[No public or meaningful name is available]

Endpoint summary

Description of key information

Due to the inconsistency of the results in silico (no alert), in chemico (negative) and in vitro (positive) no final conclusion for skin sensitization could be drawn on the basis of alternative methods. Thus, a local lymph node assay was initiated. This test showed a weak skin sensitising potency of the registered substance, leading to classification with Skin Sens 1B.

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:

supporting study

Study period:

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))

Version / remarks:

of February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
The Direct Peptide Reactivity Assay (DPRA) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.
For comparison, tests were performed with the test item, the vehicle (solvent control = negative control) and the known sensitizer Cinnamic aldehyde (positive control).

The DPRA quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25 +/-2.5 ºC. Relative peptide concentration is measured by reversed phase (C18) high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 and 258 nm. The synthetic peptides contain phenylalanine to aid in the detection.
The test item was dissolved and tested according to the given test procedure. Cinnamic aldehyde was used as positive control at a concentration of 100 mmol/L in acetonitrile.

Cysteine and lysine peptide solutions were incubated at 1:10 and 1:50 ratio in glass auto sampler vials with the test item solution for 24 hours at 25 ± 2.5 °C in the dark. Samples were visually inspected for precipitation or phase separation before HPLC analysis. The test item was analyzed in triplicate for both peptides. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours. HPLC analysis for the cysteine and lysine peptides were performed on separate days with the test item solutions freshly prepared for both assays on each day. The concentration of cysteine or lysine peptide was photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards. Percent peptide depletion is calculated according to OECD Guideline.

The following criteria should be met for a test chemical’s results to be considered valid: a) the maximum standard deviation for the test chemical replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion, b) the mean peptide concentration of three injections of the reference control C in the appropriate solvent should be 0.50 ± 0.05 mM.

According to the study protocol the mean percent cysteine and percent lysine depletion value was calculated for the test item and the positive control. Negative depletion was considered as “0” when calculating the mean. By using the cysteine 1:10/lysine 1:50 prediction model, the threshold of 6.38 % average peptide depletion can be used to support the discrimination between skin sensitizers and non-sensitizers in the framework of an IATA.

Positive control results:

The positive control cinnamic aldehyde led to a depletion of 74.07 % cysteine peptide and 56.28 % lysine peptide. The mean cysteine/lysine peptide depletion was calculated with 65.18 % leading to a positive results (high reactivity class according to the cysteine 1:10/lysine 1:50 prediction model).

Run / experiment:

other: mean of 2 runs

Parameter:

other: % depletion in the cysteine 1:10/lysine 1:50 prediction model:

Value:

0.92

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

DPRA prediction: negative

Parameter:

other: % cysteine depletion

Value:

1.83

Vehicle controls validity:

valid

Positive controls validity:

valid

Parameter:

other: % lysine depletion

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The acceptance criteria for a DPRA test to be considered valid were met.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes

The test item visually appeared a clear solution in acetonitrile at the test concentration of 100 mmol/L. Slight precipitation was observed before or after incubation.
Absorbance at 220 nm was not observed. Retention time similar to peptide was not observed. Co-elution of the test item with the peptides was not observed.

Results of the DPRA:

Test item	Mean % Cysteine peptide depletion	Mean % Lysine peptide depletion	Mean % Cysteine/Lysine peptide depletion	reactivity class	DPRA prediction
positive control (DNCB)	74.07	56.28	65.18	moderate	positive
test item	1.83	0	0.92	minimal	negative*

*Slight precipitation was observed after the incubation period. A conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result.

Interpretation of results:

other: negative

Executive summary:

The Direct Peptide Reactivity Assay (DPRA; OECD 442C) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

Since the test item visually appeared a clear solution in acetonitrile at the test concentration of 100 mmol/L this solvent was used in the DPRA. Slight precipitaton of the test item occured after incubation, thus, the peptide depletion may be underestimated. The cysteine 1:10/lysine 1:50 prediction model was applied to the test item. No relevant depletion of cysteine and lysine peptides became obvious in the DPRA (0.92 %). According to the prediction model “minimal reactivity” was derived for the test item in water, leading to a DPRA prediction of “negative“.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

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)

Version / remarks:

of February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

Specifications:
KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland as specified in OECD Test Guideline 442D.

