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Skin sensitization potential of [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids was predicted using a QSAR model battery developed by the Danish National Food Institute at the Technical University of Denmark. The model battery was queried for seven (7) individual constituents of the substance which, along with water, comprise ca. 98% of the identified constituents and ca. 77% of the total UVCB composition. All of the constituents queried are predicted to produce a NEGATIVE result in the skin sensitization test (OECD 406). None of the seven constituents are predicted to produce allergic contact dermatitis in guinea pig and human. The skin sensitization potential of a similar UVCB substance (EC/List number 943-164-7) was tested according to OECD method 429 (2010) in 2016. The study found no indication that the test item elicited a Stimulation Index ≥ 3 when tested up to 10% and the substance was determined to be NOT a skin sensitizer according to the test guidelines.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
QSAR predictive modeling
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
March, 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
See attached QPRF and QMRF documents.
Qualifier:
equivalent or similar to
Guideline:
other: The QSAR battery predicts an endpoint of skin sensitization consistent with OECD 406
Version / remarks:
Use of QSAR models is consistent with ECHA "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals".
Principles of method if other than guideline:
Use of QSAR models is consistent with ECHA "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals".
Justification for non-LLNA method:
QSAR modeling was utilized to predict skin sensitization potential in favor of conducting animal testing.
Specific details on test material used for the study:
Skin sensitization potential was predicted using a QSAR model for seven individual constituents of the UVCB substance. These constituents, along with water, comprise ca. 98% of the identified constituents and ca. 77% of the total UVCB composition.
Key result
Remarks on result:
other: QSAR model
Remarks:
Skin sensitization potential was predicted using a QSAR model for seven individual constituents of the UVCB substance.
Interpretation of results:
GHS criteria not met
Conclusions:
All seven of the constituents queried are predicted produce a NEGATIVE result in the skin sensitization test (OECD 406). These constituents are predicted to be not sensitizing via dermal exposure. [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids is predicted to be not sensitizing to skin.
Executive summary:

Skin sensitization potential of [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids was predicted using a (Q)SAR model battery developed by the Danish National Food Institute at the Technical University of Denmark. All three models predict a skin sensitization endpoint consistent with OECD 406. Skin sensitization potential was predicted for seven (7) individual constituents of the UVCB substance. These constituents, along with water, comprise ca. 98% of the identified components and ca. 77% of the total UVCB composition. All seven of the constituents are within the applicability domain for each of the three model systems.

Skin sensitization is an immunological response to a hapten compound formed between a foreign, low molecular weight substance and the amino acid nucleophilic side chains in the skin. Haptens can be recognized as an antigen by the immune system resulting in inflammatory pruritic, eczematous lesions. The sensitizing ability of haptens is influenced by lipophilicity, tridimensional chemical structure, and protein‐binding affinity. The long-chain linear organic fatty acids that characterize the composition of [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids are not known to produce an immunological effect via dermal exposure by this mechanism. All of the queried constituents are organic long-chain fatty acids with a relatively simplistic linear structure consisting of only carbon, hydrogen, and oxygen. None of the model outputs flagged any sub-structures or fragments that are not represented in the model training sets. No reports of allergic contact dermatitis to the long-chain fatty acid constituents of [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids were identified in the literature.

All seven of the constituents queried are predicted to produce a NEGATIVE result in the skin sensitization test (OECD 406). None of the seven constituents are predicted to produce allergic contact dermatitis by any of the three (Q)SAR model systems. Given the well-defined chemical structures of the substance constituents in relation to the mechanism of skin sensitization, the predictive modeling results are considered appropriate to support a weight of evidence determination regarding the skin sensitization potential of [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
27 September 2016 - 25 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008, including amendments
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 20.4 - 23.3 g
- Housing: Animals were group housed in labeled Makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
dimethyl sulphoxide
Concentration:
0, 5%, 10%, and 25%
No. of animals per dose:
5
Details on study design:
WEIGHT OF EVIDENCE ANALYSIS: In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to the start of this study. All available information was evaluated (e.g. existing human and animal data, literature, item data supplied by the Sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity)). It was concluded by the Study Director that no severe effects were to be expected.

