Calcium(2+) 12-hydroxyoctadecanoate

Endpoint summary

Description of key information

In vitro and in vivo studies on dermal and ocular irritation have been conducted on C14, C16, C18 fatty acid lithium salts, and in vitro/in vivo eye irritation studies with calcium myristate. No classifiable irritant responses were observed relevant to calcium salts of C14-22 fatty acids.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion

Remarks:

other: in vitro

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

The study was performed between 30 March 2010 and 05 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

other: EU Guideline Testing of Chemicals B46

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Draft Test Guideline

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: reconstituted human epidermis model

Strain:

other: reconstituted human epidermis model

Details on test animals or test system and environmental conditions:

Not applicable

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Controls:

no

Amount / concentration applied:

TEST MATERIAL

- The test Material was applied neat.

- Amount(s) applied (volume or weight with unit):
Approximately 10 mg of the test material was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test material and the epidermis.

- Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used

Duration of treatment / exposure:

15 Minutes & 42 hour post exposure incubation

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

TEST SITE
- Area of exposure:
Approximately 10 mg of the test material was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test material and the epidermis.

- % coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

- Type of wrap if used:
None used

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material.

- Time after start of exposure:
15 Minutes post exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
For the test material the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is =50% : Irritant (I) R38

Mean tissue viability is >50% : Non-Irritant (NI)

Irritation parameter:

other: Viability of cells

Basis:

mean

Remarks:

Viability of cells

Time point:

other: Day 6

Reversibility:

other: Not Applicable

Remarks on result:

other: See relative mean viability below.

Irritant / corrosive response data:

The relative mean viability of the test material treated tissues was 93.1% after a 15 minute exposure.

Other effects:

No

RESULTS

Direct MTT Reduction

An assessment found the test material was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed during the determination of skin irritation potential. The test material was adequately rinsed from the tissues, however the results obtained showed that during rinsing no degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD₅₄₀ values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test material treated tissues was 93.1% after a 15 -minute exposure.

The qualitative evaluation of tissue viability is given in Table 2.

Following the 15-minute exposure the test material treated tissues appeared blue/white which was considered indicative of viable tissue.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was =40% relative to the negative control treated tissues and the standard deviation value of the percentage viability was =20%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₄₀ for the negative control treated tissues was =0.6 and the SD value of the percentage viability was =20%. The negative control acceptance criterion was therefore satisfied.

Table 1 : Mean OD₅₄₀ Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	OD540of tissues	Mean OD540of triplicate tissues	±SDof OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Material	1.016	0.962	0.002	105.6	100*	5.4
	0.958			99.6
	0.913			94.9
Positive Control Material	0.068	0.064	0.009	7.1	6.7	0.9
	0.054			5.6
	0.070			7.3
Test Material	0.877	0.896	0.021	91.2	93.1	2.2
	0.919			95.5
	0.892			92.7

SD=    Standard deviation

*=     The mean viability of the negative control tissues is set at 100%

Table 2 : Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative Control Material	-	-	-
Positive Control Material	++	++	++
Test Material	-	-	-

MTT visual scoring scheme
-          =         blue tissue (viable)
+         =         blue/white tissue (semi-viable)
++       =         tissue is completely white (dead

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

The test material was considered to be Non-Irritant (NI)

Executive summary:

Introduction: The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN^TM reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1a in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Methods:

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Results: 

The relative mean viability of the test material treated tissues was 93.1% after a 15-minute exposure.

Quality criteria: 

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion:  The test material was considered to be Non-Irritant (NI).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The substances in the category are considered to be similar on the basis that they have common structures of a calcium ion varying only by the length of the fatty acid chain and the presence of unsaturated and/or hydroxyl functional groups. As a result it is expected that the substances will have similar, predictable properties. REACH Annex V, Entry 9, groups fatty acids and their potassium, sodium, calcium and magnesium salts, including C6 to C24, predominantly even-numbered, unbranched, saturated or unsaturated aliphatic monocarboxylic acids. Provided that they are obtained from natural sources and are not chemically modified, the substances included in REACH Annex V, Entry 9 are exempt from registration, unless they are classified as dangerous (except for flammability, skin irritation or eye irritation) or they meet the criteria for PBT/vPvB substances. The metal fatty acid substances in the category are therefore not expected to be hazardous. Due to the close structural similarity and the narrow range of carbon chain numbers covered in this category, the irritation properties are expected to be predictable across the category.

