the 50% dose group on Day 3. At the measurement day, test substance precipitate was present on the ears of the animal, the two ears showed largely different values, and the effect disappeared on day 6, indicating that this increased value was most probably due to test substance remaining on the ear.

For three animals on Day 3, and for three animals of the positive control group on Day 6, increased ear thickness values (≥ 25%) were measured. All measured ear punch weights were outside the historical control range, with one animal showing positive response. This would suggest a slightly stronger local response in the positive control group animals, but these positive control values were considered not to adversely affect the results or integrity of the study.

PROLIFERATION ASSAY

Lymphocyte proliferation in test groups is compared to that in the vehicle treated control. The ratio of the proliferation in test groups to that in the control, termed Stimulation Index (SI), is determined and must be at least equal or greater than three, for a test substance to be classified as a skin sensitizer.
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5% (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.

Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

The appearance of the lymph nodes was normal in the negative control group and in 2% (w/v) test substance treated dose group, slightly larger than normal in the 10 and 25% (w/v) dose groups. Larger than normal lymph nodes were observed in the 50% (w/v) and positive control group.

No mortality or signs of systemic toxicity were observed during the study. Rigid ears or slightly rigid ears were observed on Day 2 and Day 3 in all of the 50 and 25% (w/v) dose group animals. Test substance precipitate or minimal amount of test substance precipitate were present up to Day 5 in the 50 and 25% (w/v) dose groups, and up to Day 3 in the 10% (w/v) dose group. No marked body weight loss (≥ 5%) was observed on the mean body weight changes, although the body weight loss was ≥5% for 1/4 animals in the 10% (w/v) dose group. This change was considered incidental.The stimulation index values were 1.5, 1.2, 1.1, and 1.0 at concentrations of 50, 25, 10, and 2% (w/v), respectively.The result of the positive control substance HCA was used to demonstrate the appropriate performance of the assay.

Preliminary irritation / toxicity test:

- no mortality or signs of systemic toxicity were noted. A visible amount of test substance precipitation was observed from Day 1 up to Day 4 on the ears of the animals (4 out of 4 animals). There were no indications of any irritancy at the site of application,

- no marked body weight loss (>5% reduction of body weight) was observed on the average animal body weight per group,

- the ear thickness values on Day 6 were significantly (more than 25% compared to the Day 1 values) larger in the 50% (w/v) dose group, and in one animal in the 25% dose group (mean value). Ear punch weights were within the acceptable range,

 - the draining auricular lymph nodes of the animals were visually examined: they were larger than normal in the 50% (w/v) dose group, and normal in the 25% (w/v) dose group (subjective judgement by analogy with observations of former experiments).

 

Based on these results, 50% (w/v) dose was selected as top dose for the main test. Based on ear thickness values alone, and noting that (not visible) test substance precipitate might influence these results, it has been decided that four dose groups would be tested in the main study.

Interpretation of results:

other: CLP criteria not met

Conclusions:

Under the study conditions, the test substance did not show a sensitisation potential.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance PBBA according to OECD Guideline 429 and EU Method B.42 (local lymph node assay), in compliance with GLP. The preliminary irritation/toxicity test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 50 and 25% (w/v) in DMSO. Based on the observations recorded in the preliminary test, the 50% (w/v) was selected as top dose for the main test. In the main assay, 24 female mice were allocated to 6 groups of 4 animals each: 4 groups received test substance(formulated in DMSO) at 50, 25, 10 and 2% (w/v), respectively. The negative control group received the vehicle only and the positive control group received 25% (w/v) HCA inDMSO. The test substance solutions (25 µL/ear) were applied on the dorsal surface of ears of experimental animals for 3 consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring disintegrations per minutes after incorporation of tritiated methyl thymidine (³HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group. No mortality or signs of systemic toxicity were observed during the study. Rigid or slightly rigid ears were seen on Days 2 and 3 in all of the 50 and 25% (w/v) dose group animals. Minimal amount of test substance precipitate were present up to Day 5 in the 50 and 25% (w/v) dose groups, and up to Day 3 in the 10% (w/v) dose group. The stimulation index values were 1.5, 1.2, 1.1, and 1.0 at 50, 25, 10, and 2% (w/v), respectively.The result of the positive control substance HCA was used to demonstrate the appropriate performance of the assay. Under the study conditions, the test substance did not show a sensitisation potential (Oroszlány, 2017).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DPRA assay