RANGE FINDING TESTS: Two test item concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines. The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per conce ntration 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. Animals were sacrificed after the final observation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: 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 item may be regarded as a skin sensitizer.

ANIMAL ASSIGNMENT: Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION: The test item preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed. The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at Charles River Den Bosch.

STUDY ADMINISTRATION:
Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.
Observations:
- Mortality/Viability: Twice daily.
- Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
- Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
- Irritation: Once daily on Days 1-6 (on Days 1 - 3 within 1 hour after dosing) according to a numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.
- Necropsy: No necropsy for gross macroscopic examination was performed according to the study plan.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis not performed
Positive control results:
The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Laboratories Den Bosch B.V. is an appropriate model for testing for contact hypersensitivity.
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.9
Test group / Remarks:
10%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test item concentrations 5% and 10% were 1321 and 2176 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1144 DPM.
Key result
Parameter:
EC3
Value:
> 10
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

DETAILS ON STIMULATION INDEX CALCULATION: 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. The SI values calculated for the test item concentrations 5% and 10% were 1.2 and 1.9, respectively.

EC3 CALCULATION: It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%.

CLINICAL OBSERVATIONS: At 25%, signs of systemic toxicity (hunched posture and/or piloerection) were noted for the majority of animals between Days 2 and 5. These signs could have affected the sensitization response of the animals and therefore this group was excluded from interpretation. Sufficient dose groups were available to warrant the study integrity. No further clinical signs of systemic toxicity were observed in the remaining animals of the main study. No mortality occurred.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

SKIN REACTIONS/IRRITATION: Very slight erythema and/or scaliness were noted for the majority of vehicle and test item animals dosed at 5 and 10%. This erythema was considered not to have an effect on the activity of the lymph nodes.
Interpretation of results:
GHS criteria not met
Remarks:
Not sensitising according to Regulation (EC) No. 1272/2008
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD 429 test guideline and GLP principles, the substance was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 10% v/v.
Executive summary:

An LLNA skin sensitisation study was performed according to OECD 429 test guideline and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 5%, 10% and 25% v/v. Reliable negative and positive controls were included. At 25%, signs of systemic toxicity (hunched posture and/or piloerection) were noted for the majority of animals between Days 2 and 5. These signs could have affected the sensitization response of the animals and therefore this group was excluded from interpretation. Sufficient dose groups were available to warrant the study integrity. No further clinical signs of systemic toxicity were observed in the remaining animals of the main study. No mortality occurred. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Very slight erythema and/or scaliness were noted for the majority of vehicle and test item animals dosed at 5% and 10%. This erythema was considered not to have an effect on the activity of the lymph nodes. Mean DPM/animal values for the experimental groups treated with test item concentrations 5% and 10% were 1321 and 2176 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1144 DPM. The SI values calculated for the test item concentrations 5 and 10% were 1.2 and 1.9, respectively. As the SI appeared not to be ≥ 3 when tested up to 10% v/v, the substance is considered to NOT be a skin sensitiser and does not meet the criteria for classification as skin sensitisation according to Regulation (EC) No. 1272/2008.

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

Skin sensitization is an immunological response to a hapten compound formed between a foreign, low molecular weight substance and the amino acid nucleophilic side chains in the skin.  The sensitizing ability of haptens is influenced by lipophilicity, tridimensional chemical structure, and protein‐binding affinity.  The long-chain linear organic fatty acids that characterize the composition of [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids are not known to produce an immunological effect via dermal exposure by this mechanism.

Justification for classification or non-classification

[ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids is a UVCB substance of biological origin. The substance is a complex mixture of long-chain fatty acids produced and excreted by an engineered Escherichia coli K-12 organism through an aqueous microbial fermentation process. The substance is predominantly comprised of linear unbranched long-chain organic fatty acids differentiated only by saturation and number of terminal carboxyl or hydroxyl groups.

