2-hydroxy-4-(trifluoromethyl)benzoic acid

Endpoint summary

Description of key information

Weight of evidence: Based on the available data, it was concluded that the test item is not sensitising.

- OECD 442C, GLP study. The test item shows mean depletion of 2% for lysine and 20% for cysteine, i.e. an overall average of 11%, reflecting low reactivity and thus, a positive prediction of DPRA skin sensitization test.

- OECD 442D, GLP study. From the results of this study, under the specified experimental conditions, test item was deemed as sensitiser in the KeratinoSens assay.

- OECD 442B, GLP study. The test item showed no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay, with

stimulation indexes of 0.8, 1.0, and 0.7 at concentrations of 10%, 5%, and 2.5% (w/v) test item in DMSO, respectively.

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:

weight of evidence

Study period:

07 March 2018 - 28 May 2018

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

TEST ITEM SOLVENT
Initial election of test item solvent: solubility of the test item in an appropriate solvent was assessed before performing the assay. The chosen solvent was Milli-Q water .

TEST SYSTEM
Cysteine peptide (supplier RS Synthesis, Louisville, KY 40270, USA)
Lysine peptide (supplier RS Synthesis, Louisville, KY 40270, USA)

CONTROLS
Positive control: cinnamaldehyde (CAS 104-55-2; batch no MKBT8955V; purity 99.1%)
Co-elution control: test item in phosphate buffer (750 µL of phosphate buffer (pH 7.5) + 200 µL of acetonitrile + 50 µL test item) for cysteine peptide and ammonium acetate buffer (750 µL of ammonium acetate buffer (pH 10.2) + 250 µL of test item) for lysine peptide.
Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy (750 µL Cysteine/Lysine peptide (0.667 mM) + 250 µL of acetonitrile).
Reference control B: prepared with acetonitrile and included at the beginning and at the end of the sequence in order to check the stability of peptide over time (same as preparation for reference control A, but run before and after 24 ± 2 hours incubation at 25 ± 2.5ºC in dark).

PREPARATION OF THE TEST ITEM AND POSITIVE CONTROL SOLUTIONS
Test item solution: To achieve a concentration of 100 mM, 61.41 mg of test item was weighed and volume was made up to 3 mL with acetonitrile. Precipitation was visually inspected for test item solution of 100 mM.
Positive control solution: 38.10 µL of Cinnemaldehyde was dissolved in 2961.9 µL of acetonitrile for the preparation of 100 mM solution (3 mL) for both Cysteine and Lysine peptides.

PREPARATION OF THE PEPTIDE SOLUTIONS
Peptide solutions were prepared at 0.667 mM:
Cysteine solution: 9.025 mg of cysteine peptide were pre-weighted then dissolved, right before the incubation, in 18013.97 µl of phosphate buffer (pH 7.5).
Lysine solution: 9.328 mg of lysine peptide were pre-weighted then dissolved, right before the incubation, in 18007.72 µl of ammonium acetate buffer (pH 10.2).

TEST SOLUTIONS
Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively.
1 ml of each solution was prepared according to the following quantities:
-Cysteine test solution: 750 µl of cysteine peptide solution (buffer only to check co-elution) + 50 µl of sample or solvent for Reference controls + 200 µl of acetonitrile.
-Lysine test solution: 750 µl of lysine peptide solution (buffer only to check co-elution) + 250 µl of sample or solvent for Reference controls.

Vials were caped, vortexed and were placed in the HPLC auto sampler at 25 ± 2.5ºC in dark for 24 ± 2 hours. HPLC analysis of the batch of samples was started 24 ± 2 hours after the test item was added to the peptide solution.

Replicates: Samples were prepared in triplicate for both peptides.

HPLC ANALYSIS
-Apparatus: Shimadzu with UV Detector. Column: ZorbaX SB-C18 (2.1 mm x 100 mm x 3.5 micron) with Guard Column Phenomenex Security Guard C18 (4 mm x 2 mm)
-Calibration curve: Six peptides standards solutions between 0.534 mM and 0.0167 mM (dilution factor 2) were prepared in 20% acetonitrile in phosphate buffer for cysteine peptide and ammonium acetate for lysine peptide. The dilution buffer was also included as blank in the standard calibration curve.
-Volume injected: 5 µl of each sample
-Run time: 20 min
-Sequence (gradient): see table 1 on “Any other information on materials and methods incl. tables”
-Analysis sequence:
The analysis was programmed according to the following principles:
The reference controls B were placed at the beginning and at the end of the analysis (3 repetitions).
The standards of the calibration curve and the reference controls A were placed in order to be analyzed progressively throughout the sequence.
Lysine and cysteine analysis were conducted on separate day. Samples were visually inspected for precipitation prior to HPLC analysis. Precipitation was not observed with test item and positive control.

DEVIATIONS FROM OECD GUIDELINE: No.

OTHER:
Some batches of acetonitrile have a negative impact on Cysteine peptide stability. Hence, stability was assessed by injecting the Cysteine reference control A at 0, 24, and 48 hours in three replicates.

Positive control results:

The depletion mean rate was 79% for cysteine peptide and 45% for lysine peptide.