Preparation of Cultures:
For testing, cells were 80-90 % confluent, and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested and distributed into 96-well plates (10 000 cells/well). Attention was paid to avoid sedimentation of the cells during seeding to ensure homogeneous cell number distribution across wells. For each repetition, three technical replicates were used for the luciferase activity measurements, and three parallel technical replicates used for the cell viability assay.
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing 1 % serum and Geneticin.

Treatment:
In 96-well plates, incubated at 37±1 °C, 5 % (v/v) CO2, for about 48 hours in medium with serum but without Geneticin. For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at two independent repetitions each containing three replicates of each concentration. Each independent repetition was performed on a different day with fresh stock solution of test chemicals and independently harvested cells. Cells may come from the same passage however.

Luciferase Activity Measurements:
After the 48 hour exposure time with the test and control items, cells were washed with a phosphate buffered saline, and the relevant lysis buffer (One GlowTM Luciferase Assay System)6 for luminescence readings added to each well for an adequate time at room temperature. Plates with the cell lysate will then be placed in the luminometer for reading.

Cytotoxicity Assessment:
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cells were incubated for 4 hours at 37 °C in the presence of 5 % CO2. The MTT medium was removed and cells were lysed by adding 10 % aqueous SDS solution to each well overnight or for up to 3 days at 37 °C. After shaking to ensure homogeneity of the solution in the wells and then absorption read at 620 nm using a photometer.

Positive controls:
Cinnamic aldehyde (CAS No. 14371-10-9), supplied by Sigma-Aldrich Chemie GmbH. was used as the positive control. A series of 5 master concentrations ranging from 0.4 to 6.4 mM was prepared in DMSO and diluted as described for the master concentrations, so that the final concentration of the positive control range from 4 to 64 μM.

Negative controls:
The negative control was diluted into culture medium containing serum so that the final concentration was 1 %. The solvent DMSO was used as the negative control, which is known to not affect cell viability and corresponds to the same concentration of DMSO found in the test item and in the positive control.

Positive control results:

The assay aceptance criteria for the positive controls were met:
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 32 to 64 µM in Experiments 1 and 2.
The EC1.5 values for the positive control were 20.19 and 25.22 µM in Experiments 1 and 2, respectively. The average induction for the positive control at 64 µM was 4.97 in Experiment 1 and 17.47 in Experiment 2.

Run / experiment:

other: Exp. 1

Parameter:

other: Imax value

Value:

2.8

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Exp. 2

Parameter:

other: Imax value

Value:

2.92

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: proven

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 7.13 % and 6.41 % in Experiments 1 and 2, respectively.

All assay acceptance criteria were met.

Since the Imax in both experiments was above 1.5-fold, the test article was considered to be positive in the ARE-Nrf2 Luciferase Test.

Table 1: Numerical results for the test item

	Luciferase determinations	Luciferase determinations	Cytotoxicity determinations	Cytotoxicity determinations
parameter	Imax	EC 1.5 [µM]	IC 50 [µM]	IC30 [µM]
test item, Exp. 1	2.80*	32.13*	259.60	217.42
test item, Exp. 2	2.92*	34.47*	374.02	323.39
average ± SD	2.86 ± 0.09	34.30  ± 0.24	311.60  ± 80.91	265.17  ± 74.93
positive control (64 µM) Exp. 1/2	2.25*/2.37*	20.19/25.22

* = significant compared to the negative control (p<0.05)

For the positive control cinnamic aldehyde the average fold induction in the two replicates at 64 μM should be between 2 and 8, the EC1.5 value should be between 7 μM and 30 μM.

Executive summary:

The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The ARE-Nrf2 Luciferase Test Method (KeratinoSens) was performed acording to OECD 442D.

The test substance was solved in DMSO and tested at final concentrations of 0.98 to 2000 µM. The assay acceptance criteria for the positive control cinnamic aldehyde and the negative control DMSO were met in this test. Treatment of the cell cultures for 48 hours with the test item led to the following results: The mean maximal average fold induction of the luciferase activity (Imax) value observed at any concentration of the test item was 2.86 and the mean EC1.5 value, representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50 % enhanced luciferase acitivity), was 34.30 µM.

