Castor oil, hydrogenated, lithium salt

Endpoint summary

Description of key information

In vitro studies on dermal irritation have been conducted on C14, C16, and C18 fatty acid lithium salts. No irritant responses leading to classification were observed.
In vitro and in vivo studies on ocular irritation were conducted on C14, C16 and C18 fatty acid lithium salts. No classifiable irritant responses were observed for C16 and C18 fatty acid lithium salts, but irritation was seen for C14 (lithium myristate). This result was not read across to the category since the C16 and C18 results were negative.

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:

experimental study

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-1α 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

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant, guideline study, available as an unpublished report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

- Selection of animals: Immediately prior to test initiation, both eyes of the provisionally selected test rabbits were examined and any animals showing evidence of ocular irritation or defect were rejected
- Age at test initiation: 12 to 20 weeks
- Acclimatisation period: At least 5 days
- Housing: Individually in suspended cages
- Feeding: Free access to mains drinking water and commercial food
- Temperature: 17 to 23°C
- Relative humidity: 30 to 70 %
- Air flow: At least fifteen changes per hour
- Photoperiod: Twelve hours continuous light (06:00 to 18:00) and twelve hours darkness

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

- Volume: 0.1 mL of lithium palmitate, equivalent to approximately 66 mg, was applied to the conjunctival sac of the right eye and the upper and lower eyelids were held together for about one second immediately after treatment

Duration of treatment / exposure:

- Duration of exposure: The rabbits' eyes were not washed

Observation period (in vivo):

- Observations of eyes: Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment
- Observations of reversibility: An observation was made on Day 7 to assess the reversibility of the ocular effects

Number of animals or in vitro replicates:

- Number of animals: Initially, a single rabbit was treated and after consideration of the ocular response, a second animal was treated

Details on study design:

- Control: The left eye remained untreated and was used for control purposes
- Observations of rabbits: Any other ocular effects and any clinical signs of toxicity were also noted and individual bodyweights were recorded on the day of dosing and at the end of the observation period
- Assessment method: Occular irritation potential results were interpreted according to the Globally Harmonized System of Classification and Labelling of Chemicals

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24, 48, 72h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Remarks:

mean

Basis:

animal #1

Time point:

other: 24, 48, 72h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible

Remarks:

within 48 h

Irritation parameter:

iris score

Remarks:

mean

Basis:

animal #2

Time point:

other: 24, 48, 72h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

mean

Basis:

animal #1

Time point:

other: 24, 48, 72h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible

Remarks:

within 7 days

Irritation parameter:

conjunctivae score

Remarks:

mean

Basis:

animal #2

Time point:

other: 24, 48, 72h

Score:

1.33

Max. score:

3

Reversibility:

fully reversible

Remarks:

within 7 days

Irritation parameter:

chemosis score

Remarks:

mean

Basis:

animal #1

Time point:

other: 24, 48, 72h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible

Remarks:

within 7 days

Irritation parameter:

chemosis score

Remarks:

mean

Basis:

animal #2

Time point:

other: 24, 48, 72h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible

Remarks:

within 7 days

Irritant / corrosive response data:

- Corneal effects: None were noted during the study
- Iridial effects: Inflammation was noted in both treated eyes one hour after treatment and in one treated eye at the 24 Hour observation
- Conjunctival effects: Moderate irritation in both treated eyes at 1 and 24 hours after treatment, with minimal irritation in both treated eyes at 48 hours and in 1 treated eye at 72 hours
- Reversibility: Both treated eyes appeared normal at 7 days
- Conclusion: Lithium palmitate produced a maximum group mean score of 17.0 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. Lithium palmitate does not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals or Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.

Other effects:

- Body weight: Both animals showed expected gain in bodyweight during the study

Individual scores and individual total scores for occular irritation

Rabbit Number and Sex	72629Male					72644Male
	IPR = 0					IPR = 0
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	1 Hour	24 Hours	48 Hours	72 Hours	7 Days
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0	0	0
IRIS
D	1	1	0	0	0	1	0	0	0	0
Score (D x 5)	5	5	0	0	0	5	0	0	0	0
CONJUNCTIVAE
A = Redness	2	2	2	1	0	2	2	1	1	0
B = Chemosis	2	2	1	1	0	2	2	1	1	0
C = Discharge	2	1	0	0	0	2	1	0	0	0
Score (A + B + C) x 2	12	10	6	4	0	12	10	4	4	0
Total Score	17	15	6	4	0	17	10	4	4	0

IPR = Initial pain reaction

Individual total scores and group mean scores for occular irritation

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours	7 Days
72629Male	17	15	6	4	0
72644Male	17	10	4	4	0
Group Total	34	25	10	8	0
Group Mean Score	17.0	12.5	5.0	4.0	0.0

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Lithium palmitate does not meet the criteria for classification according to the Globally Harmonised System of Classification and Labelling of Chemicals or Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Dangerous Substances.

Executive summary:

The eye irritation of lithium palmitate was assessed in a GLP-compliant, in vivo eye irritation study following OECD guideline 405 (Harlan 2013). A single treatment of lithium palmitate was applied to the non-irrigated eye of two rabbits and observations made at 1, 24, 48 and 72 hours and 7 days for effects on conjunctivae, iris and cornea and for reversibility of effects. No irritation classifiable under CLP/GHS was observed. The study is considered relevant and reliable for use for this endpoint.

