Dilithium azelate

Endpoint summary

Description of key information

It is considered from the results that all the substances in the category of lithium salts of C6-C10 dicarboxylic acids exhibit similar lack of irritant and corrosive toxicity potential across the entire category. There is no evidence of a relevant intrinsic irritant or corrosive properties requiring classification or substance specific risk mitigation measures (RMMs). 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

07 April 2015 to 10 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant, guideline study, available as an unpublished report.

Justification for type of information:

For read across justification, see Section 13 of IUCLID

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

yes

Remarks:

The mean relative tissue viability to the positive control was up to 33% and the max difference in % between the mean viability of two tissues and one of the two tissues was up to 20% . The study integrity was not adversely affected by the deviations.

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS (In vitro skin corrosion, human skin model test)

Deviations:

yes

Remarks:

The mean relative tissue viability to the positive control was up to 33% and the max difference in % between the mean viability of two tissues and one of the two tissues was up to 20% . The study integrity was not adversely affected by the deviations.

GLP compliance:

yes (incl. QA statement)

Species:

other: EpiDerm Skin Model (EPI-200, Lot no.: 21997 kit M)

Strain:

other: Not applicable

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:

unchanged (no vehicle)

Controls:

other: Not applicable

Amount / concentration applied:

The test was performed on a total of 4 tissues per test substance together with a negative control and positive control.

- Treatment group: Test carried out on a total of 4 tissues. 25.6 to 26.5 mg of the test item was applied topically with a small glass weight boat, ensuring an even covering, to the epidermis surface which had previously been moistened with 25 µL sterile, distilled water to improve contact between the solid test item and the tissue.
- Negative control: Tissue treated with 50 µL Mili-Q water
- Positive control: 50 µL 8N KOH

Duration of treatment / exposure:

Two tissues were used for a 3-minute exposure to Dilithium adipate and two for a 1-hour exposure.

Observation period:

Not applicable.

Number of animals:

Not applicable.

Details on study design:

Test for the interference of the test substance with the MTT endpoint:
A test substance may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test substance is present on the tissues when the MTT viability test is performed.

Test for colour interference by the test substance:
Some non-coloured test substances may change into coloured substances in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, approximately 25 mg of Dilithium adipate or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
In case the test substance induces colour interference in aqueous conditions, in addition to the normal procedure, two tissues must be treated with test substance for 3 minutes and two tissues for 1-hour. Instead of MTT solution these tissues will be incubated with medium.

Test for reduction of MTT by the test substance:
To assess the ability of the test substance to reduce MTT, approximately 25 mg of Dilithium adipate was added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple colour change was observed.
In case the test substance reacts with the MTT medium in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test substance and two freeze-killed non treated tissues must be used for the cytotoxicity evaluation with MTT.

Treatment of the test substance:
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM medium per well. The plates were incubated for 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before Dilithium adipate was applied. The skin was moistened with 25 μL Milli-Q water to ensure close contact of the test substance to the tissue and 26.5 to 29.0 mg of the solid test substance (with a small glass weight boat) was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μL Milli-Q water (negative control) and with 50 μL 8N KOH (positive control), respectively. After the exposure period, the tissues were washed with PBS (phosphate buffered saline) to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 μL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

Cell viability measurement
The DMEM medium was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

93

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

71

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability obtained after 3 minutes and 1 hour treatment with Dilithium adipate compared to the negative control tissues was 93 % and 71 %, respectively. The mean optical density measured at 570 nm after 3 minutes and 1 hour was 1.924 (SD = 0.244) and 1.404 (SD = 0.082), respectively.
The test item is considered to be non-corrosive in the in vitro skin corrosion test under the experimental condition described in the report.
- Positive control: The relative mean tissue viability after 3 minutes and 1 hour application was 10 % relative to the negative control. The mean optical density after 3 minutes and 1 hour application was 0.205 (SD = 0.048) and 0.197 (SD = 0.055), respectively.
- Negative control: The mean optical density after 3 minutes and 1 hour application was 2.059 (SD = 0.152) and 1.984 (SD = 0.122), respectively.