All assay acceptance criteria were met. Since the Imax values in both experiments was above 1.5-fold, the test article was considered to be positive in the ARE-Nrf2 Luciferase Test.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

of February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

Specifications:
KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland as specified in OECD Test Guideline 442D.

Preparation of Cultures:
For testing, cells were 80-90 % confluent, and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested and distributed into 96-well plates (10 000 cells/well). Attention was paid to avoid sedimentation of the cells during seeding to ensure homogeneous cell number distribution across wells. For each repetition, three technical replicates were used for the luciferase activity measurements, and three parallel technical replicates used for the cell viability assay.
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing 1 % serum and Geneticin.

Treatment:
In 96-well plates, incubated at 37±1 °C, 5 % (v/v) CO2, for about 48 hours in medium with serum but without Geneticin. For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at two independent repetitions each containing three replicates of each concentration. Each independent repetition was performed on a different day with fresh stock solution of test chemicals and independently harvested cells. Cells may come from the same passage however.

Luciferase Activity Measurements:
After the 48 hour exposure time with the test and control items, cells were washed with a phosphate buffered saline, and the relevant lysis buffer (One GlowTM Luciferase Assay System)6 for luminescence readings added to each well for an adequate time at room temperature. Plates with the cell lysate will then be placed in the luminometer for reading.

Cytotoxicity Assessment:
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cells were incubated for 4 hours at 37 °C in the presence of 5 % CO2. The MTT medium was removed and cells were lysed by adding 10 % aqueous SDS solution to each well overnight or for up to 3 days at 37 °C. After shaking to ensure homogeneity of the solution in the wells and then absorption read at 620 nm using a photometer.

Positive controls:
Cinnamic aldehyde (CAS No. 14371-10-9), supplied by Sigma-Aldrich Chemie GmbH. was used as the positive control. A series of 5 master concentrations ranging from 0.4 to 6.4 mM was prepared in DMSO and diluted as described for the master concentrations, so that the final concentration of the positive control range from 4 to 64 μM.

Negative controls:
The negative control was diluted into culture medium containing serum so that the final concentration was 1 %. The solvent DMSO was used as the negative control, which is known to not affect cell viability and corresponds to the same concentration of DMSO found in the test item and in the positive control.

Positive control results:

The assay aceptance criteria for the positive controls were met:
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 32 to 64 µM in Experiments 1 and 2.
The EC1.5 values for the positive control were 13.68 and 26.56 µM in Experiments 1 and 2, respectively. The average EC1.5 value of the positive control of 19.06 μM is within two standard deviations of the historical mean. The average induction in the two replicates for cinnamic aldehyde at 64 μM is 3.86 and 2.16, and thus as required between 2 and 8;

Run / experiment:

other: Exp. 1

Parameter:

other: Imax value

Value:

2.52

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Exp. 2

Parameter:

other: Imax value

Value:

2.64

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: proven

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

PES PRECURSOR XP 2542 (TLE) was tested at 12 concentrations in the range from 0.98 to 2000 μM in DMSO. Cinnamic aldehyde tested at five concentrations from 4 – 64 μM was used as the positive control. Two independent repetitions with three parallel technical replicates were run with this same set-up, and one parallel plate was prepared for cytotoxicity determination.
For the MTT data the % viability was then calculated for each well in the test plate in relation to average of the six solvent control wells.
For the luciferase data the average value of the six solvent control wells was set to 1, and for each well in the test plate the fold induction was calculated in relation to this value. The parameters of luciferase induction and cytotoxicity determinations were calculated for the test item-treated cells (see Table 1).