 

Many of the UVCB constituents are either naturally produced or obtained through the food chain of a broad range of organisms and have been positively identified in algae [4], plant cuticular waxes [2], seeds [4], fish [10], avian eggs [11], edible nuts [8], and berries [12]. Palmitic acid (C16:0) is synthesized endogenously and serves essential cellular functions in humans [6]. Palmitic acid and oleic acid (C18:1) comprise more than half of the fatty acid content in human breast milk [9]. There is a significant body of ongoing clinic research investigating the potential benefits of long-chain fatty acids in reducing inflammatory conditions [1]. No reports of allergic contact dermatitis to the fatty acid constituents of [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids were identified in the literature.

 

With respect to sensitization potential, a similar UVCB test substance (EC/List number 943-164-7) and the [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids substance are considered to be within the same category of chemical substances. The two substances are produced by the same E. coli fermentation and have at least 8.5% of their compositions in common. The substances differ only in the extraction solvent, resulting in the esterification of some of the fatty acid constituents. The chain lengths of the constituents and their relative ratios within the UVCB composition are otherwise very similar. Therefore, a chemical category approach is adopted as part of a weight of evidence approach to characterize sensitization potential. A qualitative read-across approach is employed in using the previous test results to support the determination predicted by the QSAR model.

Given the chemical structures of the constituents, the ubiquity of long-chain fatty acids in biological systems, the predictive modeling results, and the test results of a similar UVCB substance [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids is determined to be not sensitizing to the skin.

 

References:

1.      Artemis P. Simopoulos. Omega-3 Fatty Acids in Inflammation and Autoimmune Diseases, Journal of the American College of Nutrition. 2013; 21:6, 495-505

2.      Fernandes, A. M. Silva, Baker, E.A., Martin, J.T. Studies on plant cuticle. Annals of Applied Biology 1964, 53(1):43-58

3.      Frago LM, Canelles S, Freire-Regatillo A, et al. Estradiol Uses Different Mechanisms in Astrocytes from the Hippocampus of Male and Female Rats to Protect against Damage Induced by Palmitic Acid. Frontiers in Molecular Neuroscience. 2017;10:330

4.      Hajghanbari et al. Essential oil constituents and fatty acids in Echium amoenum grown wild in Iran. International Journal of Biosciences 2015, 6(1): 156-161

5.      Hassain, J, et al. Effects of Different Biomass Drying and Lipid Extraction Methods on Algal Lipid Yield, Fatty Acid Profile, and Biodiesel Quality. Appl Biochem Biotechnol 2015, 175: 3048

6.      Innis, Sheila. Palmitic Acid in Early Human Development. Critical Reviews in Food Science and Nutrition 2016, 56(12): 1952-1959

7.      Kakimoto, Pamela and Kowaltowski, Alicia. Effects of high fat diets on rodent liver bioenergetics and oxidative imbalance. Redox Biology 2016, 8 (2016) 216–225

8.      Maguire LS, O’Sullivan SM, et al. Fatty acid profile, tocopherol, squalene and phytosterol content of walnuts, almonds, peanuts, hazelnuts and the macadamia nut. International Journal of Food Science Nutrition 2004, 5:171-178

9.      Martin, Camilia, Ling Pei-Ra, Blackburn, George. Review of Infant Feeding: Key Features of Breast Milk and Infant Formula. Nutrients 2016, 8(5), 279

10.   Ozogul Y, Ozogul F, Cicek E, Polat A, Kuley E: Fat content and fatty acid compositions of 34 marine water fish species from the Mediterranean Sea. International Journal of Food Science Nutrition 2008, 29:1-12

12.   Toledo, A., Andersson, M.N., Wang, HL. et al. Fatty acid profiles of great tit (Parus major) eggs differ between urban and rural habitats, but not between coniferous and deciduous forests. Sci Nat 2016, 103: 55

13.   Yang B, Kallio HP. Fatty acid composition of lipids in sea buckthorn (Hippophaë rhamnoides L.) berries of different origins. J Agricultural Food Chemistry 2001, 49:1939-1947