Key result

Run / experiment:

other: mean of 3 repetitions

Parameter:

other: %depletion in lysine peptide

Value:

2

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Mean of 3 repetitions

Parameter:

other: %depletion in cysteine peptide

Value:

20

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Mean of lysine and cysteine peptides

Parameter:

other: mean %depletion in peptides

Value:

11

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Test item was found to be soluble in acetonitrile at the concentration of 100 mM. Precipitation was not observed at 100 mM.
- The relative coefficient of variability (RCV) for the stability of Cysteine peptide in acetonitrile was 1.85% against set standard of <15%. This showed that Cysteine peptide was stable in the solvent, i.e., acetonitrile.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recommended in the OECD guideline 442C were performed, obtaining values that fall within the respective reference range for 8 out of the 10 for each peptide.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for calibration curve: yes, coefficient r2 was higher than 0.99 for lysine and cysteine.
- Acceptance criteria met for reference control A: yes, the mean concentration of peptide was 0.45 mM for lysine and 0.52 mM for cysteine which are equal to 0.50± 0.05 mM
- Acceptance criteria met for reference control B: yes, the CV (coefficient of variation) of the controls B was 1.60% for cysteine peptide and 3.78% for Lysine peptide which are lower than 15%.
- Acceptance criteria met for positive control: Yes, RCV of the 3 repetitions for each peptide was 6.43% for cysteine and 1.60% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively. The depletion mean rate was 79% for cysteine peptide and 45% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.4% for the lysine.
- Acceptance criteria met for variability between replicate measurements: Yes, SD of the 3 repetitions of the test item for each peptide was 1.45% for cysteine and 0.28% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively.

Table 3: Positive control and test item percent peptide depletion

Percent Peptide Depletion (Cysteine)
Positive Control Name/ Test Item Code	Rep-1 Area (AU)	Rep-2 Area (AU)	Rep-3 Area (AU)	Average	% Depletion	SD	RCV	Expected Depletion
Cinnamic aldehyde (28.04.2018)	514670	459015	463859	479181	76	30829.37	6.43	60.8-100
Test item (28.04.2018)	1656913	1637695	1611119	1635242	20	22995.31	1.41	-
Co-elution control - Test item (28.04.2018)	ND	-	-	-	-	-	-	-
Percent Peptide Depletion (Lysine)
Cinnamic aldehyde (29.04.2018)	837414	860283	861899	853199	45	13693.78	1.60	40.2-69
Test item (29.04.2018)	1509906	1524043	1507220	1513723	2	9037.72	0.60	-
Co-elution control - Test item (29.04.2018)	ND	-	-	-	-	-	-	-

Keys: Rep = Replicate, AU = Arbitrary Units, SD = Standard Deviation, RCV = Relative Coefficient of Variability, ND = Not Detected, - = Not applicable.

No co-elution occurred of the test item neither with lysine nor with cysteine peptides.

Table 4: Reference controls A and B Peak Area with Retention Time for Cysteine and Lysine

Peptide (Date)	Cysteine (28.04.2018)		Lysine (29.04.2018)
Reference Control A	Area (AU)	RT (min)	Area (AU)	RT (min)
Rep-1	2040199	9.489	1537702	6.475
Rep-2	2042282	9.503	1558279	6.453
Rep-3	2102955	9.494	1500063	6.431
Average	2061812	-	1532015	-
SD	35646.10	-	29521.77	-
RCV (%)	1.73	-	1.93	-

Peptide (Date)	Cysteine (28.04.2018)		Lysine (29.04.2018)
Reference Control B	Area (AU)	RT (min)	Area (AU)	RT (min)
Rep-1	2023555	9.508	1458731	6.412
Rep-2	2014956	9.505	1505197	6.443
Rep-3	2084995	9.494	1619085	6.497
Rep-4	2020629	9.445	1557041	6.431
Rep-5	2013676	9.455	1560745	6.468
Rep-6	2076538	9.420	1592798	6.467
Average	2039058	-	1548933	-
SD	32619.26	-	58480.67	-
RCV (%)	1.60	-	3.78	-

Keys: Rep = Replicate, AU = Arbitrary Units, RT = Retention Time, min = Minute, SD = Standard Deviation, RCV = Relative Coefficient of Variability, - = Not applicable.

Note: Reference control A was prepared with acetonitrile and used to verify the accuracy of the calibration curve for peptide quantification. Reference control A run after 24 ± 2 hours incubation at 25 ± 2.5 °C in dark is considered as Reference Control B.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item shows mean depletion of 2% for lysine and 20% for cysteine, i.e. an overall average of 11%, reflecting low reactivity and thus, a positive prediction of DPRA skin sensitization test.

Executive summary:

A DPRA skin sensitization test was performed for dipotassium methanedisulphonate as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item was prepared at 100 mM in Milli-Q water and Cinnamaldehyde (positive control) was prepared at 100 mM in acetonitrile. Reference controls A and B were prepared with acetonitrile and Milli-Q water (control C). Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item and positive control were incubated for 24 ± 2 hours at 25°C ± 2.5°C (in dark) with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, before HPLC analysis. Each sample was tested in triplicate for both peptides. Relative peptide concentration was measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. HPLC system suitability was determined by the standard calibration curve and value of r2 obtained was 0.99987 for cysteine and 0.99909 for Lysine peptides, against set standard of r2 > 0.99. Values of the mean percent peptide depletion value of positive control was 76% for cysteine peptide and 45% for Lysine peptide. The Relative Coefficient of Variability (RCV) for the reference control B was 1.60 for Cysteine peptide and 3.78 for Lysine peptide. The mean peptide concentration of reference control A and B was 0.50 ± 0.05 mM. The relative coefficient of variability (RCV) for the stability of Cysteine peptide in acetonitrile was 1.85 against set standard of <15%, indicating that Cysteine is stable in acetonitrile. Percent peptide depletion values of test item for Cysteine and Lysine were 20% and 2%, respectively. Mean percent depletion for test item was 11%.