Table 1 Mean absorption in the in vitro skin corrosion test with Dilithium adipate

3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)	SD	A (OD570)	B (OD570)	Mean (OD570)	SD
Negative control	2.167	1.951	2.059	± 0.152	2.070	1.898	1.984	± 0.122
Dilithium adipate	2.097	1.752	1.924	± 0.244	1.462	1.346	1.404	± 0.082
Positive control	0.171	0.239	0.205	± 0.048	0.236	0.158	0.197	± 0.055

SD = Standard deviation

Duplicate exposures are indicated by A and B.

Table 2 Mean tissue viability in the in vitro skin corrosion test with Dilithium adipate

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Dilithium adipate	93	71
Positive control	10	10

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that the test is valid and that Dilithium adipate is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.

Executive summary:

The possible corrosive potential of Dilithium adipate was tested in in vitro skin corrosion test according to OECD Guideline 431 and EU guideline B.40 Bis using a human skin model.

The test consists of topical application of Dilithium adipate for 3 minutes and 1 hour on a human three dimensional epidermal model (EpiDerm EPI-200). Skin corrosion is expressed as the remaining cell viability after exposure to the test substance.

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Dilithium adipate compared to the negative control tissues was 93% and 71%, respectively. Dilithium adipate is considered to be not corrosive.

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:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

30 April 2015 to 14 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For read across justification, see Section 13 of IUCLID

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF Guidelines (2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: 24 - 26 weeks old
- Weight at study initiation: 4322 - 4981 g
- Housing: Individually housed in labeled cages with perforated floors (dimensions: 67 x 62 x 55 cm, Ebeco, Germany) and shelters (dimensions: 40 x 32 x 23 cm, Ebeco, Germany).
- Diet: Pelleted diet for rabbits (Global Diet 2030, Harlan Tekland, Mucedola, Milanese, Italy), approximately 100 g per day. Hay (Tecnilab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study period.
- Water: Tap water, ad libitum.
- Acclimation period: At least 5 days before test start, under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 24 °C
- Humidity: 40 - 70 %
- Air changes: at least 10 air changes/hour
- Photoperiod: 12 hours dark: 12 hours light

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: Animals were treated by instillation of 27.8 mg (range 27.4 - 28.3 mg) of the test substance (a volume of approximately 0.1 mL) in the conjunctival sac of one eye after gently pulling the lower lid away from the eyeball. The second eye remianed untreated and served as the control.

Duration of treatment / exposure:

Single application.

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 were made twice daily for mortality/viability and at least once daily for toxicity. Body weight was measured prior instillation of test item and after the final observation.

Number of animals or in vitro replicates:

3 females. The study was performed in a stepwise manner and was started by treatment of a single rabbit. Two other animals were treated in a similar manner 11 days later, after considering the degree of eye irritation observed in the first animal.

Details on study design:

PREEMPTIVE PAIN MANAGEMENT
One hour prior to instillation of the test substance, buprenorphine (Buprenodale®, Dechra Ltd., Stoke-on-
Trent, United Kingdom) 0.01 mg/kg was administered by subcutaneous injection in order to provide
a therapeutic level of systemic analgesia.

Five minutes prior to instillation of the test substance, two drops of the topical anesthetic alcaine 0.5%
(SA Alcon-Couvreur NV, Puurs, Belgium) were applied to both eyes.

Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic
analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH,
Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection.

REMOVAL OF TEST SUBSTANCE: None.

SCORING SYSTEM: The irritation was assessed according to the numerical scoring system. At each observation, the highest scores given were recorded.

TOOL USED TO ASSESS SCORE: 2 % fluorescein (Merck, Germany) in water (adjusted to pH 7.0)

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 24 h

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 72 h

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 72 h

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 72 h

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.7

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.7

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 72 h

Irritant / corrosive response data:

- Corneal effects: None were noted during the study
- Iridial effects: No iridial irritation observed during the study.
- Conjunctival effects: Irritation of the conjunctivae, which consicted of redness, chemosis and discharge. The irritation had completely resolved within 72 hours in all animals.

