Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol

Endpoint summary

Description of key information

- In vitro skin sensitisation (OECD 442D, GLP): inconclusive

- In vivo skin sensitisation (OECD 429, GLP): not sensitising

 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Sep - 08 Oct 2021

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 KeratinoSens™ test method)

Version / remarks:

adopted in 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item was dissolved in DMSO at 10 mg/mL
(clear colourless solution).
- Preparation of the test chemical serial dilutions: 11 spike solutions in DMSO were prepared from the stock solution (2-fold dilution series). The stock and spike solutions were diluted 25-fold with exp osure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 0.049, 0.098, 0.20, 0.39, 0.78, 1.6, 3.1, 6.3, 13, 25, 50, and 100 μg/mL (final concentration DMSO of 1%).
- Preparation of the positive controls: For the positive control ethylene dimethacrylte glycol (EDMG) a 2-fold dilution series was prepared in DMSO ranging from 0.78 to 25 mM. The final concentrations of EDMG in the culture medium was 7.8 to 250 μM (1% final DMSO concentration).
- Preparation of the solvent, vehicle and negative controls: The vehicle control was 1% DMSO in exposure medium.
- Stable dispersion obtained: All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates.

DOSE RANGE FINDING ASSAY:
- Highest concentration used: The highest test concentration was considered to be the limit of solubility.
- Solubility in solvents: The test item was suspended in DMSO to a final concentration of 40 mg/mL (homogeneous translucent suspension). The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. The 100-fold dilution in DMEM glutamax of 40 and 20 mg/mL showed moderate precipitate and were therefore not suitable to test. The 100-fold dilution of the 10 mg/mL DMSO stock showed slight precipitation. This concentration was selected as highest concentration for the main assay (limit of solubility). At concentration of 40 mg/mL the test item formed a suspension in dimethyl sulfoxide whereas at 20 mg/mL and lower it was fully soluble.
- Solubility in incubation medium: not tested
- Cytotoxicity assessment performed: For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh DMEM Glutamax containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 h at 37 ± 1.0 °C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
- Final concentration range selected on basis of: solubility test (highest dose based on limit of solubility).

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES:
- Number of replicates: 3 (vehicle control: 18 wells/plate)
- Number of repetitions: 2 experiments were performed
- Test chemical concentrations: 0.049, 0.098, 0.20, 0.39, 0.78, 1.6, 3.1, 6.3, 13, 25, 50, and 100 μg/mL
- Application procedure: The medium was removed and replaced with fresh exposure medium
containing the test item, vehicle or positive control. Three wells per plate were left empty to assess background values. The plates were covered with foil and incubated at 37.0 ± 1.0 °C in the presence of 5% CO2.
- Exposure time: 48 h
- Study evaluation and decision criteria used:
In compliance with the OECD Guideline TG 442D. In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method. (Adopted June, 2018), a KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is equal or higher than (≥) 1.5-fold and statistically significantly different as compared to the vehicle (negative) control (as determined by a two-tailed, unpaired Student’s t-test).
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity ≥ 1.5-fold (i.e. at the EC1.5 determining concentration).
3. The EC1.5 value is less than (<) 1000 μM (or < 200 μg/mL for test chemicals with no defined MW).
4. There is an apparent overall dose-response for luciferase induction.
Negative results obtained with concentrations < 1000 μM or 200 μg/mL and which do not
reach cytotoxicity (< 70% viability) at the maximal tested concentration should be considered
as inconclusive.
- Description on study acceptance criteria:
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, ethylene dimethacrylate glycol, should be statistically significant equal or above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 μM).
b) The EC1.5 of the positive control should be within two standard deviations of the historical mean. Moreover, the induction for ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose response of ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded. If the variability is higher, a maximum of three of the eighteen wells may be excluded based on the Dixon’s Q-test. If the variability is still higher, the results should be discarded.

SEEDING AND INCUBATION:
One day prior to testing cells were harvested, and seeded at a density of 10000 cells / well in basic medium in conventional 96-well plates. One plate was used for the luciferase activity measurements, and one parallel replicate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator.

MAINTAINANCE: Cells were subcultured upon reaching 80 - 90% confluency.

