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Description of key information

In a modified OECD 429 GLP study under the presented test conditions the test item isooctadecanoic acid, potassium salt at concentrations of 1%, 5% or 10% (w/w) in coconut oil did not reveal any skin sensitising properties in the local lymph node assay. The test item is classified as not sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A modified OECD 429 LLNA study was conducted as a last resort according to Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017 section 7.3.7.2 Application of the Testing and Assessment Strategy and in accordance with Commission Regulation (EU) 2016/1688 (replacement of Point 8.3 of Annex VII of Regulation (EC) No 1907/2006).

The rationale behind conducting the in vivo test as a last resort can be summarized as follows based on the scheme provided in figure 7.3-2 of Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017:

1. Existing data on physico-chemical properties - the test item is neither a strong acid (pH≤ 2.0) nor base (pH≥ 11.5), corrosive to the skin or (spontaneously) flammable in air or in contact with water or moisture at room temperature

2. No adequate existing human data, which provide evidence that the test item is a skin sensitiser, is available to the registrant.

3. There were no data from existing studies on skin sensitisation in laboratory animals (LLNA, GPMT, or Buehler test, EU B.42, B.50, B.51 and B.6/OECD TGs 429, 442A, 442B and 406) available, which provide sound conclusive evidence that the substance is a sensitiser or non-sensitiser.

4. No “read-across” from structurally and mechanistically related substances was possible (no data available) and a (Q)SAR prediction (VEGA QSAR on Skin Sensitization model (CAESAR) 2.1.6 done for isostearic acid) indicated a skin sensitisation potential which was considered not sufficient for classification and labelling (based on the training set of substances used by the QSAR software - see atached VEGA ouptput file).

5.a/b/c

No reliable evidence/hypothesis of dermal bioavailability based on physico-chemical, in silico, in vitro or in vivo data for the test item is available.

No data on peptide/protein binding properties of the test item in an EU/OECD adopted in chemico test (e.g. OECD TG 442c, key event 1 of the AOP) are available.

No data on activation of the Nrf2-Keap1-ARE toxicity pathway of the test item in an EU/OECD adopted in vitro test (e.g. OECD TG 442d, key event 2 of the AOP) are availavle.

No data on induction of the cell surface markers (CD54 and/or CD86) on monocytic cells of the test item in a validated in vitro test (e.g. h-CLAT, key event 3 of the AOP) are available.

No data of non-validated in vitro test(s) conducted withthe test item, which provide evidence that the substance may be a skin sensitiser are available.

6. Taking all available existing and relevant data (elements 1-5) into account, there is not sufficient information available for classification and labelling.

Generation of new non-animal data

7a. Key event 1 of the AOP - OECD TG 442c:
The test item is considered a complex mixtures of unknown composition, substances of unknown or variable composition, complex reaction products or biological materials (i.e. UVCB substance). Hence the OECD TG 442c test method is considered not applicable (see limitations of OECD TG 442c method).

7b. Key event 2 of the AOP - OECD 442d (activation of the Nrf2-Keap1-ARE toxicity pathway):
According to OECD TG 442d mainly mono-constituent substances were tested according to the guidance and only a limited amount of data also exists on the testing of mixtures. The test method is nevertheless technically applicable to the testing of multiconstituent substances and mixtures. However, before use of this Test Guideline on a mixture for generating data for an intended regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose. In this regard the test item could be used in the OECD TG 442d test.
Nevertheless taking into account the limitations with regard to the test item of OECD TG 442c and OECD TG 442e described, the possbile results on activation of the Nrf2-Keap1-ARE toxicity pathway are of limited use regarding classification and labelling. The OECD 442d test is not considered a stand-alone method (Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017, section 7.3.4.1)

7c. Key event 3 of the AOP - OECD 442e (induction of the cell surface markers (CD54 and/or CD86)):
The test item has a logKow > 3.5. Lipophilic substances tend to produce a higher rate of false negative results. Therefore the OECD TG 442c test method is considered not applicable (see limitations of OECD TG 442e).

7d. Additional testing/generation of data is considered necessary in order to conclude on classification, or e.g. to explain the inconsistent data obtained by VEGA QSAR analysis (positive indication for skin sensitization - see attachement).

8. Based on the available information classification as Skin Sensitiser Cat. 1A or no classification is considered not reliable and not sufficient to meet the respective information requirement of Section 8.3 of Annex VII.