From the results of this study, under the specified experimental conditions, test item was concluded as non-sensitizer in DPRA assay.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 25, 2018 - May 2, 2018

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:

TEST SYSTEM
The parental spontaneously immortalized human keratinocyte cell line (HaCaT) was developed through long-term culture of normal human adult skin keratinocytes at reduced calcium concentration and elevated temperature. The genetically modified HaCaT cell line (KeratinoSensTM) obtained from Givaudan Schweiz AG, was used in this study. KeratinoSensTM cell line contains a stable insertion of the luciferase gene under the transcriptional control of a constitutive SV40 promoter fused with an antioxidant response element (ARE) of the human AKR1C2 gene. KeratinoSens culture was maintained at the Mutagenicity section, Jai Research Foundation. The batch of the cell line was tested for mycoplasma contamination before using the cells in the experiment. Cultures free from any contamination were used in the study. KeratinoSensTM cell line passage number 21 (Experiment-1 and 2) and 24 (Experiment-3) were used in the study. Cell line with lot number JRF/HaCaT/2016/01 was used in the study.

CULTURE MEDIUM
The medium for culturing cells consists of D-MEM containing Glutamax supplemented with 9.1% fetal bovine serum and 500 μg/mL geneticin. The medium for exposure of cells with test item consists of D-MEM supplemented with 1% foetal bovine serum (495 mL D-MEM + 5 mL FBS).

CONTROLS
Concurrent positive and negative controls were included in each set of experiment.
Positive control: Trans cinnamaldehyde (stock concentrations from 0.4 to 6.4 mM were prepared in DMSO, which were further diluted to achieve final concentration of positive control ranging from 4 to 64 µM).
Negative control: DMSO

CULTURE VESSELS
Disposable tissue culture flasks of 75 cm2 culture (Nunc during main study experiment) area with vented neck were used to culture the cell line and the treatment was performed in 96-well white assay plates (for Luciferase assay) in triplicate and one 96-well transparent plates (for cytotoxicity assay).

PREPARATION OF CELL CULTURES
Cells were maintained in D-MEM containing Glutamax supplemented with 9.1 % fetal bovine serum and 500 μg/mL geneticin. After cells attained 80-90% confluency, they were washed twice with DPBS containing 0.05% of EDTA. To each flask 2-3 mL of 0.05% Trypsin-EDTA was added and flasks were incubated at 37 ± 1 oC (usually 5–10 minutes). After cells were detached completely, they were resuspended in DMEM with 9.1% FBS without geneticin. Cell count was taken and cell density was adjusted to 8 x 10E4 cells/mL. 125 µL of this culture (containing approx. 10,000 cells) was then dispensed in each well of 96 well plates, leaving one cell empty (to assess background values). Plates were incubated for 24 hours in 5 ± 1 % CO2 at 37 ± 1 ºC. Test item stock solution concentrations were between 19.53125 and 40000 µg/mL.

TEST ITEM EXPOSURE PROCEDURE
After 24 ± 2 hours of incubation, medium from all the 4 plates was aspirated and discarded. It was replaced with 150 µL of DMEM containing 1% FBS without geneticin. The 25 fold dilution of the 100X master plate was performed into a fresh plate (10 µL test solution + 240 µL of DMEM with 1% FBS without geneticin). Resulting 4X plate was further distributed to assay plates: 50 µL of this stock solutions were added 3 white assay plates and one cytotoxicity plate already containing 150 µL of culture medium. Final test concentrations used for the exposure were 0.1953125, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 µg for both the experiments. Plates were then covered with foil or petri-seal and incubated for 46 hours in 5 ± 1 % CO2 at 37 ± 1 °C.

LUCIFERASE ACTIVITY:
After 48 ± 2 hours of incubation, the medium from 96 well white assay plates was aspirated and discarded. Cells were washed once with 150 µL of DPBS. After washing, 20 µL of passive lysis buffer was added in each well. Cells were then incubated for 30 ± 10 minutes at 37 °C. After incubation, the plates with cell lysate were placed in luminometer. 50 µL of 1X Luciferase substrate was added and luciferase activity was recorded for 2 seconds.

CYTOTOXICITY EVALUATION:
Master mix was prepared by combining 27 µL of MTT (5 mg/mL in DPBS) and 200 µL of DMEM containing 1% FBS without geneticin. After 48 ± 2 hours of incubation, the medium from 96 well transparent plate was replaced with 227 µL above prepared master mix. The plate was covered with foil and incubated for 4 hours. After incubation, the medium was removed and 200 μL of a 10% SDS solution was added to each well. The plate was sealed and covered with a foil and placed in the incubator for overnight incubation. Following incubation the absorption at 570 nm was measured.

