[No public or meaningful name is available]

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

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.

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of adipic acid and neodecanoic acid, 2-​oxiranylmethyl ester (C31H56O10)+(C32H58O10)+(C33H60O10)

Molecular formula:

C32H58O10

IUPAC Name:

Reaction product of adipic acid and neodecanoic acid, 2-​oxiranylmethyl ester (C31H56O10)+(C32H58O10)+(C33H60O10)

Constituent 2

Reference substance name:

not yet assigned

Molecular formula:

C50H88O16 C51H90O16 C52H92O16

IUPAC Name:

not yet assigned

Constituent 3

Reference substance name:

2,3-epoxypropyl neodecanoate

EC Number:

247-979-2

EC Name:

2,3-epoxypropyl neodecanoate

Cas Number:

26761-45-5

Molecular formula:

C13H24O3

IUPAC Name:

(oxiran-2-yl)methyl 2,2-dimethyloctanoate

Test material form:

liquid: viscous

Details on test material:

Former EC 615-318-0 / CAS: 716336-43-5: changed to EC 825-846-5 / CAS 876528-25-5

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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.

Results and discussion

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)

In vivo (LLNA)

Results

Results 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

Results 2

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

Results 3

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.