The maximal average fold induction of the luciferase activity (Imax) value observed at any concentration of the test item was 2.58 ± 0.09 and the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was 26.79 ± 6.04 μM.
The IC50 value was 218.66 ± 25.42 μM and the IC30 value was 196.88 ± 28.09 μM for 50% and 30% reduction of cellular viability, respectively.
The KeratinoSensTM prediction of the test item is considered positive as the following 4 conditions are all met in 2 of 2 repetitions:
• the Imax of 2.58 is higher than 1.5 fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test);
• the cellular viability is higher than 70% at the lowest concentration (26.79 μM) with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration);
• the EC1.5 value of 26.79 μM is less than 1000 μM;
• there is an apparent overall dose-response for luciferase induction (Spearman's rank correlation coefficient of p<0.01).
The positive control cinnamic aldehyde was run in both repetitions. The detailed results for the positive control are reported in Text table 7.2 Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This was the case in both repetitions. The induction at 64 μM and the EC1.5 for cinnamic aldehyde were also calculated. The targets are:
• the average induction in the two replicates for cinnamic aldehyde at 64 μM should be between 2 and 8;
• the EC1.5 value should be between 7 μM and 30 μM or within two standard deviations of the historical mean value (see Appendix 2).

At least one of these two numerical criteria must be met in order to accept a repetition. In the experiments performed both criteria were fulfilled in both repetitions. Thus both repetitions were valid for the positive control.
In addition, the EC1.5 value of the positive control of 19.06 μM is within two standard deviations of the historical mean (see Appendix 2).

As further performance criterion the variability of the luminescence reading for the negative control DMSO must be below 20% in each repetition which consists of 6 wells tested in triplicate. The average coefficients of variation (CV) for the negative control were 7.79% or 5.65% for the first or second repetition, respectively.
All quality criteria required (see section 6.2) were fulfilled.

All assay acceptance criteria were met.

Since the Imax in both experiments was above 1.5-fold, the test article was considered to be positive in the ARE-Nrf2 Luciferase Test.

Table 1: Numerical results for the test item

	Luciferase determinations	Luciferase determinations	Cytotoxicity determinations	Cytotoxicity determinations
parameter	Imax	EC 1.5 [µM]	IC 50 [µM]	IC30 [µM]
test item, Exp. 1	2.52	22.86*	237.38	217.73
test item, Exp. 2	2.64	31.40*	201.43	178.02
average ± SD	2.58 ± 0.09	26.79  ± 6.04	218.66  ± 25.42	196.88  ± 28.09
positive control (64 µM) Exp. 1/2	3.86*/2.16*	13.68/26.56

* = significant compared to the negative control (p<0.05)

For the positive control cinnamic aldehyde the average fold induction in the two replicates at 64 μM should be between 2 and 8, the EC1.5 value should be between 7 μM and 30 μM.

Executive summary:

The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The ARE-Nrf2 Luciferase Test Method (KeratinoSens) was performed acording to OECD 442D.

The test substance was solved in DMSO and tested at final concentrations of 0.98 to 2000 µM. The assay acceptance criteria for the positive control cinnamic aldehyde and the negative control DMSO were met in this test. Treatment of the cell cultures for 48 hours with the test item led to the following results: The mean maximal average fold induction of the luciferase activity (Imax) value observed at any concentration of the test item was 2.58 and the mean EC1.5 value, representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50 % enhanced luciferase acitivity), was 26.79 µM.

All assay acceptance criteria were met. Since the Imax values in both experiments was above 1.5-fold, the test article was considered to be positive in the ARE-Nrf2 Luciferase Test.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

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)

Version / remarks:

Guideline adopted 22 July 2010; Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).

Deviations:

yes

Remarks:

Measurement of cell counts instead of radioactive labelling. In addition, ear swelling and ear weights are determined to discriminate the irritating potential from the sensitizing potential of the test substance.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

Principles of method if other than guideline:

The study was performed according to OECD 429. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.
Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 26 - 33 g

- Housing: The animals were kept singly in MAKROLON cages (type II) with a basal surface of approximately 360 cm² cm and a height of approximately 14 cm at a room temperature. Animals were not group-housed to prevent contact of the application sites.
Deviations from the maximum range caused for example during cleaning procedures and change of cages are dealt with in SOPs.
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week.
Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted by LUFA-ITL (see Appendix 4: Limitation for contaminants in the bedding material).