9. A modified OECD 429 LLNA study was conducted as a last resort according to Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017 section 7.3.7.2 Application of the Testing and Assessment Strategy and in accordance with Commission Regulation (EU) 2016/1688 (replacement of Point 8.3 of Annex VII of Regulation (EC) No 1907/2006).
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
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.
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
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.
Deviations:
no
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.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Product description: Potassium isooctadecanoate/potassium isostearate
Name: Isooctadecanoic acid, potassium salt
CAS No.: 66469-15-6
Physical state: off white to yellowish solid at 20 °C
Batch No.: PFS-755-169
Re-certification date of batch: 12 December 2017
Purity: 100 % (UVCB)
pH, 10% in DI water 8.0 - 9.0
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light
Details on the study design:
The alternative method used for the LLNA study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European interlaboratory 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. The local lymph node assay (LLNA) and modifications thereof were recently recognized by the OECD as stand-alone methods for the detection of skin-sensitising potential. However, although the validity of the LLNA was acknowledged by the ICCVAM, attention was drawn to one major problem, i.e., the possibility of false positive results caused by non-specific cell activation as a result of inflammatory processes in the skin (irritation). This is based on the fact that inflammatory processes in the skin may lead to non-specific activation of dendritic cells, cell migration and non-specific proliferation of lymph node cells. Measuring cell proliferation by radioactive or non-radioactive methods, without taking the irritating properties of test items into account, leads thus to false positive reactions. 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 of the test item treated animals by the vehicle treated ones. The cut-off threshold value for ear weight was set at 1.1.
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
Animals / Animal maintenance

Species: Mice (non-pregnant, nulliparous, healthy)
Strain / Stock: NMRI / Crl:NMRI
Breeder: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Selection of species: The mouse is a rodent commonly used for such a study. The NMRI strain was chosen as statet stated in the literature for modifications.
Sex: Female
Number of animals: 36 (6 groups of 6 animals each)
Body weight (on test day 1): 27 - 32 g
Age (on test day 1): Approx. 9 weeks
Identification of animals: By cage label; ear tags were not used for identification of animals
Adaptation period: At least 5 days

Diet

Commercial diet ssniff® R/M-H V1534 served as food (ssniff Spezialdiäten GmbH, 59494 Soest, Germany).
This food was offered ad libitum. Food residue was removed.

Housing

The animals were kept singly in MAKROLON cages (type II) with a basal surface of approx. 360 cm2 and a height of approx. 14 cm at a room temperature of 22°C +- 3°C (maximum range) and a relative humidity of 55% +- 10% (maximum range). 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.
The rooms were alternately lit (150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each.
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 3: Limitation for contaminants in the bedding material).

Drinking water

Tap water was offered ad libitum.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
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. Three concentrations of 1%, 5% and 10% suspended or dissolved in coconut oil were examined.
Isooctadecanoic acid, potassium salt (1:1) was an off white to yellowish solid. Hence, a 10% suspension was the highest feasible concentration of Isooctadecanoic acid, potassium salt in coconut oil. 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.

As a result of the preliminary experiment three concentrations of Isooctadecanoic acid, potassium salt (1%, 5% and 10%), suspended or dissolved in coconut oil (w/w), were tested in six female NMRI mice per group and compared to a vehicle control group. A 10% concentration (w/w) of the test item in coconut oil was the maximum feasible concentration.
No. of animals per dose:
6
Details on study design:
The vehicle coconut oil was used as negative reference item. The positive control was dissolved in acetone / olive oil (4:1, v/v).
The test item suspension was administered to the dorsum of both animal's ears at an application volume of 25 µL/ear.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight was examined by linear regression analysis employing PEARSON's correlation coefficient. An U-test was performed also for cell count. Outliers were determined according to the Nalimov test.
Positive control results:
Positive control

Designation: alpha-hexyl cinnamic aldehyde (HCA)
Vehicle: Acetone / olive oil (4:1, v/v)
Route of administration: Open application to the dorsum of both ears
Administration volume: 25 µL/ear
Concentration: 20% (v/v)