A study was conducted to determine the reactivity potential of the test substance PBBA with synthetic cysteine and lysine peptides according to OECD Guideline 442 C (direct peptide reactivity assay (DPRA)), in compliance with GLP. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test substance for 24 h at the ratios 1:10 cysteine: test substance and 1:50 lysine: test substance. The test substance was dissolved at 100 mM in a 1:9 mixture of DMSO : acetonitrile. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with UV detection at 220 nm. Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide area in the three relevant reference control C samples. Results of the test substance reactivity indicated for the cysteine peptide, the mean depletion value of 1.21% and for the lysine peptide, the mean depletion value of 0.72%. The mean of the percent cysteine and percent lysine depletions was equal to 0.97%. However, since precipitates were observed at the end of the incubation with the peptides, depletion may be underestimated. Since the mean of the percent cysteine and percent lysine depletions was <6.38%, the test substance was considered to have no/minimal peptide reactivity. The acceptance study criteria were satisfied, the study was therefore considered to be valid. Under the study conditions, the test substance was considered to have no/minimal peptide reactivity, although with limitations due to test substance precipitation with the peptides. Therefore, the test substance was considered to produce negative results in the DPRA assay (Chevallier, 2017).

ARE-Nrf2 Luciferase assay

A study was conducted to determine the potential of the test substance PBBA to activate the Nrf2 transcription factor according to OECD Guideline 442 D (in vitro skin sensitisation: ARE-Nrf2 Luciferase), in compliance with GLP. In this study, the KeratinoSens cell line and an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line was used. The KeratinoSens cells were first plated on 96-well plates and grown for 24 h at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test substance and of positive controls. The treated plates were then incubated for 48 h at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed. The following concentrations of the test substance were investigated in Experiments I and II (without analytical verification): 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO. At these tested concentrations: no precipitate/emulsion were observed in any wells at the end of the 48 -h treatment period. In the first run a decrease in cell viability to less than <70% was noted at concentrations ≥500 μM. The corresponding IC30 and IC50 were calculated to be 302.30 and 414.78 μM, respectively. Statistically significant gene inductions above the threshold of 1.5 were noted in comparison to the negative control at all tested concentrations (except at the too cytotoxic dose-levels) with an apparent overall dose-response for luciferase induction up to the cytotoxic concentrations. In the second run a decrease in cell viability to less than < 70% was noted at concentrations ≥250 μM. The corresponding IC30 and IC50 were calculated to be 237.24 and 388.36 μM, respectively. Statistically significant gene inductions above the threshold of 1.5 were noted in comparison to the negative control at concentrations ≥1.95 μM with an apparent overall dose-response for luciferase induction up to the cytotoxic concentrations. The geometric means IC30 and IC50 of the two validated runs were calculated to be 267.80 and 401.35 μM, respectively. The evaluation criteria for a positive response were met in both runs, the final outcome was therefore positive. Under the study conditions, the test substance showed positive results in the KeratinoSens assay and was therefore considered to activate the Nrf2 transcription factor (Chrystel, 2017).

LLNA assay

A study was conducted to determine the skin sensitisation potential of the test substance PBBA according to OECD Guideline 429 and EU Method B.42 (local lymph node assay), in compliance with GLP. The preliminary irritation/toxicity test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 50 and 25% (w/v) in DMSO. Based on the observations recorded in the preliminary test, the 50% (w/v) was selected as top dose for the main test. In the main assay, 24 female mice were allocated to 6 groups of 4 animals each: 4 groups received test substance(formulated in DMSO) at 50, 25, 10 and 2% (w/v), respectively. The negative control group received the vehicle only and the positive control group received 25% (w/v) HCA inDMSO. The test substance solutions (25 µL/ear) were applied on the dorsal surface of ears of experimental animals for 3 consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring disintegrations per minutes after incorporation of tritiated methyl thymidine (³HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group. No mortality or signs of systemic toxicity were observed during the study. Rigid or slightly rigid ears were seen on Days 2 and 3 in all of the 50 and 25% (w/v) dose group animals. Minimal amount of test substance precipitate were present up to Day 5 in the 50 and 25% (w/v) dose groups, and up to Day 3 in the 10% (w/v) dose group. The stimulation index values were 1.5, 1.2, 1.1, and 1.0 at 50, 25, 10, and 2% (w/v), respectively.The result of the positive control substance HCA was used to demonstrate the appropriate performance of the assay. Under the study conditions, the test substance did not show a sensitisation potential (Oroszlány, 2017).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available weight of evidence from in vitro and in vivo studies, the test substance is not required to be classified for skin sensitization according to EU CLP (EC 1272/2008) criteria.