For the test item three experiments were conducted and out of these two valid experiments (experiment 1 and 3) were considered for final evaluation. Reason for conducting the repeating the experiments was due to failure of negative control (i.e., variation in 3*6 solvent control well was higher than 20%) to meet the acceptance criteria (experiment 2).

CALCULATIONS:
The following parameters were calculated in the KeratinoSensTM test method:
1. The maximal fold gene-induction (Imax) value observed at any concentration of the test item and positive control.
2. The EC1.5 value representing the concentration for which a gene induction above the 1.5-fold threshold (i.e., 50% enhanced gene activity).
3. The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Key result

Run / experiment:

other: Mean (run 1 and 3)

Parameter:

other: Imax

Value:

2.7

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Mean (run 1 and 3)

Parameter:

other: EC1.5 (µg/mL)

Value:

8.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Mean (run 1 and 3)

Parameter:

other: IC50 (µg/mL)

Value:

306.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency of the facility in performing KeratinosensTM Assay was demonstrated by performing validation study (JRF Study N° 628-1-06-15806) .

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The coefficient of variation observed for the negative control during experiment 1 and 3 were 8.59% and 12.24%, respectively, which was below 20%. Since variation between the replicates was less than 20%, results of this run were considered as valid.
- Acceptance criteria met for positive control:
The gene induction for positive control was found to be >1.5 at concentrations of 32 µM and 64 µM in both the repetitions. The E.C1.5 value for positive control was found to be 35.84 µM and 12.79 µM in experiment 1 and 3, respectively. The average gene induction for positive control at 64 µM was found to be 1.93 and 6.31 for experiment 1 and 3, respectively. Clear dose response with increasing gene induction at increasing dose levels was observed in experiment 1 and experiment 3.
- Range of historical values if different from the ones specified in the test guideline:
Positive contro: Mean EC1.5 = 22.77 (SD ± 5.26) (Min 16.61 - Max. 32.12)
95% Control Limits: 12.24 - 33.30

The EC1.5 mean value was 8.80 µg/mL, which was less than 200 µg/mL. The value of maximum induction (Imax) was 1.76 and 1.68 at the tested concentration of 25 and 50 µg/mL in experiment 1 and 1.68, 1.86, 2.30, 2.39 and 1.94 at the tested concentration of 6.25, 12.5, 25, 50 and 100 µg/mL, respectively in experiment 3, which was higher than 1.5 fold. The cellular viability was 96.90 and 77.59 % at the test concentration of 25 and 50 µg/Ml in experiment 1 and 96.63, 95.70, 97.22, 89.27 and 87.52 % at the tested concentration of tested concentration of 6.25, 12.5, 25, 50 and 100 µg/mL, respectively, in experiment 3 with induction of luciferase activity above 1.5 fold. Observed dose response for luciferase induction was also statistically significant.

For the test item three experiments were conducted and out of these two valid experiments (experiment 1 and 3) were considered for final evaluation. Reason for conducting the repeating the experiments was due to failure of negative controls (i.e., variation in 3*6 solvent control well was higher than 20%) to meet the acceptance criteria (experiment 2).

Results of the present study indicate that, test item met all the evaluation criteria to conclude as sensitisers in KeratinoSens assay. Negative and positive controls met the acceptance criteria for the controls and were correctly identified as non-sensitiser and sensitiser, respectively. This showed the suitability of test system and procedures used in the test facility.

TEST ITEM:

Imax

Rep1	Rep3	Average
1.76*	2.39*	2.07

 

EC1.5

Rep1	Rep3	GeoMean
14.54µg/mL	5.32µg/mL	8.80µg/mL

 

IC50

Rep1	Rep3	GeoMean
316.31µg/mL	297.58µg/mL	306.80µg/mL

Keys: * = Significant induction was observed, Rep = Replicate/Experiment.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

From the results of this study, under the specified experimental conditions, test item was concluded as sensitisers in KeratinoSens assay.

Executive summary:

