Registration Dossier

Administrative data

Description of key information

No data is available for the substance reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide. Read across from dilithium azelate and reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide is used to complete the skin irritation and corrosion, eye irritation and corrosion endpoints. 

Two key studies conducted with read across substances, dilithium azelate and reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide are available for the skin irritation endpoint. Both substances are not irritant to skin.

Also, two key studies with read across substances, dilithium azelate and reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide are available for the eye irritation endpoint. Based on the results of these studies, the substances were not classified for eye irritancy.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
other: 92/69 EEC, B.4 OECD 404 OPPTS 870.2500
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
other: NZW Crl:KBL BR
Type of coverage:
semiocclusive
Vehicle:
other: Carboxymethylcellulose (1 % aqua bidest)
Amount / concentration applied:
500 mg
Duration of treatment / exposure:
4 h
Number of animals:
3
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Max. score:
0
Remarks on result:
other: Max. duration: 0 d; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Max. score:
0
Remarks on result:
other: Max. duration: 0 d; Max. value at end of observation period: 0 (related to all animals)
Other effects:
none
Interpretation of results:
other: not classified
Endpoint:
skin irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: 92/69 EEC, B.4 OECD 404 OPPTS 870.2500
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
other: NZW Crl:KBL BR
Type of coverage:
semiocclusive
Vehicle:
other: Carboxymethylcellulose (1 % aqua bidest)
Amount / concentration applied:
500 mg
Duration of treatment / exposure:
4 h
Number of animals:
3
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Max. score:
0
Remarks on result:
other: Max. duration: 0 d; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Max. score:
0
Remarks on result:
other: Max. duration: 0 d; Max. value at end of observation period: 0 (related to all animals)
Other effects:
none
Interpretation of results:
other: not classified
Conclusions:
The test item is not classified as a skin irritant.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose for cross-reference:
read-across: supporting information
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:
other: other: % relative tissue viability
Value:
107
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes . Reversibility: no data. (migrated information)
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:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
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:
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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
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:
other: other: % relative tissue viability
Value:
107
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes . Reversibility: no data. (migrated information)
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:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
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

Referenceopen allclose all

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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
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:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
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:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
other: 92/69 EEC, B.5 OECD 405 OPPTS 870.2400
GLP compliance:
yes
Specific details on test material used for the study:
Nature of substance: powder
Species:
rabbit
Strain:
other: NZW Crl:KBL BR
Vehicle:
not specified
Amount / concentration applied:
100 mg
Number of animals or in vitro replicates:
3
Irritation parameter:
conjunctivae score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
0.33
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
conjunctivae score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
1
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
conjunctivae score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
0.33
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
48 h
Score:
<= 2
Max. score:
2
Reversibility:
not specified
Remarks on result:
other: Max. duration: 48 h; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
0.33
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
0.33
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24 h
Score:
<= 1
Max. score:
1
Reversibility:
not specified
Remarks on result:
other:
Remarks:
Max. duration: 24 h; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 0 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other:
Remarks:
Max. duration: 0 h; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 0 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other:
Remarks:
Max. duration: 0 h; Max. value at end of observation period: 0 (related to all animals)
Other effects:
-The application of the test substance at a dose of 100 mg caused minimal blepharospasm in all animals immediately after the application.
-1h after application a slight erythema of conjunctivae was visible in all animals.
- Slight signs of irritation were visible at the 24 h reading in all of the 3 test animals. In one animal slight erythema was also visible at the 48 hour reading.
-No changes were visible at the 72 h reading in any of the 3 test animals.
-No corneal lesions were found upon fluorescein examination at the 72 h reading.
-The test item produced no corrosion or irreversible effects in any of the animals.
-No toxic effects were observed.
Interpretation of results:
other: not classified
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose for cross-reference:
read-across: supporting information
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:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
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:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: 92/69 EEC, B.5 OECD 405 OPPTS 870.2400
GLP compliance:
yes
Specific details on test material used for the study:
Nature of substance: powder
Species:
rabbit
Strain:
other: NZW Crl:KBL BR
Vehicle:
not specified
Amount / concentration applied:
100 mg
Number of animals or in vitro replicates:
3
Irritation parameter:
conjunctivae score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
0.33
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
conjunctivae score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
1
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
conjunctivae score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
0.33
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
48 h
Score:
<= 2
Max. score:
2
Reversibility:
not specified
Remarks on result:
other: Max. duration: 48 h; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
0.33
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
0.33
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24 h
Score:
<= 1
Max. score:
1
Reversibility:
not specified
Remarks on result:
other:
Remarks:
Max. duration: 24 h; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 0 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other:
Remarks:
Max. duration: 0 h; Max. value at end of observation period: 0 (related to all animals)
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
(mean score 1)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
(mean score 2)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
(mean score 3)
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
other: No further details available.
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 0 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other:
Remarks:
Max. duration: 0 h; Max. value at end of observation period: 0 (related to all animals)
Other effects:
-The application of the test substance at a dose of 100 mg caused minimal blepharospasm in all animals immediately after the application.
-1h after application a slight erythema of conjunctivae was visible in all animals.
- Slight signs of irritation were visible at the 24 h reading in all of the 3 test animals. In one animal slight erythema was also visible at the 48 hour reading.
-No changes were visible at the 72 h reading in any of the 3 test animals.
-No corneal lesions were found upon fluorescein examination at the 72 h reading.
-The test item produced no corrosion or irreversible effects in any of the animals.
-No toxic effects were observed.
Interpretation of results:
other: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

An in vivo skin irritation study in three New Zealand white rabbits under semi-occlusive condition was conducted on the reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide

(BIOSERVICE SCIENTIFIC LABORATORIES, 1999). The study was conducted according to EU guideline (92/69 EEC B.4) and OECD Test Guideline (OECD 404, OPPTS 870.250) and is GLP compliant. Based on the study, the material was not classified for this endpoint.

A reliable in vitro skin irritation study (OECD 439; EU B46) is also available for dilithium azelate (Eurlings 2015) which finds the substance is not irritating.

 

Eye irritation

An in vivo ocular irritation study in three New Zealand white rabbits was conducted on the reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide according

to EU (92/69 EEC, B.4), OECD Test Guideline (OECD 404, OPPTS 870.250) and is GLP compliant. Application of the test substance (100 mg) caused involuntary tight closure of the eyelids (blepharospasm) in all rabbits immediately after application. Slight conjunctival rednessand chemosis was observed, but this was reversible and there was no classification for eye irritancy.

 

Conducted to internationally validated guidelines, an eye irritation study (OECD 405; EU B5; OPPTS 870.2400) is available for dilithium azelate (Latour 2015). A single treatment of dilithium azelate resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. However, the irritation had completely resolved within 72 hours. Based on the results, dilithium azelate is not classified for eye irritation.

 

The studies with reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide have been used as key studies in the NONS dossier for the source substance and have been used to conclude on the hazard assessment and classification of the substance. Therefore, the data are considered to be reliable and adequate for assessment of the hazards of the substance. No Klimisch scores have been assigned by the original registrants and little detail is included in the robust study summary provided for the source substance but the studies are GLP-compliant, guideline studies, and therefore are considered reliable.

 

The studies with dilithium azelate are GLP-compliant, guideline studies, and have been assigned a Klimisch score of 1.

Justification for classification or non-classification

Based on the data with structural analogues, Reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide is not expected to be skin or eye irritating and no classification is required.