LUCIFERASE ASSAY:
- Assay: Steady-Glo Luciferase Assay (Promega, Leiden, The Netherlands)
- Incubation time: Prior to measurement, the plates were shaken on a plate shaker for at least 5 min at room temperature.
- Luminometer: TECAN Infinite® M200 Pro Plate Reader

MTT VIABILITY ASSAY:
- Incubation time: Cells were exposed to MTT for 3 – 4 h at 37.0 ± 1.0 °C in the presence of 5% CO2. Thereafter, the MTT medium was removed and the cells were lysed by adding 10% SDS solution.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored in the ultra-low freezer set to maintain -150°C as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium (Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1 % (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum and geneticin (500 μg/mL)). Cells were subcultured upon reaching 80 - 90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25). For testing, cells were 80 - 90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium (Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands)). One plate was used for the luciferase activity measurements, and one parallel replicate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+11 in experiment 1 and P+12 in experiment 2.
- Incubation conditions: All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 42 – 94%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.7 – 40.2 °C).
- Washing conditions: not washed after incubation
- Precipitation noted: no

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: TECAN Infinite® M200 Pro Plate Reader
- Plate used: One plate per repetition
- Lysate preparation: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium was removed. Then 100 µL of the SteadyGlo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 min at room temperature. Plates with the cell lysates were placed in the plate reader to assess the quantity of luciferase (integration time two seconds).

DATA EVALUATION
- Cytotoxicity assessment: For the KeratinoSens™ cell viability assay, medium was replaced after the 48 h exposure time with fresh DMEM Glutamax containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 h at 37 °C ± 1.0 °C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
- Prediction model used:
The following parameters are calculated in the KeratinoSens™ test method:
- The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
- The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5-fold threshold (i.e. 50% enhanced luciferase activity) was obtained
- The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.
Equation 1: Fold induction = (Lsample − Lblank) / (Lvehicle − Lblank)
Where:
Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and no treatment
Lvehicle is the average luminescence reading in the wells containing cells and vehicle (negative) control

The EC1.5 is calculated by linear interpolation according to Equation 2, and the overall EC1.5 is calculated as the mean of the individual repetitions.
Equation 2: EC1.5 = (Cb − Ca) × ((1.5 - Ia) / (Ib - Ia)) + Ca
Where:
Ca is the lowest concentration in μM (or µg/mL) with > 1.5-fold induction
Cb is the highest concentration in μM (or µg/mL) with < 1.5-fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5-fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5-fold induction (mean of three replicate wells)

Viability is calculated by Equation 3:
Equation 3: Viability = (Vsample − Vblank) / (Vvehicle − Vblank) × 100
Where:
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vvehicle is the average MTT-absorbance reading in the wells containing cells and vehicle (negative) control

Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.
Equation 4: ICx = (Cb – Ca) x (((100 - x) - Va) / (Vb − Va)) + Ca
x is the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca is the lowest concentration in μM (or µg/mL) with > x% reduction in viability
Cb is the highest concentration in μM (or µg/mL) with < x% reduction in viability
Va is the % viability at the lowest concentration with > x% reduction in viability
Vb is the % viability at the highest concentration with < x% reduction in viability

In case the luciferase activity induction is equal or higher than 1.5-fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the vehicle (negative) control wells to determine whether the luciferase activity induction is statistically significant (p < 0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

Vehicle / solvent control:

DMSO

Negative control:

other: blank wells (no cells and no treatment)

Positive control:

other: ethylene dimethacrylate glycol (EDMG)

Positive control results:

The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be statistically significant equal or above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 μM).
b) The EC1.5 of the positive control should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.05

Cell viability:

The test item showed no toxicity. The viability of the cells was higher than 70% at all test
concentrations and therefore no IC30 and IC50 values could be calculated.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

EC1.5 could not be calculated

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.36

Cell viability:

The test item showed no toxicity. The viability of the cells was higher than 70% at all test
concentrations and therefore no IC30 and IC50 values could be calculated.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

EC1.5 could not be calculated

Outcome of the prediction model:

data inconclusive

Other effects / acceptance of results:

OTHER EFFECTS:
- Precipitation: There was no precipitation observed up to the highest concentration tested.
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: No demonstration of proficiency shown in the study report. However, the acceptance criteria are met (see below), indicating that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes. The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration. In addition, the EC1.5 was within two standard deviations of the historical mean (122 and 79 μM in experiment 1 and 2, respectively). A dose-response was observed and the induction at 250 μM was higher than 2-fold in experiment 2 (1.87- and 2.25-fold in experiment 1 and 2, respectively) (refer to table 2 and 3 in 'Any other information on results incl. tables').
- Acceptance criteria met for variability between replicate measurements: The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (9.8 and 20% in experiment 1 and 2, respectively).