An In Vitro Skin Sensitization Keratinocyte-Based ARE-Nrf2 Luciferase Reporter Gene Test was performed according to OECD Guideline 442D (GLP study). Test item was found to be soluble at 40 mg/mL in DMSO. Test item was tested in two independent experiments. KeratinosensTM (HaCaT) cells were exposed to test item between test concentrations of 400 µg to 0.1953125 µg and with positive control between 4 to 64 µM for 48 ± 2 hours in 5 + 1% CO2 at 37 ± 1 o C.  After incubation cells were analyzed for luciferase activity. Cell viability of the concurrently treated cells was also evaluated using MTT test with separate set of plate. Imax and EC1.5 values were calculated based on luciferase activity i.e., luminescence measured (reading of three plates) while IC50 was calculated based on results of cytotoxicity (OD values, reading of one plate). For positive control trans cinnamldehyde, EC1.5 value was found to be 21.41 µM, when run concurrently. The IC50 value for test item was found to be 306.80 µg. The EC1.5 mean values for test item was 8.80 µg/mL, which was less than 200 µg/mL. The value of maximum induction (Imax) for for test item was 1.76 and 1.68 at the tested concentration of 25 and 50 µg/mL in experiment 1 and 1.68, 1.86, 2.30, 2.39 and 1.94 at the tested concentration of 6.25, 12.5, 25, 50 and 100 µg/mL, respectively in experiment 3, which was higher than 1.5 fold. The cellular viability was 96.90 and 77.59 % at the test concentration of 25 and 50 µg in experiment 1 and 96.63, 95.70, 97.22, 89.27 and 87.52 % at the tested concentration of tested concentration of 6.25, 12.5, 25, 50 and 100 µg/mL, respectively, in experiment 3 with induction of luciferase activity above 1.5 fold. Observed dose response for luciferase induction was also statistically significant. All criteria for a valid study were met as described in the study plan. From the results of this study, under the specified experimental conditions, test item was concluded as sensitiser in KeratinoSens assay.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 24, 2019 - May 14, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals (Bioneeds India Private Ltd., Devarahosahalli Sompura Hobli, Taluk, Nelamangala, Karnataka 562111, India)
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals: not reported
- Age at study initiation: 8 weeks
- Weight at study initiation: Pre-screen test: 21.38 g to 22.78 g; Main test: 22.13 g to 23.15 g
- Housing: A maximum of four animals were housed in a standard polypropylene cage (Size: L 290 X B 220 X H 140 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material
- Diet: Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum.
- Water: Deep bore-well water passed through Reverse Osmosis Unit was provided in plastic water bottles with stainless steel sipper tubes, ad libitum.
- Acclimation period: 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 23.1
- Humidity (%): 42 - 68
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: Pre screen test: From: 24/04/2019 – 04/05/2019; Main test: 04/05/2019 – 14/05/2019

Vehicle:

dimethyl sulphoxide

Concentration:

Pre-screen test: 2.5, 5, 10, 25, 50 and 100% (w/v) test item in DMSO; Main test: 2.5, 5 and 10% (w/v) test item in DMSO.

No. of animals per dose:

Pre-screen test: 2 females/dose; Main test: 4 females/group

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: a solubility/suspensibility test was carried out in-house using vehicles recommended. The test item did not mix well with Acetone:olive oil (4:1 ratio) and propylene glycol. The test item was clearly soluble in DMSO. Hence, DMSO was selected as vehicle for dose formulation preparation of test item.
- Irritation: Concentrations above 10% caused local skin irritation, whereas 10%, 5% and 2.5% (w/v) TFMSA in DMSO did not cause any local skin irritation.
- Systemic toxicity: Clinical signs of reduced activity and abdominal breathing were observed at 100% w/v) and 50% w/v test item. Reduced activity was observed at 25% w/v) test item. In Group G1 (100% w/v) and G2 (50% w/v), all animals were found dead on Day 3. In Group G3 (25% w/v), one animal was found dead on Day 3.
- Ear thickness measurements: Ear thickness measurements were performed using a thickness gauze (e.g. Digital Vernier Calipers) on Day 1 (Pre-dosing), Day 3 (approx. at 48 hours after the first dose) and on Day 6. On Day 6, animals were weighed and then humanely euthanized by CO2 asphyxiation and ear thickness was determined by ear punch weight determination. There was no within-group change in mean ear thickness noted in groups treated with 10%, 5% and 2.5% TFMSA in DMSO. At doses of 100%, 50% and 25% TFMSA in DMSO there was a progressive increase in ear thickness within each group which did not decline by Day 6.
- Erythema scores: In the 100% w/v dose group, well defined erythema [Grade 2] was observed beginning on Day 2. Moderate to severe erythema [Grade 3] and severe erythema to eschar formation [Grade 4] was observed beginning on Day 3. In the 50% w/v dose group, well defined erythema [Grade 2] was observed beginning on Day 2. Moderate to severe erythema [Grade 3] and severe erythema to eschar formation [Grade 4] was observed beginning on Day 3. In the 25% w/v dose group well defined erythema [Grade 2] was observed beginning only on Day 4. Severity decreased to slight [Grade 1] by Day 6. No erythema was observed in mice at doses of 10% w/v, 5% w/v and 2.5% w/v TFMSA in DMSO.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay: BrdU - ELISA.
- Criteria used to consider a positive response: as outlined by OECD TG 442B, SI ≥ 1.6, is considered to indicate sensitisation.
- Parameters observed: clinical signs, body weight, ear thickness, ear punch weights, determination of cellular proliferation.

TREATMENT PREPARATION AND ADMINISTRATION:
- Each day prior to dosing, the required quantity of test item and positive control 25% (v/v) α-Hexylcinnamaldehyde in Acetone:Olive oil (4:1 v/v) were prepared. Groups of four mice were treated with the test item diluted at 2.5%, 5% and 10% (w/v) in DMSO, based on the results of the pre-screen tests. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item or control to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- On Day 5, 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route. On day 6 (end of the test), the abody weight, ear thickness and clinical signs of every animal in all the groups were recorded. Approximately 24 hours after BrdU injection, animals were humanely euthanized by CO2 asphyxiation. The draining lymph nodes from all test animals were carefully dissected, trimmed of fascia and fat, and then transferred to labelled 2 mL centrifuge tube. Ear punch weights were recorded for each individual ear of all animals using punch method.
To prepare the cell suspensions, 0.3 mL of phosphate buffered saline (PBS) was added to the centrifuge tube that contained the collected lymph nodes. The lymph nodes were crushed with a plunger of the syringe followed by passage through a 70 µm cell strainer fitted to a 50 mL graduated centrifuge tube. The single cell suspension was finally made up to 15 mL with phosphate buffered saline (PBS). NrdU contents in DNA were measured with a commercially available ELISA kit (Roche Applied Science, Manheim, DE).
- Observations: Mice were observed daily for clinical signs and for local irritation at the application site, and twice daily for mortality and morbidity; body weights were recorded on day 1 and day 6; ear thickness measurements were performed with a thickness gauge on days 1, 3 and 6. After euthanising, all animals were subjected to ear punch weight determination and lymph node collection.