Other effects:

No signs of systemic toxicity observed in the animals during the test period and no mortality occured.

Table 1. Individual eye irritation scores

Animal	Time after dosing (h)	Cornea			Iris	Conjunctivae			Comments
		Opacity (0-4)	Area (0-4)	Fluor area (%)^2	(0-2)	Redness (0-3)	Chemosis (0-4)	Discharge (0-3)
43	1	0	0	0	0	2	2	1	-
	24	0	0		0	2	1	0	-
	48	0	0		0	1	0	0	-
	72	0	0		0	0	0	0	-
2	1	0	0	0	0	2	1	1	-
	24	0	0		0	1	0	0	-
	48	0	0		0	1	0	0	-
	72	0	0		0	0	0	0	-
84	1	0	0	0	0	2	1	1	-
	24	0	0		0	1	0	0	-
	48	0	0		0	1	0	0	-
	72	0	0		0	0	0	0	-

Table 2. Animal specification

Animal	Sex	Age at start (weeks)	Body weights (g)
			Prior to application	At termination
43	female	24	4867	4918
2	female	25	4322	4359
84	female	26	4991	4985

Interpretation of results:

GHS criteria not met

Conclusions:

Dilithium adipate 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 dilithium adipate was assessed in a GLP-compliant, in vivo eye irritation study following OECD guidelines 405 (WIL Research 2015). A single treatment of dilithium adipate was applied to the non-irrigated eye of three rabbits and observations made at 1, 24, 48 and 72 hours for effects on conjunctivae, iris and cornea and for reversibility of effects.

Instillation of the dilithium adipate resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 72 hours in all animals. Based on the results, dilithium adipate does not have to be classified for eye irritation according to GHS and Regulation EC No. 1272/2008.

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 acid chain. 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.  

  

Key in vitro skin irritation studies on dilithium adipate (C6), dilithium azelate (C9) and dilithium sebacate (C10) gave negative results. Since under REACH, in vitro studies are sufficient for compliance with Annexes VII and VIII, no key in vivo study has been conducted. EPISKIN, the 3D-model of human epidermal keratinocytes seeded onto a dermal collagen matrix substitute, has been used to evaluate the in vitro irritation of fatty acid (C6-C10) lithium salts (OECD Guideline 439). Triplicate human epidermal tissues were treated with the test item (12.4-16.7 mg) for a period of 15 minutes and evaluated after 42 hours. The relative mean tissue viability of epidermal keratinocytes exposed to dilithium adipate was 103%, to dilithium azelate was 107% and to dilithium sebacate was 107%, which are above the 50% threshold for skin irritancy. The lithium salts of dicarboxylic acids C6-C10 are considered to be not irritants.

  

Corrosivity has been evaluated in vitro using the human EpiDerm Skin Model (OECD Guideline 431). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts and exposed to the test item, skin corrosion was evaluated on the basis of cell viability. Compared to the negative control tissues, the relative mean tissue viability obtained after 3-minute and 1-hour treatments with dilithium adipate was 93% and 71%, respectively, and comparable results were also observed for dilithium azelate and dilithium sebacate. The substances are considered to be not corrosive.

 

Anin vitrobovine corneal opacity and permeability (BCOP) test according to OECD 437 was conducted and generatedin vitroirritation scores (IVIS) of 4.3, 7.4 and 7.6 for dilithium adipate, dilithium azelate and dilithium sebacate, respectively. Since the IVIS score was > 3 ≤ 55, no prediction on the classification could be made. Dilithium adipate (C6), dilithium azelate (C9) and dilithium sebacate (C10) were then subjected toin vivoeye irritation studies in rabbits according to OECD 405 (key studies). A single treatment of the test item resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge, but the irritation had completely resolved within 72 hours. Based on the results, no classifiable irritation was observed for any substance and the substances do not have to be classified for eye irritation according to GHS and Regulation EC No. 1272/2008.

Justification for classification or non-classification

Skin irritation: Not classified. All studies were negative. 

Eye irritation: Not classified based on available data.