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 lithium ion varying only by the length of the fatty acid chain and the presence of unsaturated and/or hydroxyl functional groups. 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 fatty acid components of the category members are therefore not expected to be hazardous. As all category members are lithium salts, any toxicity is expected to be driven by the lithium ion. Due to the close structural similarity and the narrow range of carbon chain numbers covered in this category, irritation potential is expected to be similar or show a predictable trend across the category.

Although fatty acids C18 (unsaturated) lithium salts is not in the list of substances being registered, this substance falls within the definition of the lithium salts of fatty acids C14-C22 category (see Appendix 1 – Category Justification Document) by virtue of its chemical structure and therefore read across from data on fatty acids C18 (unsaturated) lithium salts to other members of the category is considered to be justified (see below).

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 skin irritation studies on lithium myristate (C14) and Fatty acids C18 (unsaturated) lithium salts gave negative results. Since under REACH, in vitro studies are sufficient for compliance with Annexes VII and VIII, no key in vivo study(ies) has been conducted. A number of in vivo supporting studies in rabbits for skin irritation using greases formulated with lithium 12-hydroxystearate have been conducted. However, since these were performed several decades previously on formulated greases containing other (unknown) performance additives, the results from the in vitro skin irritation tests on isolated C14 and C18 fatty acid lithium salts are considered to be more relevant and indicative of no classification for this endpoint.

Lithium palmitate (C16), and Fatty acids C18 (unsaturated) lithium salts were subjected to in vitro eye irritation studies using the isolated rabbit eye or reconstituted human corneal epithelium models, andin vivoeye irritation studies in rabbits according to OECD 405 (key studies). For both substances, no classifiable irritation was observed. Several supporting studies where grease containing approximately 5 to 13% lithium 12-hydroxystearate was tested in rabbit eyes are reported, and where the test materials were found to be ‘not irritating’.

One reliable study (Klimisch 1) is referenced in the API Robust Summary document (2008) where lithium complex grease (containing 13.1% lithium 12-hydroxystearate and 2.6% dilithium azelate) was applied to the right eye of six rabbits. This was reported as non-irritant in the API hazard summary. The Cosmetic Ingredient Review Panel (CIR 1982) concludes that certain metal stearates (Mg, Na, Zn) were non-irritant to rabbit eye. No quality review of the data was reported and the data were either unpublished proprietary studies or from literature publications before 1960.

Lithium myristate (C14) was also tested in an OECD 405 rabbit eye irritation study. The results from this study showed ocular irritation, reversible within the 14-day observation period. Since both C16 and C18 fatty acid lithium salts gave negative results in the ocular irritation tests, the positive irritant response from lithium myristate (C14) was further reviewed. The initial substance for testing was prepared by reacting lithium hydroxide with myristic acid and washing the reaction product with isopropyl alcohol. Following substance analysis, the presence of small concentrations of residual lithium hydroxide (a strong ocular irritant) could not be excluded, and the aqueous pH (10% preparation) taken before and after the rabbit eye irritation study showed values of 10.1 drifting to 11.1 after 20 minutes. A new sample of lithium myristate was prepared, taking care to remove residual lithium hydroxide using water to wash the final product. The pH of a 10% preparation of this sample was ca 9.5 (and pH ca 8 from a sample moistened with water). Because of the modification of the synthesis, elemental analysis, and the consequent lower pH values (indicating little or no residual lithium hydroxide), it was considered appropriate to subject this sample to repeat in vitro and in vivo eye irritation studies. However, the results obtained were very similar to those from the first sample, and consequently lithium myristate is considered to be classified as an ocular irritant.

The irritant/corrosivity properties of free fatty acids are chain length dependent, where the lower carbon chain length < C9 are corrosive, C10 – C12 are irritant and C14 above are non-irritant. No ocular irritation was observed in the studies with C16 and C18 lithium salts. It is therefore concluded that the positive eye irritation results obtained with lithium myristate (C14) represents a worst case and are not indicative of a category response for the longer chain length substances. The positive results are not read across to the other category members which are considered not to be eye irritants. 

References

API (American Petroleum Institute) (2008) Robust Summary of Information on Grease Thickeners (Creation date: October 11, 2003. Printing date: February 20, 2009. Last update: October 20, 2008. Document date: January 11, 2005)

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) (1982). Final report of the safety assessment of lithium stearate, aluminum distearate, aluminum stearate, aluminum tristearate, ammonium stearate, calcium stearate, magnesium stearate, potassium stearate, sodium stearate and zinc stearate. Journal of the American college of toxicologists, vol. 1, issue 12, pp. 143-177.

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 representative of the lithium salts of C14 - C22 fatty acids and can be read across to other category members

Justification for selection of eye irritation endpoint:
This substance is representative of the lithium salts of C14 - C22 fatty acids and can be read across to other category members with the exception of lithium myristate (C14).

Justification for classification or non-classification

Skin irritation: Not classified. All studies were negative.

Eye irritation: C16 -C22 fatty acid lithium salts not classified (studies were negative). C14 fatty acid lithium salts classified as eye irritant category 2.