Positive control substance(s):

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

Statistics:

One-way ANOVA followed by Dunnet’s test was performed for body weight, ear thickness and ear punch weight data by using SPSS statistical software, v22.

Positive control results:

The positive control group (25% α-Hexylcinnamaldehyde in Acetone:Olive oil [4:1, v/v]) had a SI = 2.3 when compared with vehicle DMSO; and SI = 2.5 when compared to G7 group (Acetone:Olive oil [4:1, v/v]), clearly indicating a positive response (P > 0.05).

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

10% (w/v) test item in DMSO

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

5% (w/v) test item in DMSO

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

2.5% (w/v) test item in DMSO

Parameter:

other: EC1.6

Remarks on result:

not determinable

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : see 'Any other information on results' below.

DETAILS ON STIMULATION INDEX CALCULATION
BrdU labelling index and SI was calculated using the following formulae:
- BrdU Labelling Index = (ABS370-ABSblank370) – (ABS492-ABSblank492);
- SI = (Mean BrdU labelling index for each Test group/ positive control group) / (Mean BrdU labelling index for concurrent vehicle control group).

EC1.6 CALCULATION : Not determinable, as no stimulation index greater than 1.6 was recorded whatever the tested concentration.

CLINICAL OBSERVATIONS:
- In the pre-screen test, signs of reduced activity and abdominal breathing were observed at 100% w/v) and 50% w/v test item. Reduced activity was observed at 25% w/v) test item. In Group G1 (100% w/v) and G2 (50% w/v), all animals were found dead on Day 3. In Group G3 (25% w/v), one animal was found dead on Day 3.
- No mortality and no signs of systemic toxicity were noted in the treated and control animals during the main test. Very slight erythema (barely perceptible) was observed in two animals of group G8 (Positive Control) on day 1 and in all animals of this group from Day 2 to Day 5.

BODY WEIGHTS
No treatment related effects on the body weights were observed throughout the study and all animals showed a normal trend in body weight increase.

OTHER:
- In the pre-screen test, a progressive increase in ear thickness was observed at doses of 25, 50 and 100% (w/v) test item, which did not decline by Day 6. No such changes were noted in any group up to 10% (w/v) test item. In the main test, no significant change in mean ear thickness was noted in treated groups when compared with the vehicle control group.
- In the pre-screen test an increase in ear punch weight was observed at doses of 25, 50 and 100% (w/v) test item; no changes were observed at doses up to 10% (w/v). In the main test, no significant change in mean ear punch weights was noted in treated groups when compared with the vehicle control group.

Main study

Table 1. Main study results.

Treatment	BrdU Labelling Index	Stimulation Index
Vehicle - Acetone:Olive oil [4:1, v/v]	0.318	1.0
Positive control (25% α-Hexylcinnamaldehyde in Acetone:Olive oil [4:1, v/v])	0.811*	2.5
Vehicle control - DMSO	0.353	1.0
Positive control (25% α-Hexylcinnamaldehyde Acetone:Olive oil [4:1, v/v])	0.811*	2.3
10% w/v TFMSA in DMSO	0.277	0.8
5% w/v Acetone:Olive oil [4:1, v/v]	0.344	1.0
2.5% w/v Acetone:Olive oil [4:1, v/v]	0.250	0.7

* Statistically significant; A stimulation index (SI) of ≥1.6, is considered to indicate sensitization.

 

Table 2. Summary of clinical signs and mortality of mice during themain test.

Group	Treatment	Sex	Number of animals	Clinical Signs	Mortality (No. of Mortality/ No. of Animals Dosed)
G7	Acetone: Olive oil (4:1 v/v)	Female	4	N	0/4
G8	25% (v/v) α-Hexylcinnamaldehyde in Acetone: Olive oil (4:1 v/v)	Female	4	N	0/4
G9	DMSO	Female	4	N	0/4
G10	10% (w/v) TFMSA in DMSO	Female	4	N	0/4
G11	5% (w/v) TFMSA in DMSO	Female	4	N	0/4
G12	2.5% (w/v) TFMSA in DMSO	Female	4	N	0/4

N: Normal

 