Table 1: Overview Luminescence Induction and Cell Viability in Experiment 1 and 2

Concentration (μg/mL)	0.049	0.098	0.20	0.39	0.78	1.6	3.1	6.3	13	25	50	100
Exp 1 luminescence	0.91	1.05	0.99	0.97	0.98	0.99	1.03	1.02	0.92	0.94	0.92	0.92
Exp 1 viability (%)	110	115	111	107	106	110	109	106	105	107	106	108
Exp 2 luminescence	1.36	1.06	1.10	1.17	1.17	1.17	1.21	1.10	1.17	1.15	1.07	0.96
Exp 2 viability (%)	76	78	79	77	77	74	74	76	76	77	76	81

 

Table 2: Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (μM)	7.8	16	31	63	125	250
Exp 1 luminescence	0.93	0.97	1.05	1.23	1.51***	1.87***
Exp 1 viability (%)	111	110	101	104	101	97
Exp 2 luminescence	0.87	1.03	1.11	1.43	1.70***	2.25***
Exp 2 viability (%)	77	76	76	76	76	76
*** p < 0.001 Student’s t test

 

Table 3: Overview EC_1.5, I_max, IC₃₀ and IC₅₀ Values

	EC1.5 (μg/mL)	Imax	IC30 (μg/mL)	IC50 (μg/mL)
Test item Exp 1	NA	1.05	NA	NA
Test item Exp 2	NA	1.36	NA	NA
	EC1.5 (μM)	Imax	IC30 (μM)	IC50 (μM)
Positive control Exp 1	122	1.87	NA	NA
Positive control Exp 2	79	2.25	NA	NA
NA = Not applicable

 

Table 4: Historical Control Data for the KeratinoSens^TM Studies

	Positive control
	EC1.5 (μM)	Imax
Range (mean ± 2x SD)	-8.4 - 115	-5.67 – 12.54
Mean	53.5	3.43
SD	31.0	4.55
n	531	531
SD = Standard deviation n = Number of observations The above-mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2018 to May 2021.

Interpretation of results:

other: Inconclusive based on the key event 'keratinocyte activation'

Conclusions:

There is regulatory acceptance in the EU for the application of the KeratinoSens™ test method to address key event 2, keratinocyte activation, in the skin sensitisation Adverse Outcome Pathway.
Under the conditions of the test, Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol did not show a keratinocyte activating potential (no biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes). However, the negative results were obtained with concentrations < 200 μg/mL and the maximal tested concentration did not reach cytotoxicity (< 70% viability), therefore, the results are considered as inconclusive.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Apr - 04 May 2022

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:

Adopted in 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 weeks
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 23.5 - 26.7 g
- Housing: On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany); ad libitum (except during designated procedures)
- Water: tap water; ad libitum
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
- Indication of any skin lesions: Not reported. However, before the initiation of dosing, a health inspection was performed, and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target: 20 - 24; Actual: 22 - 23
- Humidity (%): Target: 40 - 70; Actual: 42 - 47
- Air changes (per hr): ≥ 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES:
Initiation of dosing to completion of In-Life: 13 Apr - 02 May 2022

Vehicle:

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

Concentration:

Pre-screen test: 25%, 50% (maximum concentration as required in the test guidelines)
Main study: 10%, 25%, 50%

No. of animals per dose:

Pre-screen test: 4 animals (2 per dose group)
Main study: 20 animals (5 per dose group and negative solvent control)

Details on study design:

PRE-SCREEN TESTS
- For the pre-screen test, acetone/olive oil (4 : 1 v / v) solutions with the test substance at 25% and 50% were applied to both ears of the mice at 25 μL / ear for 3 consecutive days (2 mice / concentration). The day that application was started was calculated as day 1 of the study. Erythema and eschar formation observations were performed once daily on days 1 - 6 (on days 1 - 3 within 1 h after dosing)
- Compound solubility: not reported, but vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor.
- Irritation: At a 25% and 50% test material concentration, no irritation was observed (Erythema score 0 on Day 1 - Day 6 for both concentrations).
- Systemic toxicity: At a 25% and 50% test material concentration, no signs of systemic toxicity were noted.
- Ear thickness measurement: The increase in ear thickness was < 25% (threshold for selection for use in the main study) for all time points and concentrations.
Refer to the respective results tables under 'Any other information on results incl. tables'.
- The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
- Based on the results of the pre-screen test a 50% concentration was selected as highest concentration for the main study