Positive controls were used to demonstrate appropriate performance of the assay and competence of the laboratory to successfully conduct the assay. An index for the lymph node cell count above 1.4 is considered positive.
Key result
Parameter:
SI
Value:
1.166
Test group / Remarks:
Group 4, 10 %
Remarks on result:
other: Lymph node cell count
Key result
Parameter:
SI
Value:
1.333
Test group / Remarks:
Group 4, 10 %
Remarks on result:
other: Lymph node weight
Key result
Parameter:
SI
Value:
0.929
Test group / Remarks:
Group 4, 10 %
Remarks on result:
other: Ear weight
Key result
Parameter:
SI
Value:
1.055
Test group / Remarks:
Group 4, 10 %
Remarks on result:
other: Ear thickness, TD 4
Key result
Parameter:
SI
Value:
1.148
Test group / Remarks:
Group 3, 5 %
Remarks on result:
other: Lymph node cell count
Key result
Parameter:
SI
Value:
1.271
Test group / Remarks:
Group 3, 5 %
Remarks on result:
other: Lymph node weight
Key result
Parameter:
SI
Value:
0.918
Test group / Remarks:
Group 3, 5 %
Remarks on result:
other: Ear weight
Key result
Parameter:
SI
Value:
1.008
Test group / Remarks:
Group 3, 5 %
Remarks on result:
other: Ear thickness, TD 4
Key result
Parameter:
SI
Value:
0.995
Test group / Remarks:
Group 2, 1 %
Remarks on result:
other: Lymph node cell count
Key result
Parameter:
SI
Value:
1.188
Test group / Remarks:
Group 2, 1%
Remarks on result:
other: Lymph node weight
Key result
Parameter:
SI
Value:
0.978
Test group / Remarks:
Group 2, 1%
Remarks on result:
other: Ear weight
Key result
Parameter:
SI
Value:
1.033
Test group / Remarks:
Group 2, 1%
Remarks on result:
other: Ear thickness, TD 4
Parameter:
SI
Value:
1.595
Test group / Remarks:
positive control - significantly different from control at p ≤ 0.01
Remarks on result:
other: Lymph node cell count
Parameter:
SI
Value:
1.426
Test group / Remarks:
positive control - significantly different from control at p ≤ 0.01
Remarks on result:
other: Lymph node weight
Parameter:
SI
Value:
1.076
Test group / Remarks:
positive control - significantly different from control at p ≤ 0.01
Remarks on result:
other: Ear weight
Parameter:
SI
Value:
1.074
Test group / Remarks:
positive control
Remarks on result:
other: Ear thickness, TD 4

Observations

Dated and signed records of all activities relating to the day by day running and maintenance of the study within the animal unit as well as to the group observations and examinations outlined in the study plan were recorded in the appropriate documentation. In addition, observations related to individual animals were made throughout the study and were recorded.

Clinical signs

Animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. Observations were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous mem¬branes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded. In addition, animals were checked regularly throughout the working day from 7:30 a.m. to 4:30 p.m. On Saturdays and Sundays animals were checked regularly from 8:00 a.m. to 12:00 noon with a final check performed at approximately 4:00 p.m., if applicable. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

Body weight

The weight of each mouse was recorded at the time of allocation of animals to groups (test day 1) and at the time of necropsy (test day 4).

Analysis of results

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.

For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight was examined by linear regression analysis employing PEARSON's correlation coefficient. An U-test was performed also for cell count. Outliers were determined according to the Nalimov test. 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 of the test item treated animals by the vehicle treated ones. The cut-off threshold value for ear weight was set at 1.1.

Findings

Three concentrations of Isooctadecanoic acid, potassium salt (1:1) (1%, 5% and 10%), suspended or dissolved in coconut(w/w) were tested in six female NMRI mice per group and compared to a vehicle control group. A 10% suspension was the highest feasible concentration ofIsooctadecanoic acid, potassium salt (1:1)in coconut oil. The vehiclecoconut oilwas used as negative reference item. In addition, a positive control group(20% solution (v/v) ofa-hexyl cinnamic aldehyde inacetone / olive oil(4:1, v/v)) and one group with the vehicle of the positive control were employed. In a preliminary experiment, concentrations of 1%, 5% and 10%ofIsooctadecanoic acid, potassium salt, employing 1 animal per concentration, were examined. No irritating properties were observed in this preliminary experiment, no differences in ear weight and ear thickness were noted.

Main study results: stimulation indices (SI):

 Parameter  Group 1, negative control  Group 2, 1%  Group 3, 5%  Group 4, 10%  Group 5, positive control  Group 6, vehicle of positive control
 Lymph node cell count  1.000  0.995  1.148  1.166  1.595*  1.000
 Lymph node cell weight  1.000  1.188  1.271  1.333  1.426*  1.000
 Ear weight  1.000  0.978  0.918  0.929  1.076*  1.000
 Ear thickness, TD 4  1.000  1.033  1.008  1.050  1.074  1.000

* significantly different from control at p ≤ 0.01

In the main study treatment with Isooctadecanoic acid, potassium salt at concentrations of 1%, 5% or 10% did not reveal any statistical significantly increased values for the lymph node cell count. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. Hence, the test item is classified as not sensitising. 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.

Interpretation of results:
GHS criteria not met
Conclusions:
In a modified OECD 429 GLP study under the presented test conditions the test item isooctadecanoic acid, potassium salt at concentrations of 1%, 5% or 10% (w/w) in coconut oil did not reveal any skin sensitising properties in the local lymph node assay. The test item is classified as not sensitising.
Executive summary:

In a modified OECD 429 GLP study under the presented test conditions the test item isooctadecanoic acid, potassium salt at concentrations of 1%, 5% or 10% (w/w) in coconut oil did not reveal any skin sensitising properties in the local lymph node assay.

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). No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment. The test item is classified as not sensitising.

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

Justification for classification or non-classification

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