Table 3.Summary of mouse body weights (g) recorded during the main test

Group	Treatment	Sex		Body Weight (g) on Days		Percent Change in Body weight
				1	6	Day 1-6
G7	Acetone: Olive oil (4:1 v/v)	Female	Mean	24.23	27.93	15.27
			±SD	0.28	0.27	0.53
			n	4	4	4
G8	25% (v/v) α-Hexylcinnamaldehyde in Acetone: Olive oil (4:1 v/v)	Female	Mean	23.88	27.56	15.43
			±SD	0.47	0.54	0.78
			n	4	4	4
G9	DMSO	Female	Mean	24.08	27.77	15.31
			±SD	0.36	0.51	0.48
			n	4	4	4
G10	10% (w/v) TFMSA in DMSO	Female	Mean	24.21	27.89	15.22
			±SD	0.33	0.37	0.82
			n	4	4	4
G11	5% (w/v) TFMSA in DMSO	Female	Mean	24.12	27.76	15.09
			±SD	0.44	0.54	0.55
			n	4	4	4
G12	2.5% (w/v) TFMSA in DMSO	Female	Mean	23.92	27.58	15.28
			±SD	0.36	0.41	0.45
			n	4	4	4

n: Number of animals; SD: Standard Deviation 

 

Table 4. Summary of skin erythema scores of mice recorded during main test

Group	Treatment	Sex		Ear Thickness (mm) on Days
				1		3		6
				Left	Right	Left	Right	Left	Right
G7	Acetone: Olive oil (4:1 v/v)	Female	Mean	0.188	0.185	0.188	0.188	0.188	0.186
			±SD	0.003	0.004	0.003	0.003	0.003	0.003
			n	4	4	4	4	4	4
G8	25% (v/v) α-Hexylcinnamaldehyde in Acetone: Olive oil (4:1 v/v)	Female	Mean	0.185	0.184	0.193*	0.193	0.203*	0.205*
			±SD	0.004	0.003	0.003	0.003	0.003	0.004
			n	4	4	4	4	4	4
G9	DMSO	Female	Mean	0.185	0.188	0.185	0.188	0.185	0.188
			±SD	0.004	0.003	0.004	0.003	0.004	0.003
			n	4	4	4	4	4	4
G10	10% (w/v) TFMSA in DMSO	Female	Mean	0.185	0.188	0.188	0.186	0.186	0.188
			±SD	0.004	0.003	0.003	0.005	0.005	0.003
			n	4	4	4	4	4	4
G11	5% (w/v) TFMSA in DMSO	Female	Mean	0.188	0.185	0.188	0.188	0.188	0.188
			±SD	0.003	0.004	0.003	0.003	0.003	0.003
			n	4	4	4	4	4	4
G12	2.5% (w/v) TFMSA in DMSO	Female	Mean	0.188	0.188	0.188	0.188	0.188	0.188
			±SD	0.003	0.003	0.003	0.003	0.003	0.003
			n	4	4	4	4	4	4

LE: Left Ear; RE: Right Ear; F: Female

* Data are presented as follows: Erythema Grade (Number of animals with the given erythema grade.)

Scoring values: Erythema and Eschar formation:0= No erythema; 1= Very slight erythema (barely perceptible); ()=Values in parenthesis are number of animals with the given erythema grade.

 

Table 5.Summary of ear thickness of mice recorded during main test

Group	Treatment	No. of Animals	Sex	Scores on Day
				1		2		3		4		5		6
				LE	RE	LE	RE	LE	RE	LE	RE	LE	RE	LE	RE
G7	Acetone: Olive oil (4:1 v/v)	4	F	0	0	0	0	0	0	0	0	0	0	0	0
G8	25% (v/v) α-Hexylcinnamaldehyde in Acetone: Olive oil (4:1 v/v)	4	F	1 (2)*	1 (2)*	1 (4)*	1 (4)*	1 (4)*	1 (4)*	1 (4)*	1 (4)*	1 (4)*	1 (4)*	0	0
G9	DMSO	4	F	0	0	0	0	0	0	0	0	0	0	0	0
G10	10% (w/v) TFMSA in DMSO	4	F	0	0	0	0	0	0	0	0	0	0	0	0
G11	5% (w/v) TFMSA in DMSO	4	F	0	0	0	0	0	0	0	0	0	0	0	0
G12	2.5% (w/v) TFMSA in DMSO	4	F	0	0	0	0	0	0	0	0	0	0	0	0

n: Number of animals; SD: Standard Deviation

*: Statistically significant when compared to both the control groups (p<0.05)  

 

Table 6. Summary of ear punch weights (mg) of mice recorded during the main test

Group	Treatment	Sex		Ear Punch Weight (mg)
				Left Ear	Right Ear	Mean
G7	Acetone: Olive oil (4:1 v/v)	Female	Mean	5.98	6.05	6.01
			±SD	0.05	0.13	0.06
			n	4	4	4
G8	25% (v/v) α-Hexylcinnamaldehyde in Acetone: Olive oil (4:1 v/v)	Female	Mean	7.80*	7.75*	7.78*
			±SD	0.32	0.42	0.35
			n	4	4	4
G9	DMSO	Female	Mean	5.98	5.95	5.96
			±SD	0.13	0.13	0.10
			n	4	4	4
G10	10% (w/v) TFMSA in DMSO	Female	Mean	5.95	5.88	5.91
			±SD	0.17	0.25	0.19
			n	4	4	4
G11	5% (w/v) TFMSA in DMSO	Female	Mean	5.88	5.93	5.90
			±SD	0.17	0.10	0.13
			n	4	4	4
G12	2.5% (w/v) TFMSA in DMSO	Female	Mean	5.68	5.60	5.64
			±SD	0.28	0.26	0.26
			n	4	4	4

n: Number of animals; SD: Standard Deviation

*: Statistically significant when compared to both the control groups (p<0.05).