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION
For the main study, concentrations of the test substance were set to on the basis of the results of the preliminary study (10%, 25% and 50% acetone/olive oil (4 : 1, v / v) solutions) and were applied open to both ears of the mice at 25 μL / ear for 3 consecutive days (5 mice / concentration) for induction. In addition, there was a group to which acetone/olive oil (4 : 1 v / v) only was applied, as a control group. The day that application was started was calculated as day 1 of the study, and the presence or absence of irritation (erythema) was confirmed once daily. On day 6 of the study, 3H-methyl thymidine prepared to a concentration of 20 µCi by using PBS was administered intravenously at 0.25 mL / animal, and 5 hours after administration auricular lymph nodes were recovered from the animals and the relative size of the nodes was estimated by visual examination. Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS. LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 min at 4 ºC. To precipitate the DNA, the LNC were exposed to 5 % trichloroacetic acid (TCA) and then stored in the refrigerator until the next day. On day 7, precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid. Radioactivity (DPM) was measured using a Packard scintillation counter (2910TR).
- Post-dose observations were performed once daily on days 1 - 6 (on days 1 - 3 at least 3 h after dosing). All the animals were examined for reaction to dosing. The onset, intensity and duration of
these signs was recorded (if appropriate), particular attention being paid to the animals during
and for the first hour after dosing.
- Animals were weighed individually on Day 1 (pre-dose) and 6 (prior to necropsy).
- Erythema and eschar formation observations were performed once daily on Days 1 - 6 (on
Days 1 - 3 within 1 h after dosing)

EVALUATION CRITERIA:
- Criteria used to consider a positive response: Disintegrations per minute (DPM) values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test material may be regarded as a skin sensitizer.
- According to CLP/ GHS, if the results indicate a SI ≥ 3, the test material may be regarded as a skin sensitizer (Category 1). Additionally, if it is considered a skin sensitizer (Category 1), if the EC3 value is ≤ 2% it is in sub-category 1A and if the EC3 value is > 2% it is in sub-category 1B.

Positive control substance(s):

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

Statistics:

DPM values were measured for each animal and for each dose group. A mean SI and Standard Error of the Mean (SEM) was calculated for each group using the individual SI values.

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques. In the last study, performed in November 2021, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Alpha-Hexylcinnamaldehyde, technical grade (HCA) at concentrations of 5, 10 and 25% in Acetone/Olive oil (4:1 v/v). The SI values calculated for the material concentrations 5, 10 and 25% were 1.5, 2.8 and 5.7, respectively. An EC3 value of 12.2% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 12.8, 9.0, 10.9, 8.0 and 13.5. Based on the results, it was concluded that the LLNA as performed is an appropriate model for testing for contact hypersensitivity.

Key result

Parameter:

EC3

Value:

> 50

Key result

Parameter:

SI

Value:

1.9

Test group / Remarks:

50%

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

10%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The DPM values of the 10%, 25% and 50% treatment groups were 642, 341, and 860, respectively (refer to table 4 in 'Any other information on results incl. tables').

DETAILS ON STIMULATION INDEX CALCULATION:
The SI of the 10%, 25% and 50% treatment groups were 1.4, 0.8, and 1.9, respectively (refer to table 4 in 'Any other information on results incl. tables').

EC3 CALCULATION:
The EC3 was determined to be greater than 50%.

CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.

BODY WEIGHTS:
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss noted in one animal dosed at 10% was considered not related to treatment with the test item since the changes were slight in nature and no dose-related incidence was apparent.

SIGNS OF TOXICITY:
No skin irritation was observed in any of the animals (refer to table 1 and 3 in 'Any other information on results incl. tables').

LYMPH NODE WEIGHTS:
The majority of auricular lymph nodes were considered normal in size, except the nodes in
four animals treated at 50% which were slightly enlarged (refer to table 4 in 'Any other information on results incl. tables'). No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Table 1: Pre-Screen Test: Body Weights and Skin Reactions

