Azelaic acid, calcium salt (1:1)

Endpoint summary

Description of key information

Data on skin and eye irritation and corrosion have been read across from dilithium azelate to calcium azelate. It is concluded that the substance is not irritating or corrosive to skin or eyes and does not meet the criteria for classification. Data are taken from GLP-compliant study reports following OECD guidelines.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

key study

Study period:

21 April 2015 to 24 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other: GLP-compliant, guideline study, available as an unpublished report.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:

Please see attached read across justification for full details of the read across approach.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EPISKIN-SM Human epidermis model (Lot no: 15-EKIN-016)

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:

- Treatment group: Test carried out in triplicate. 10.5 to 13.0 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 5 µL sterile, distilled water to improve contact between the solid test item and the epidermis.
- Negative control: Triplicate tissues treated with 25 µL Phosphate buffered saline (PBS)
- Positive control: Triplicate tissues. 25 µL Sodium Dodecyl Sulphate (SDS) at 5% w/v aqueous solution spread over entire surface of the epidermis using a pipette tip with the process being repeated after 7 minutes.

Duration of treatment / exposure:

- Treatment period: 15 minutes
- Post-exposure incubation: At the end of the exposure period, tissues were rinsed with PBS to remove any residual test item. The rinsed tissues were dried carefully and transferred to a new well on 2 mL pre-warmed maintenance medium, and incubated for 42 hours, at 37°C and 5 % CO2 in air.

Observation period:

Not applicable.

Number of animals:

Not applicable.

Details on study design:

Preincubation:
The tissues were transferred to 12-well plates and preincubated with pre-warmed Maintaince medium for 26 hours at 37°C, 5 % ± 0.5 Co2 in air.

Application/Treatment of the test substance
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 μL Milli-Q water and the solid test substance (10.5 to 13.0 mg; with a small glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with PBS to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Cell vaibility measurement
Cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol. Tubes were stored at room temperature and protected from light for 4 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes

Value:

107

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 15 ± 0.5 minutes treatment with Dilithium azelate compared to the negative control tissues was 107%. The mean optical density measured at 570 nm was 1.062 with standard deviation of 0.051.
The test item is considered to be non-irritant using the EPISKIN-SM (TM) human epidermis model.
- Positive control: The relative mean tissue viability was 8 % relative to the negative control. The mean optical density was 0.083 and the standard deviation was 0.029.
- Negative control: The mean optical density was 0.992 and the standard deviation was 0.108.

Table 1. Mean absorption (OD570) values and percentage viabilities for the negative control, positive control and test items.

Item	OD570of tisues	Mean OD570 of triplicate tissues	± SD of OD570	Relative mean viability (%)
Negative control item	0.917	0.992	0.108	100
	0.944
	1.116
Positive control item	0.076	0.083	0.029	8
	0.115
	0.058
Dilithium azelate	1.035	1.062	0.051	107
	1.030
	1.120

OD - optical density

SD - standard deviation

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that this test is valid and that Dilithium azelate is non-irritant in the in vitro skin irritation test under the experimental conditions described in the report.

Executive summary:

The purpose of the test was to evaluate the skin irritation potential of the Dilithium azelate using the EPISKIN-SM reconstructed human epidermis model according to the OECD 439 guideline. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Dilithium azelate compared to the negative control tissues was 107%. Since the mean relative tissue viability for Dilithium azelate was above 50% after 15 ± 0.5 minutes treatment Dilithium azelate is considered to be non-irritant.

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 from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

30 April 2015 to 14 May 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:

Please see attached read across justification for full details of the read across approach.

Reason / purpose for cross-reference:

read-across source

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: 23 - 24 weeks old
- Weight at study initiation: 3812 - 4172 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 18.5 mg (range 18.1 - 18.8 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: 72 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:

0.7

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

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 was observed in each of three rabbits at 1 hour after treatment (redness, chemosis and discharge), with minimal irritation at 48 hours (redness). 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)
53	1	0	0	0	0	2	1	1	c
	24	0	0		0	1	0	0	-
	48	0	0		0	1	0	0	-
	72	0	0		0	0	0	0	-
2A	1	0	0	0	0	2	1	1	-
	24	0	0		0	1	0	0	-
	48	0	0		0	1	0	0	-
		0	0		0	0	0	0	-
61	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	-

C – Remnants of the test item on the outside of the eyelids

Table 2. Animal specification

Animal	Sex	Age at start (weeks)	Body weights (g)
			Prior to application	At termination
53	Female	24	4172	4160
2A	Female	23	3864	3903
61	Female	23	3812	3850

Interpretation of results:

GHS criteria not met

Conclusions:

Dilithium azelate 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 azelate was assessed in a GLP-compliant, in vivo eye irritation study following OECD guidelines 405 (WIL Research 2015). A single treatment of dilithium azelate 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 azelate 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 azelate 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

Conclusions for skin and eye irritation and corrosion for calcium azelate are based on data read across from the structural analogue dilithium azelate.

The corrosive potential of dilithium azelate was tested in an in vitro skin corrosion test according to OECD Guideline 431 using a human skin model. Skin corrosion is assessed after exposure to the test substance by topical application for 3 minutes and 1 hour on a human 3D epidermal model (EpiDerm EPI-200). The relative mean tissue viability compared to the negative control tissues was 90% and 93%, respectively so the substance is considered to be not corrosive. The skin irritation potential of dilithium azelate was assessed using the EPISKIN-SM reconstructed human epidermis model according to the OECD 439 guideline. Tissues were exposed for 15 minutes followed by a 42 hour post-exposure incubation period before determination of the cytotoxic (irritancy) effect. The relative mean tissue viability compared to the negative control tissues was 107% so the substance is considered to be non-irritant.

Dilithium azelate was tested for eye corrosion using the Bovine Corneal Opacity and Permeability test (BCOP test) according to OECD Guideline 437. The test item was applied as a 20 % (w/v) suspension in physiological saline (750 μL) directly on top of the corneas for 240 minutes and opacity and permeability were assessed to generate an In Vitro Irritancy Score (IVIS). The test substance has an IVIS of 7.4 and therefore was considered to be a potential ocular irritant as the IVIS was > 3 ≤ 55, no prediction on the classification can be made. The eye irritation of dilithium azelate was assessed in a GLP-compliant, in vivo eye irritation study following OECD guidelines 405. A single treatment 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. The test substance resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge but irritation had completely resolved within 72 hours in all animals so the substance is not classified for eye irritation.

Justification for classification or non-classification

Data on skin and eye irritation and corrosion have been read across from dilithium azelate to calcium azelate. It is concluded that the substance is not irritating or corrosive to skin or eyes and does not meet the criteria for classification.