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Based on an alert obtained in silico (OECD Toolbox), a negative result in the DPRA (in chemico testing) and technical problems (solubility) in testing the substance in vitro (KeratinoSens and h-CLAT) in vivo testing was initiated to gain information on potential in vivo skin sensititization activities. Macrolex Rot 5B did not reveal any skin sensitising properties in the mouse local lymph node assay and can thus be regarded as non-sensitizer under the conditions of this test.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
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 +/-2.5ºC. 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 72.64% cysteine peptide and 65.70% lysine peptide. The mean cysteine/lysine peptide depletion was calculated with 69.17% 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; percent peptide depletion of the suspension
Parameter:
other: reactivity in the cysteine 1:10/lysine 1:50 prediction model
Value:
0.24
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable because of methodological limitations
Remarks:
DPRA prediction: negative; however, a negative prediction at a concentration < 100 mmol/L does not allow a firm conclusion on the lack of reactivity
Run / experiment:
other: mean of 2 runs; percent peptide depletion of the filtrate
Parameter:
other: reactivity in the cysteine 1:10/lysine 1:50 prediction model
Value:
0.57
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable because of methodological limitations
Remarks:
DPRA prediction: negative; however, a negative prediction at a concentration < 100 mmol/L does not allow a firm conclusion on the lack of reactivity
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 negative control: yes
- Acceptance criteria met for positive control: yes

Since the test item was poorly soluble in any solvent acceptable for the DPRA the required test concentration of 100 mmol/L could not be reached. The test was therefore performed with

a suspension at the concentration of 100 mmol/L in acetone and with the filtrate of this suspension (saturated solution). In both cases the concentration of solved test item was < 100

mmol/L. According to OECD Guideline a negative result obtained at a concentration of < 100 mmol/L does not allow a firm conclusion on the lack of reactivity.

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 was poorly soluble in any solvent acceptable for the DPRA the required test concentration of 100 mmol/L could not be reached. The test was therefore performed with a suspension at the concentration of 1 00 mmol/L in acetone and with the filtrate of this suspension (saturated solution). In both cases the concentration of solved test item was < 100 mmol/L. According to OECD Guideline a negative result obtained at a concentration of < 100 mmol/L does not allow a firm conclusion on the lack of reactivity.

The cysteine 1:1 0/lysine 1:50 prediction model was applied to the test item. Relevant depletion of cysteine and lysine peptides was not detectable in the DPRA (< 1 %).

Taking the test concentration of < 100 mmol/L into account the negative outcome of the DPRA for Macrolex Rot 5B is not reliable.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
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 10 weeks
- Weight at study initiation: 27 - 33 g
- Housing: The animals were kept singly in MAKROLON cages (type II). Animals were not group-housed to prevent contact of the application sites.
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C
- Humidity (%): 55% +/- 10%
- Photoperiod (hrs dark / hrs light): 12hrs dark / 12 hrs light
- IN-LIFE DATES: From: 22 May 2018 To: 21 June 2018
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Three concentrations of Macrolex Rot 5B (2.5%, 5% and 10% (w/w)), suspended in acetone/olive oil (4:1, v/v) were tested in six female NMRI mice per group and compared to a vehicle control group. A 10% (w/w) concentration was the highest feasible concentration of Macrolex Rot 5B in acetone/olive oil (4:1, v/v), see Table 1.
No. of animals per dose:
6
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: suspension in acetone/olive oil (A/O, 4:1, v/v), see Table 1
- Irritation: no effects
- Systemic toxicity: no effects
- Ear thickness measurements: no effects
- Erythema scores: 0
A preliminary experiment was carried out in 3 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 2.5%, 5% and 10% (w/w) suspended in acetone / olive oil (4:1, v/v) 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 test item suspension was 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 PBS /0.5% 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:
1) The analytical method was validated by LPT before in vivo testing. The following parameters were determined:
- Linearity, Accuracy, Precision, Sensitivity, Specificity and Stability (6 hours at room temperature)
2) 3 Samples of approximately 5 mL were taken during the in-life phase from the prepared formulations of 2.5%, 5% and 10% (w/w). The samples were stored at ≤ 20°C until analysis for concentration.