Since REACH Annex V groups together calcium, potassium, sodium and magnesium salts of C6 to C24 fatty acids as being potentially exempt from registration, these metal cations are therefore not considered to contribute to any health hazard. On this basis, relevant published or proprietary data on any potassium, sodium or magnesium salt within the fatty acid category range of C14 to C22 can be used to read across to the calcium salts of C14-C22 fatty acid category.

Lithium salts of fatty acids are not included in REACH Annex V as being potentially exempt from registration. For these salts it is expected that the lithium cation would be the species with the potentially higher toxicity profile when compared to calcium, potassium, sodium, and magnesium cations. However, the substance fatty acids C18 (unsaturated) lithium salts contains a fatty acid anion that falls within the C14-C22 category. Experimental data for the mammalian toxicity Annex VIII endpoints have been generated on this substance and the results obtained are relevant to read across to the calcium salts of C14-C22 fatty acids either in a weight of evidence approach or as key studies due to the structural similarity and its position within the category fatty acid range. Additionally, ocular irritation studies have been conducted on lithium palmitate.

Published reviews consider that the irritant/corrosivity properties of fatty acids are chain length dependent, where the lower carbon chain lengths, < C9 are corrosive, C10 – C12 are irritant, and chain lengths from C14 are not irritating (HERA 2002), referencing Briggs et al, 1976 and CIR, 1987). Key in vitro and in vivo ocular irritation studies have been conducted on the category member with the shortest chain length, calcium myristate, since that would be considered the ‘worst case’ substance for irritation based on the relationship to chain length. The in vitro study (Rabbit Eye Enucleation Test - REET) showed no evidence of severe irritation/corrosivity, and the in vivo OECD Guideline 405 study demonstrated no classifiable eye irritation. Since the fatty acid salts with longer chain lengths are considered to have proportionately lower irritation potential, this negative result can be read across to the other category members. This is also supported by read across data from ocular irritation studies on relevant lithium fatty acid salts with longer carbon chain lengths. Negative in vitro and in vivo (OECD Guideline 405) studies were conducted on lithium palmitate (C16) and fatty acids, C18 (unsaturated), lithium salts. Since the lithium cation might be expected to demonstrate higher irritation potential than the calcium cations, reading across the negative results to the latter category is validated. Further supporting evidence is shown by a negative published eye irritation study on magnesium stearate, taken from a peer-reviewed article.

 

The dermal corrosion and irritant properties of magnesium stearate to the intact and abraded skin of rabbits were evaluated in a published supporting study. A primary irritation index of 0.0 was obtained after four hours exposure under occlusive dressing. Although no information on the test methods is available in the publications, it is a peer-reviewed article and considered reliable and relevant for assessment of this endpoint. A supporting study in rabbits for skin irritation using a grease formulated with calcium 12-hydroxystearate has been conducted and the formulation was non-irritant.

 

Key in vitro skin irritation studies on lithium myristate (C14) and fatty acids C18 (unsaturated) lithium salts gave negative results. The negative results from the studies with the lithium fatty acid salts are suitable for reading across to the calcium fatty acid salts for the reasons mentioned above. Since the irritation properties of fatty acids and their salts are inversely proportional to carbon chain length, the negative results are applicable for the whole category from C14 to C22.

 

Overall, the calcium fatty acid salts of C14 to C22 fatty acids are not considered to be skin or ocular irritants.

 

References

Briggs GB, Doyle RL, Young JA (1976) Safety studies on a series of fatty acids. American Industrial Hygiene Association Journal, vol. 37, pp. 251-252

CIR (Cosmetics Ingredients Review) (1987) Final report on the safety assessment of oleic acid, lauric acid, palmitic acid, myristic acid and stearic acid. Journal of American Toxicologists, vol. 6, issue 3, pp. 321-401

HERA (Human Health and Environmental Risk Assessment on ingredients of European household cleaning products) (2002) Fatty Acid Salts (Soap) Environmental and Human Health Risk Assessment

Justification for selection of skin irritation / corrosion endpoint:
This substance is a representative fatty acid salt that can be read across to the calcium salts of C14-C22 fatty acids category

Justification for classification or non-classification

Not classified under the EU CLP Regulation. All studies were negative.