- Diet (e.g. ad libitum): yes (Commercial diet ssniff® R/M-H V1534)
- Water (e.g. ad libitum): yes
- Acclimation period: 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C
- Humidity (%): 55% +/- 10%
- Photoperiod (hrs dark / hrs light): The rooms were alternately lit (about 150 lux at approximately 1.50 m room height) and darkened for periods of 12 hours each.
- IN-LIFE DATES: From: 25 October 2019 To: 23 January 2020

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

The test item was used undiluted or dissolved in acetone /olive oil (4:1, v/v).
Acetone/olive oil (4:1, v/v) was selected as recommended by the OECD guideline and it provided a most suitable solution of the test item both for administration and adherence to the mouse ear of such high concentrations. It was also used as negative reference item.
The positive control was dissolved in acetone / olive oil (4:1, v/v).

No. of animals per dose:

6

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: solution in acetone/olive oil (A/O, 4:1, v/v)
- Irritation:
no effects
- Systemic toxicity:
no effects
- Ear thickness measurements:
no effects
- Erythema scores:
0
A preliminary experiment was carried out in 4 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations out of the technically feasible concentrations (see Table below). Three concentrations of 25%, 50% and 75% (w/w) dissolved in acetone / olive oil (4:1, v/v) and the undiluted test item (100%) were examined. Doses were selected based on the OECD guideline from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% (w/w) etc.
The preliminary experiment was conducted under conditions identical to the main LLNA study, except there was no assessment of lymph node proliferation and only 1 animal per concentration was used. Both ears of each mouse were observed for erythema. The neat test item and the soutions were administered to the dorsum of both animal's ears at an application volume of 25 µL/ear.
No irritating properties were observed in this preliminary experiment at concentrations of 2.5%, 5% or 10% (w/w), and no differences in ear weight and ear thickness were noted.


MAIN STUDY
The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation, as the radioactive method proposed by the OECD guideline led to problems in various EU laboratories: such as (i) practical difficulties/complexity of the test, in particular the radiochemical steps, which sometimes resulted in loss of specimen/activity; this in turn led to variability in the results and to a poor reproducibility and (ii) radiation protection issues. However, the OECD guideline allows other endpoints for assessment of proliferation in form of lymph node cell counts and lymph node weights if justification and appropriate scientific support exist showing the validity of this method.
The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European inter-laboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs.
In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item.
By additionally measuring simple inflammatory parameters such as ear thickness or ear weight, it is possible to reliably determine the degree of response that is attributable to irritation (Vohr and Ahr, 2005 ).
Hence, in addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control. The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group to the test item treated animals by the vehicles treated ones. The cut-off threshold value for ear weight was set at 1.1.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: modified LLNA
- Criteria used to consider a positive response:
The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the interlaboratory validation of this method, for details see Ehling et al. 2005a and 2005b, page 12), were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.

TREATMENT PREPARATION AND ADMINISTRATION:
The experimental schedule of the assay was as follows:
- Day 1:
The weight of each animal was individually identified. The weights and any clinical signs were recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Open application of 25 µL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
- Days 2 and 3:
The application procedure carried out on day 1 was repeated.
- Day 4 (24 hours after the last application):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
The animals were euthanized by carbon dioxide (CO2) inhalation and laparotomised.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in Dulbecco's Phopshate Buffered SAline (DPBS)/0.5% Bovine serum albumin (BSA) and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

Test formulation analysis was carried out non-GLP in 2 steps:
The analytical method was validated by Analytisches Zentrum Biopharm Berlin GmbH (AZB) before in vivo testing. The following parameters were determined:
- Linearity, Accuracy, Precision, Sensitivity, Specificity and Stability (6 hours at room temperature)
1) Samples of approximately 2 x 0.5 mL were taken on 20 January 2020 at the following times and stored at -20 °C ± 10 % until analysis for the dose levels of 50 % (w/w), 75 % (w/w) and 100 % (w/w).
Analysis of homogeneity and concentration:
At the start of administration, during (middle) administration and before administration to the last animal/concentration (3 samples/dose level). Number of samples: 9 (18)
(2) Formulation samples were sent on 27 February 2020 on dry ice to Analytisches Zentrum Biopharm Berlin.
The samples were labelled with study number, type of sample, concentration, test day, sampling time and date.
Observations of the animals daily for clinical signs, local irritation or systemic toxicity and body weight (day 1 and day 4) were included in the study.