TI (%)	Animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
		bw (g)	Erythema^1		Erythema		Erythema		Erythema		Erythema		Erythema		bw (g)
			L	R	L	R	L	R	L	R	L	R	L	R
25	81	22.8	0	0	0	0	0	0	0	0	0	0	0	0	22.7
	82	21.4	0	0	0	0	0	0	0	0	0	0	0	0	22.4
50	83	23.3	0	0	0	0	0	0	0	0	0	0	0	0	24.9
	84	23.4	0	0	0	0	0	0	0	0	0	0	0	0	25.2
L = Left (dorsal surface of left ear) R = Right (dorsal surface of right ear) TI = test item (% w / w) bw = body weight (grams) ^1 Grading erythema and eschar formation: 0 = No erythema

 

Table 2: Pre-Screen Test: Ear Thickness Measurements

TI (%)	Animal	Day 1		Day 3				Day 6
		Left (mm)	Right (mm)	Left (mm)	%^1	Right (mm)	%	Left (mm)	%	Right (mm)	%
25	81	0.205	0.210	0.225	10	0.220	5	0.215	5	0.220	5
	82	0.200	0.220	0.215	8	0.220	0	0.210	5	0.210	-5
50	83	0.230	0.225	0.260	13	0.255	13	0.240	4	0.240	7
	84	0.225	0.225	0.245	9	0.240	7	0.245	9	0.230	2
Left (mm) = thickness of left ear in millimeters Right (mm) = thickness of right ear in millimeters TI = test item (% w / w) ^1 Percent increase compared to Day 1 pre-dose value. A 25% value is used as the threshold for selection for use in the main study.

 

Table 3: Main Study: Body Weights and Skin Reactions

Group	TI (%)	Animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
			bw (g)	Erythema^1		Erythema		Erythema		Erythema		Erythema		Erythema		bw (g)
				L	R	L	R	L	R	L	R	L	R	L	R
1	0	1	24.2	0	0	0	0	0	0	0	0	0	0	0	0	27.2
		2	26.1	0	0	0	0	0	0	0	0	0	0	0	0	26.2
		3	23.5	0	0	0	0	0	0	0	0	0	0	0	0	27.4
		4	26.7	0	0	0	0	0	0	0	0	0	0	0	0	29.3
		5	23.8	0	0	0	0	0	0	0	0	0	0	0	0	26.3
2	10	6	25.6	0	0	0	0	0	0	0	0	0	0	0	0	26.5
		7	26.5	0	0	0	0	0	0	0	0	0	0	0	0	24.8
		8	25.0	0	0	0	0	0	0	0	0	0	0	0	0	26.1
		9	23.6	0	0	0	0	0	0	0	0	0	0	0	0	24.3
		10	25.5	0	0	0	0	0	0	0	0	0	0	0	0	26.4
3	25	11	26.1	0	0	0	0	0	0	0	0	0	0	0	0	28.1
		12	25.2	0	0	0	0	0	0	0	0	0	0	0	0	26.2
		13	26.2	0	0	0	0	0	0	0	0	0	0	0	0	27.5
		14	24.4	0	0	0	0	0	0	0	0	0	0	0	0	26.5
		15	24.4	0	0	0	0	0	0	0	0	0	0	0	0	25.4
4	50	16	24.2	0	0	0	0	0	0	0	0	0	0	0	0	25.3
		17	24.6	0	0	0	0	0	0	0	0	0	0	0	0	25.0
		18	25.4	0	0	0	0	0	0	0	0	0	0	0	0	25.7
		19	25.4	0	0	0	0	0	0	0	0	0	0	0	0	26.8
		20	24.6	0	0	0	0	0	0	0	0	0	0	0	0	25.9
L = Left (dorsal surface of left ear) R = Right (dorsal surface of right ear) TI = test item (% w / w) bw = body weight (grams) ^1 Grading erythema and eschar formation: 0 = No erythema

 

Table 4: Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

Group	TI (%)	Animal	Size Nodes^1		DPM / Animal	Mean DPM	± SEM	Mean SI	± SEM
			Left	Right
1	0	1	n	n	382	449	71	1.0	0.2
		2	n	n	247
		3	n	n	482
		4	n	n	323
		5	n	n	711
2	10	6	n	n	741	642	92	1.4	0.2
		7	n	n	650
		8	n	n	287
		9	n	n	725
		10	n	n	806
3	25	11	n	n	420	341	94	0.8	0.2
		12	n	n	125
		13	n	n	169
		14	n	n	645
		15	n	n	347
4	50	16	n	n	491	860	151	1.9	0.3
		17	n	+	1379
		18	+	+	939
		19	+	+	847
		20	+	+	645
TI = test item (% w / w) DPM = Disintegrations per minute SEM = Standard Error of the Mean ^1 Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under the present test conditions, Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol was not a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In vitro skin sensitisation