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 caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid.(see Table 2)
Key result
Parameter:
SI
Value:
0.954
Test group / Remarks:
2.5% test item in A/O: the stimulation index (SI) for lymph node cell count is well below the threshold level of 1.4
Remarks on result:
other: no indication of skin sensitization
Key result
Parameter:
SI
Value:
1.152
Test group / Remarks:
5% test item in A/O: the stimulation index (SI) for lymph node cell count is well below the threshold level of 1.4
Remarks on result:
other: no indication of skin sensitization
Key result
Parameter:
SI
Value:
1.239
Test group / Remarks:
10% test item in A/O: the stimulation index (SI) for lymph node cell count is well below the threshold level of 1.4
Remarks on result:
other: no indication of skin sensitization
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
In the main study treatment with Macrolex Rot 5B at concentrations of 2.5%, 5% or 10% (w/w) did not reveal any statistical significantly increased values for the lymph node cell count and lymph node weight. The stimulation index for the lymph node cell count did not exceed the threshold level of 1.4. (see Table 2)

The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted. (see Table 2)

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid. (see Tabel 2)

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)).

EC 1.4 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 2.5%, 5% and 10% (w/w) of Macrolex Rot 5B, employing 1 animal per concentration, were examined. 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. A 10% (w/w) concentration was the highest feasible concentration of Macrolex Rot 5B in acetone/olive oil (4:1, v/v).

Table 2: Stimulation indices (SI) in the main experiment (mean of 6 animals per group):

Parameter  negative control (A/O)  2.5% (w/w) test item in A/O  5% (w/w) test item in A/O  10% (w/w) test item in A/O  positive control (20% alpha-hexyl cinnamic aldehyde (v/v) in A/O)
 Lymph node cell count  1.000  0.954  1.152  1.239  2.174*
 Lymph node weight  1.000  1.073  1.146  1.268  1.976*
 Ear weight  1.000  1.035  1.092  1.085  1.324*
 Ear thickness  1.000  1.034  1.038  1.085  1.140

* significantly different from control at p ≤ 0.01

The analysis of the test item vehicle suspensions of the 2.5%, 5% and 10% (w/w) concentrations for the actual test item levels was carried out (non-GLP) under conditions employing a validated analytical method. The analysis resulted in actual levels of 103.4% (2.5% (w/w) concentration), 107.7 (5% (w/w) concentration) and 99.8% (10% w/w) concentration) of the nominal concentration

Interpretation of results:
GHS criteria not met
Executive summary:

The purpose of this study was to determine the skin sensitising potential of Macrolex Rot 5B 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 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)).

Thee concentrations of Macrolex Rot 5B (2.5%, 5% and 10% (w/w)), suspended in acetone/olive oil (4:1, v/v) were tested in six female NMRI mice per group and compared to a vehicle control group. Acetone/olive oil (A/O, 4:1, v/v) was selected as it provided a suitable suspension of the test item both for administration and adherence to the mouse ear. A 10% (w/w) concentration was the highest feasible concentration of Macrolex Rot 5B in A/O. Methyl ethyl ketone, N,N-dimethylformamide, propylene glycol and dimethyl sulfoxide, other recommended vehicles, did not provide higher concentrated suitable suspensions.

In the main study treatment with Macrolex Rot 5B at concentrations of 2.5%, 5% or 10% (w/w) did not reveal any statistical significantly increased values for the lymph node cell count and lymph node weight. The stimulation index for the lymph node cell count did not exceed the threshold level of 1.4. The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted. The positive control group caused the expected increases in lymph node cell count 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. The animal body weight was not affected by the treatment.

In conclusion, 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No human data are available for the substance to assess the endpoint skin sensitization. However, no skin irritating or skin sensitizing effects were reported in occupational setting.

An assessment of potential skin sensitization properties based on defined approaches and individual sources was conducted for Macrolex Rot 5B within an IATA. The outcome of this assessment is given below:

Macrolex Rot 5B is a solid with a MW of 366 g/mole, is not soluble in water (< 1 mg/L) and a log Pow of 4.8 was dermined for the substance. Based on these physico-chemical properties dermal uptake of the substance can be predicted to be low (ECHA Guidance on Information Requirements, Chapter R7c, 2017).

Macrolex Rot 5B was investigated in silico for structural activity (QSAR) and in chemico for peptide reactivity. In vitro testing of the test item in the KeratinoSens and the h-CLAT is not possible due to insolubility in all appropriate solvents. Thus, in vivo testing was performed in the LLNA as last option to assess the endpoint skin sensitization.

OECD Toolbox 4.0:

A direct acylation potential and a high keratinocyte gene expression is predicted for the test item in silico by the OECD QSAR Toolbox v.4.0 (Schlecker, 2017).

DPRA; OECD TG 442C:

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 was poorly soluble in any solvent acceptable for the DPRA the required test concentration of 100 mmol/L could not be reached. The test was therefore performed with a suspension at the concentration of 100 mmol/L in acetone and with the filtrate of this suspension (saturated solution). In both cases the concentration of solved test item was < 100 mmol/L. According to OECD Guideline a negative result obtained at a concentration of < 100 mmol/L does not allow a firm conclusion on the lack of reactivity.