Positive control substance(s):

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

Statistics:

The acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.

Positive control results:

The positive control group (20 % solution v/v of alpha-hexyl cinnamic aldehyde in acetone / olive oil (4:1, v/v)) caused the expected increases in lymph node cell count (1.638, not statistically significant but index > 1.4) and lymph node weight (1.778, statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid. (see Table 1)

Key result

Parameter:

SI

Value:

1.419

Test group / Remarks:

50% test item in A/O: the stimulation index (SI) for lymph node cell count is above the threshold level of 1.4 compared to control

Remarks on result:

other: indication of mild skin sensitization

Key result

Parameter:

SI

Value:

1.441

Test group / Remarks:

75% test item in A/O: the stimulation index (SI) for lymph node cell count is above the threshold level of 1.4 compared to control

Remarks on result:

other: indication of mild skin sensitization

Key result

Parameter:

SI

Value:

1.575

Test group / Remarks:

100% (neat test item): the stimulation index (SI) for lymph node cell count is above the threshold level of 1.4 compared to control

Remarks on result:

other: indication of mild skin sensitization

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
In the main study treatment with PES PRECURSOR XP 2542 (TLE) at the concentration of 50 % or 75 % led to an increase (not statistically significant) of the stimulation index of the lymph node cell count above the threshold level of 1.4. Undiluted test item led to a statistically significant increase of the stimulation index of the lymph node cell count above the threshold level of 1.4. Stimulation indices of 1.419, 1.441 and 1.575 were determined for the concentrations of 50 %, 75 % and 100 %, respectively. All values indicate sensitising properties.
All concentrations led to an increase (significant at p ≤ 0.01) of the lymph node weight indicating sensitising properties.
The threshold level for the ear weight of 1.1 was exceeded only in the 50 % concentration (not statistically significant). No increase of ear thickness was noted.
 
The positive control group caused the expected increases in lymph node cell count (not statistically significant but index > 1.4) and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid.

DETAILS ON STIMULATION INDEX CALCULATION
The study was performed according to OECD 429. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.
Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).


EC1.4 CALCULATION
The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the interlaboratory validation of this method, for details see Ehling et al. 2005a and 2005b, page 12), were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones. No values were above the threshold of 1.4 (lymph node cell count) or 1.1 (ear weight). Thus, under the present test conditions, Macrolex Rot 5B at concentrations of 2.5%, 5% or 10% (w/w) in acetone/olive oil (4:1, v/v) did not reveal any skin sensitising properties in the local lymph node assay. A 10% (w/w) concentration of the test item in acetone/olive oil (4:1, v/v) was the maximum feasible concentration.


CLINICAL OBSERVATIONS and BODY WEIGHTS
No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

In a preliminary experiment, concentrations of 25 %, 50 %, 75 % and 100 % of PES PRECURSOR XP 2542 (TLE), employing 1 animal per concentration, were examined. No irritating properties were observed in this preliminary experiment at concentrations of 25 %, 50 %, 75 % and 100 %, no differences in ear weight and ear thickness were noted.

Table 1: Stimulation indices (SI) in the main experiment (mean of 6 animals per group):

Parameter	negative control (A/O)	50% (w/w) test item in A/O	75% (w/w) test item in A/O	100% (neat) test item	positive control (20% alpha-hexyl cinnamic aldehyde (v/v) in A/O)
Lymph node cell count	1.000	1.419	1.441	1.575**	1.638
Lymph node weight	1.000	1.315**	1.556**	1.630**	1.778**
Ear weight	1.000	1.136	0.886	1.011	1.068
Ear thickness, TD4	1.000	1.032	1.040	1.032	1.108

** significantly different from control at p ≤ 0.01

The analysis of the test item/vehicle solutions of the 50 %, 75 % (w/w) and 100 % concentrations for the actual test item levels was carried out by Analytisches Zentrum Biopharm GmbH (AZB) (non-GLP) under conditions employing a validated analytical method. The analysis resulted in actual levels of 56.95 – 63.20 % (50 % (w/w) concentration), 87.48 – 116.26 % (75 % (w/w) concentration) and 105.96 – 130.09 % (100 % concentration) of the nominal concentration.