The potential of Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol to induce skin sensitisation was examined in vitro according to OECD 442D (ARE-Nrf2 luciferase KeratinoSens™ test method) and GLP principles (Charles River, 2021b). The objective was to evaluate the ability of the registration substance to activate the antioxidant / electrophile responsive element (ARE)-dependent pathway in the KeratinoSens™ assay. The test item was dissolved in dimethyl sulfoxide (DMSO) at 10 mg/mL. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 25-fold in the assay resulting in test concentrations of 0.049 – 100 µg/mL. The highest test concentration was considered to be the limit of solubility. No precipitate was observed at any dose level tested. Two independent experiments were performed. In experiment 1 and 2, the test item showed no toxicity (no IC30 and IC50 value) and no induction of the luciferase activity (no EC1.5 value). The maximum luciferase activity induction (Imax) in experiment 1 and 2 was 1.36-fold, and therefore, a negative result (< 1.5-fold induction) was obtained. All acceptance criteria were met (i.e. luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5-fold in at least one concentration; EC1.5 of the positive control was within two standard deviations of the historical mean (122 µM and 79 µM in experiment 1 and 2, respectively), meaning a dose-response was observed and the induction at 250 µM was higher than 2-fold (1.87-fold and 2.25-fold in experiment 1 and 2, respectively); and the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (9.8% and exactly 20% in experiment 1 and 2, respectively)). It is therefore concluded that the test conditions were adequate and that the test system functioned properly. Overall, the result of the KeratinoSens™ assay was inconclusive since negative results (< 1.5-fold induction) were observed in both experiments, but the test concentrations were < 200 µg/mL with cell viability > 70% at the maximal test concentration compared to the vehicle control.

 

Assessment of skin sensitization potential according to OECD 497

The initial objective of performing the KeratinoSens^TM assay was to reach an overall conclusion on the endpoint skin sensitization using the "2 out of 3" (2o3) defined approach described in OECD TG 497. Since the registered substance is a UVCB substance, the DEREK in silico assessment and DPRA in chemico assay (OECD TG 442C) could not be performed.

For the available KeratinoSens^TM assay no limitations as mentioned in the respective test guideline (OECD TG 442D) were identified for the registered substance. However, according to prediction models for the individual information sources used in the 2o3 approach, the result of the KeratinoSens^TM was inconclusive. As a result, the h-CLAT test (OECD TG 442E) was not initiated since at least two concordant results are required to draw an overall conclusion. In this case, regardless of the outcome of the h-CLAT, it is not possible to have more than one valid test result.

In conclusion, it will not be possible to address the skin sensitization endpoint for the registered substance conclusively based on non-animal methods. Therefore, it is considered justified to perform an in vivo study to determine the skin sensitizing properties of esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol. A detailed evaluation of the available data, the in vitro / in chemico assays, and the options to assess the skin sensitization endpoint for esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol is provided in the endpoint study record of the LLNA.

 

In vivo skin sensitisation

The potential of Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol to induce skin sensitisation was examined in vivo according to OECD 429 (mouse local lymph node assay (LLNA)) and GLP principles (Charles River, 2022d). The objective was to evaluate the ability of the registration substance to induce skin sensitisation in mice after three epidermal exposures. Test material concentrations selected for the main study were based on the results of a prescreen test. At a 25% and 50% test material concentration, no signs of systemic toxicity were noted, and no irritation was observed. Therefore, a 50% concentration was selected as highest concentration for the main study. In the main study, three experimental groups of five female CBA/J mice were treated with test material concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v) (AcOO)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) for each animal. In addition, the mean DPM/animal and mean stimulation index (SI) were subsequently calculated for each group. Mean DPM values for the experimental groups treated with test material concentrations 10, 25 and 50% were 642, 341 and 860 DPM, respectively. The mean DPM/animal value for the vehicle control group was 449 DPM. The SI values calculated for the test material concentrations 10, 25 and 50% were 1.4, 0.8 and 1.9, respectively. Since there was no indication that the test material elicits a SI ≥ 3 when tested up to 50%, it was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test material concentration that will give a SI = 3) (if any) exceeds 50%. 

 

Conclusion:
Based on the results of the in vitro and in vivo skin sensitisation studies, the registration substance is not regarded as a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available in vivo and in vitro data on skin sensitisation conducted with the registration substance do not meet the classification criteria according to the CLP Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.