The cysteine 1:1 0/lysine 1:50 prediction model was applied to the test item. Relevant depletion of cysteine and lysine peptides was not detectable in the DPRA (< 1 %).

Taking the test concentration of < 100 mmol/L into account the negative outcome of the DPRA for Macrolex Rot 5B does not allow a firm conclusion on the lack of reactivity.

KeratinoSens™; OECD TG 442D:

The objective of the study was 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. Solubility trials were performed with dimethyl sulphoxide (DMSO), dimethylformamide (DMF) and isopropanol. The test item is known to be insoluble in water/medium. Extensive vortex mixing, ultrasonication and warming at 40°C were used to try and achieve a solution.

The test article produced a dark purple suspension with particles at all concentrations in DMSO, a dark purple suspension at all concentrations in DMF and a dark purple / black suspension at all concentrations in isopropanol. The test article was insoluble in all suitable vehicles at sufficient concentrations to meet the OECD Test Guidelines for the ARE-Nrf2 Luciferase Test.

It was concluded that the test article was outside of the applicability domain for this study. The test could thus not be performed.

h-CLAT; OECD 442E:

As shown in the solubility trials for the KeratinoSens assay, the test item is insoluble in all suitable vehicles at sufficient concentrations to meet the OECD Test Guidelines for the human cell line activation test (h-CLAT).

In vivo testing in the LLNA:

Based on an alert obtained in silico (OECD Toolbox) and the technical problems (insufficient solubility/ insolubility) in testing the substance in chemico (DPRA) and in vitro (KeratinoSens and h-CLAT) in vivo testing was initiated to gain information on potential in vivo skin sensititization activities. No information on skin sensitization potency can be derived from skin sensitization hazard reported in the in vitro studies.

Macrolex Rot 5B was thus tested in the modified local lymph node assay in mice. The study was performed according to OECD 429. 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)).

Thee concentrations of Macrolex Rot 5B (2.5%, 5% and 10% (w/w)), suspended in acetone/olive oil (4:1, v/v) were tested in six female NMRI mice per group and compared to a vehicle control group. Acetone/olive oil (A/O, 4:1, v/v) was selected as it provided a suitable suspension of the test item both for administration and adherence to the mouse ear. A 10% (w/w) concentration was the highest feasible concentration of Macrolex Rot 5B in A/O. Methyl ethyl ketone, N,N-dimethylformamide, propylene glycol and dimethyl sulfoxide, other recommended vehicles, did not provide higher concentrated suitable suspensions.

In the main study treatment with Macrolex Rot 5B at concentrations of 2.5%, 5% or 10% (w/w) did not reveal any statistical significantly increased values for the lymph node cell count and lymph node weight. The stimulation index for the lymph node cell count did not exceed the threshold level of 1.4. The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted. The positive control group caused the expected increases in lymph node cell count 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. The animal body weight was not affected by the treatment.

In conclusion, 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.

Conclusion:

Based on an alert obtained in silico (OECD Toolbox), a negative result in the DPRA (in chemico testing) at test concentrations of < 100 mmol/L  and technical problems (insolubility) in testing the substance in in vitro (KeratinoSens and h-CLAT) in vivo testing was initiated to gain information on potential in vivo skin sensititization activities. Macrolex Rot 5B was thus tested in the modified local lymph node assay in mice. Solubility trials with all recommended vehicles for the LLNA were performed that revealed a 10% (w/w) concentration of the test item in acetone/olive oil (A/O, 4:1, v/v) as the maximum feasible concentration. The concentrations tested were chosen according to OECD Guideline 429 with 2.5%, 5% or 10% (w/w) in A/O. Macrolex Rot 5B did not reveal any skin sensitising properties in the mouse local lymph node assay and can thus be regarded as non-sensitizer under the conditions of this test. Additionally, based on the physico-chemical characteristics of the substance dermal absorption in vivo can be expected to be low and no skin sensitizing effects were reported in occupational settings.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on an alert for protein binding obtained in silico (OECD Toolbox), a negative result in the DPRA (in chemico testing) at test concentrations of < 100 mmol/L and technical problems (solubility) in testing the substance in vitro (KeratinoSens and h-CLAT) in vivo testing was initiated to gain information on potential in vivo skin sensitization activities. Macrolex Rot 5B was thus tested in the modified local lymph node assay in mice. Macrolex Rot 5B did not reveal any skin sensitising properties in the mouse local lymph node assay and can thus be regarded as non-sensitizer under the conditions of this test. Additionally, based on the physico-chemical characteristics of the substance dermal absorption in vivo can be expected to be low and no skin sensitizing effects were reported in occupational settings.

Consequently, no classification is warranted for skin sensitization.