Executive summary:

The purpose of this study was to determine the sensitising potential of PES PRECURSOR XP 2542 (TLE) in the modified local lymph node assay in mice.

The study was performed according to OECD 429. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.

Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones.

Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

Two concentrations of PES PRECURSOR XP 2542 (TLE) (50 % and 75 % (w/w)), dissolved in acetone/olive oil (4:1, v/v) and the undiluted test item (100 %) were tested in six female NMRI mice per group and compared to a vehicle control group.

In the main study treatment with PES PRECURSOR XP 2542 (TLE) at the concentration of 50 % or 75 % led to an increase (not statistically significant) of the stimulation index of the lymph node cell count above the threshold level of 1.4. Undiluted test item led to a statistically significant increase of the stimulation index of the lymph node cell count above the threshold level of 1.4. Stimulation indices of 1.419, 1.441 and 1.575 were determined for the concentrations of 50 %, 75 % and 100 %, respectively. All concentrations led to an increase (significant at p ≤ 0.01) of the lymph node weight indicating sensitising properties.

The threshold level for the ear weight of 1.1 was exceeded only in the 50 % concentration (not statistically significant). No increase of ear thickness was noted.

The positive control group caused the expected increases in lymph node cell count (not statistically significant but index > 1.4) and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid.

No signs of local or systemic intolerance were recorded.

In conclusion, under the present test conditions, PES PRECURSOR XP 2542 (TLE) at the concentrations of 50 %, 75 % (w/w) and undiluted test item (100 %) revealed signs of skin sensitisation potential. The stimulation indices for the concentration of 50 %, 75 % and undiluted test item (100 %) were 1.419, 1.441 and 1.575 and hence, the test item PES PRECURSOR XP 2542 (TLE) is classified to be skin sensitising in this test system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Due to the physico-chemical characteristics of 'Ester Adipic Acid and Neodecanoic acid, 2-oxiranylmethyl ester' (i.e. water solubility below 1 mg/L, log Pow of 5.5 and a molecular mass of > 600 g/mol for the main component) dermal uptake can be predicted to be low (ECHA Guidance on Information Requirements, Chapter R7c, 2017).

'Ester Adipic Acid and Neodecanoic acid, 2-oxiranylmethyl ester' was investigated in silico for structural activity relationship (QSAR), in chemico for peptide reactivity, and in vitro for keratinocyte activation (ARE-Nrf2 Luciferase Test Method).

The structure activity relationship of the substance was investigated by using the OECD QSAR Toolbox 4.3.1 (released 2019). No protein binding alerts for skin sensitization were identified in silico by the OECD QSAR Toolbox. Additionally, low skin permeability is predicted in the Toolbox (Schlecker, 2019).

‘Ester Adipic Acid and Neodecanoic acid, 2-oxiranylmethyl ester’ was experimentally investigated for the first and second key event of skin sensitization (DPRA and ARE-Nrf2 Luciferase Test Method). The experimental investigation of the third key event of skin sensitization, the h-CLAT (OECD 442D) was not initiated since the registered substance exerts a log Pow of 5.6. According to OECD 442E test chemicals with a Log Kow greater than 3.5 tend to produce false negative results and such results should thus not be considered. The registered substance is thus outside the applicability domain of the test.

The Direct Peptide Reactivity Assay (DPRA; OECD 442C) is an in chemico procedure proposed to address the first key event of skin sensitization, i.e. protein binding. Cysteine and lysine percent peptide depletion values are measured and used in a prediction model to categorize a substance in one of four classes of reactivity. Since the registered substance visually appeared a clear solution in acetonitrile at the test concentration of 100 mmol/L this solvent was used in the DPRA. Slight precipitation of the test item occurred before or after incubation, thus, the peptide depletion may be underestimated. No relevant depletion of cysteine and lysine peptides became obvious in the DPRA (0.92 %). According to the prediction model the DPRA outcome was negative (Schaub, 2018).