 

Table 7. Summary of BrdUlabelling index and stimulation index of mice during the main test

Group	Treatment	Sex		BrdU Labelling Index	Stimulation Index (SI)
G7	Acetone: Olive oil (4:1 v/v)	Female	Mean	0.318	1.0
			±SD	0.02
			n	4
G8	25% (v/v) α-Hexylcinnamaldehyde in Acetone: Olive oil (4:1 v/v)	Female	Mean	0.811*	2.5/2.3**
			±SD	0.22
			n	4
G9	DMSO	Female	Mean	0.353	1.0
			±SD	0.06
			n	4
G10	10% (w/v) TFMSA in DMSO	Female	Mean	0.277	0.8
			±SD	0.04
			n	4
G11	5% (w/v) TFMSA in DMSO	Female	Mean	0.344	1.0
			±SD	0.02
			n	4
G12	2.5% (w/v) TFMSA in DMSO	Female	Mean	0.250	0.7
			±SD	0.04
			n	4

n: Number of animals; SD: Standard Deviation; BrdU: 5-bromo-2-deoxyuridine

*: Statistically significant when compared to both the control groups (p<0.05)

**: SI of 2.5 when compared to Acetone: Olive oil (4:1 v/v) and SI of 2.3 when compared with vehicle DMSO.

Interpretation of results:

GHS criteria not met

Remarks:

(EU criteria)

Conclusions:

Under test conditions, the test item showed to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Executive summary:

The skin sensitisation potential of the test item was evaluated in accordance with OECD 422B, under GLP conditions. In a first step, a preliminary solubility study and an irritation/toxicity test were carried out. Based on the solubility test, the vehicle chosen was DMSO. The toxicity screening was performed in female CBA/J mice (2/dose) by applying the test item at doses of 2.5, 5, 10, 25, 50, and 100% (w/v) in DMSO. Signs of reduced activity and abdominal breathing were observed at 100% and 50% (w/v) test item; and reduced activity was observed at 25% (w/v) test item. Irritation was observed in these three groups as well. All animals administered 100% and 50% (w/v) test item were found dead on day 3, as well as one out of two animals administered 25% (w/v) test item. No signs were observed up to 10% (w/v) test item. Based on these results, 10% (w/v) was selected as top dose for the main test. In the main assay, 24 female CBA/J mice were used (4/dose): four groups test item at 10%, 5%, and 2.5% w/v (in DMSO), a negative/vehicle control group (DMSO) and a positive control group (25 % HCA in AOO). The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On D6,the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte using an ELISA kit and the values obtained were used to calculate stimulation indices (SI). No mortality, systemic clinical signs or indications of irritancy in the application site were observed during the main study. No increase in ear thickness and in ear weight was noted. No treatment related effects on the mean body weight changes were observed either. The stimulation index values were 0.8, 1.0, and 0.7 at concentrations of 10%, 5%, and 2.5% (w/v) test item, respectively. Under test conditions, the test item showed to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Two in-vitro/in-chemico studies are avaible. In both of them, the substance was determined to be skin sensitizer. However, for over 40 years of substance handling, there have been no known cases of skin sensitization. The obtained results might be related to false positives and therefore, an in-vivo skin sensitization LLNA test was performed to confirm the results.

In the in vivo mouse local lymph node assay (LLNA): BrdU-ELISA performed according to OECD 422B (GLP study), the test item showed no sensitisation potential, with stimulation indexes of 0.8, 1.0, and 0.7 at concentrations of 10%, 5%, and 2.5% (w/v) test item in DMSO, respectively. The vehicle (DMSO) was chosen based on a preliminary solubility study, and the highest dose tested was 10% (w/v) test item based on an irritation/toxicity screening test. The toxicity screening was performed in female CBA/J mice (2/dose) by applying the test item at doses of 2.5, 5, 10, 25, 50, and 100% (w/v) in DMSO. Signs of reduced activity and abdominal breathing were observed at 100% and 50% (w/v); and reduced activity at 25% (w/v) test item. Irritation was observed in these three groups as well. All animals administered 100% and 50% (w/v) test item were found dead on day 3, as well as one out of two animals administered 25% (w/v) test item, whereas no signs were observed up to 10% (w/v) test item. In the main assay, no mortality, systemic clinical signs or indications of irritancy in the application site were observed during the main study. No increase in ear thickness and in ear weight was noted. No treatment related effects on the mean body weight changes were observed either. The stimulation index values demonstrated that the test item is not sensitising; the irritating potential of the test item might have led to false positive results in vitro.

Regarding the screening test, it should be borne in mind that DMSO completely solubilized the test item, thus enhancing the absorption of test item on the dorsum of the ear, where the thickness is very low compared to dorso-lateral skin on the body. Therefore, the signs observed are not directly comparable to those observed in the acute dermal toxicity studies (where the test item was applied by moistening with 0.5 ml distilled water only).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data (two positive results in vitro plus one clearly negative result in vivo), the substance is classified for skin sensitisation, according to CLP Regulation (EC) No. 1272/2008.