The ARE-Nrf2 Luciferase Test Method (KeratinoSens) was performed according to OECD 442D to investigate the second key event of skin sensitization, i.e. keratinocyte activation. The potential of the test item to induce genes that are regulated by the antioxidant response element (ARE) is measured in this test. Two batches (Batch No. LEZF 140027 and Batch No. GCZ-2 9065-2) of the registered substance were dissolved in DMSO and tested at final concentrations of 0.98 to 2000 µM. Treatment of the cell cultures for 48 hours with both test item batches led to positive results: The mean maximal average fold induction of the luciferase activity (Imax) value was 2.86-fold/2.58 -fold and the mean EC1.5 value, representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50 % enhanced luciferase activity), was 34.30 µM/26.79 µM.

Since the Imax values in both experiments were above 1.5-fold, the test article was considered to be positive in the ARE-Nrf2 Luciferase Test (Spruth 2019 and 2019b).

Due to the inconsistency of the results in silico (no alert), in chemico (negative) and in vitro (positive) no final conclusion for skin sensitization could be drawn and a LLNA (local lymph node assay) was initiated.

The LLNA was performed according to OECD 429 using an alternative method, employing lymph node weight and lymph node cell count to assess sensitizing properties via proliferation of lymphocytes. In addition, a potential acute inflammatory skin reaction (skin irritation) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements. Stimulation indices were calculated for lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones.Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

The neat test item and two concentrations (75 % and 50 % dissolved in acetone/olive oil (4:1)) were tested in six female NMRI mice per group and compared to a vehicle control group. Stimulation indices of 1.575, 1.441 and 1.419 were determined for the concentrations of 100 %, 75 % and 50 %, respectively. All three concentrations thus led to SI values of > 1.4 and to an increase (significant at p ≤ 0.01) of the lymph node weight. indicating sensitizing properties.

Based on the chosen concentrations for testing, calculation of an exact EC1.4 (effective concentration reaching the positive level of SI 1.4) to assess potency of skin sensitization is not possible. Therefore, regression analysis was used to estimate the EC1.4. Linear and exponential regression analysis of the three data points was considered, both revealing EC1.4 values of about 50% (50.548% and 49.077%, respectively). Even if only the two lower data points are evaluated via linear regression (SI values of 1.419 and 1.441 for 50 and 75% concentration), the resulting EC1.4 is still above 27% (27.777%). The registered substance should thus be regarded as weak skin sensitizer, sub-categorized with Skin Sens 1B (EC1.4 value of >2%).  

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on inconsistency of the obtained results in silico (no alert), in chemico (negative) and in vitro (positive) no final conclusion on classification for skin sensitization could be drawn on the basis of alternative methods for the registered substance.

Thus, a modified version of the Local Lymph Node Assay (LLNA) following OECD 429 was initiated. In this modified LLNA stimulation indices (SI) were calculated for lymph node cell counts and lymph node weights to assess skin sensitizing properties. SI values of above 1.4 for lymph node cell counts are considered a positive response for skin sensitization in this modified LLNA. Based on this threshold level, the effective concentration value calculated for SI > 1.4 is called EC1.4.

In the modified LLNA the neat test item and two concentrations (75 % and 50 % dissolved in acetone/olive oil (4:1)) were tested and compared to a vehicle control group. Stimulation indices of 1.575, 1.441 and 1.419 were determined for the concentrations of 100 %, 75 % and 50 %, respectively. All three concentrations thus led to SI values of > 1.4 and to an increase (significant at p ≤ 0.01) of the lymph node weight, indicating sensitizing properties.

Based on the chosen concentrations for testing, calculation of an exact EC1.4 (effective concentration reaching the positive level of SI 1.4) to assess potency of skin sensitization is not possible. Therefore, regression analysis was used to estimate the EC1.4. Linear and exponential regression analysis of the three data points was considered, both revealing EC1.4 values of about 50% (50.548% and 49.077%, respectively). Even if only the two lower data points (SI values of 1.419 and 1.441 for 50 and 75% concentration) are evaluated via linear regression, the resulting EC1.4 is still above 27% (27.777%). The registered substance should thus be regarded as weak skin sensitizer, sub-categorized with Skin Sens 1B (EC1.4 value of >2%).