Disodium sebacate

Endpoint summary

Description of key information

The test article was not irritating to the skin based on the results in rabbits. In an in vitro eye irritation battery it was shown that the test item is irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

Exposure: 24 h,

GLP compliance:

no

Remarks:

pre-GLP study

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No. Oc 288

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: WIGA Versuchstierzuchtanstalt, 8741 Sulzfeld, Germany
- Age at study initiation: adult
- Weight at study initiation: 1.5 - 2.0 kg
- Housing: individually in metal cages
- Diet: ad libitum, standard rabbit food - NAFAG, Gossau SG
- Water: ad libitum
- Acclimation period: min. 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±1
- Humidity (%): 55 ±5
- Photoperiod (hrs dark / hrs light): 14/10

Type of coverage:

occlusive

Preparation of test site:

other: shaved and abraded (left) ; shaved (right)

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

0.5 g

Duration of treatment / exposure:

24 h

Observation period:

7 days

Number of animals:

6

Details on study design:

TEST SITE
- Area of exposure: 2.5 x 2.5 cm
- Type of wrap if used: impermeable material, fastend with adhesive tape

OBSERVATION TIME POINTS
24 h, 48 h, 72 h, 4 d, 7 d

SCORING SYSTEM: according to Draize et al. (1959)
Score for skin irritation in rabbits
Erythema and eschar formation
No erythema 0
Very slight erythema (barely perceptible) 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to slight eschar
formation (injuries in depth) 4
Total possible erythema score 4

Edema formation
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well defined by
definite raising) 2
Moderate edema (raised approximately 1 mm) 3
Severe edema (raised more than 1 mm and
extending beyond area of exposure) 4
Total possible ederna score 4

Calculation of the primary skin irritation index:
primary irritation index = [erythema intact skin (24 h + 72 h) + erythema abranded skin (24 h + 72 h) + edema intact skin (24 h + 72 h) + edema abranded skin (24 h + 72 h) ]/4

Assessment of primary skin irritation index:
0 = none
>0 - 1 = minimal
>1 - 2 = slight
>2 - 4 = moderate
>4 - 6 = marked
>6 - 8 = extreme

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 4 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

edema score

Basis:

mean

Remarks:

of all animals. No edema formation were observed

Time point:

24/48/72 h

Score:

0

Max. score:

4

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-06-22 to 2016-08-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test-substance preparation was solved in H2O per weight per volume (w/v) and stirred with a magnetic stirrer shortly before application.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals: age min. 12 months, max. 60 months
- indication of any existing defects or lesions in ocular tissue samples: Only Corneas free of defects were used

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution):
Treatment: 750 µL, 20 % (w/v) test substance in water
Negative control: 750 µL de-ionized water
Positive control: 750 µL, 20 % (w/v) imidazole/de-ionized water

Duration of treatment / exposure:

Corneal opacity: 4 h at 32 °C

Duration of post- treatment incubation (in vitro):

Permeability: replacement of the test substance with 1 mL sodium fluorescin solution (5 mg/mL) and 90 ±5 min. incubation at 32 °C

Number of animals or in vitro replicates:

3 corneas in each group (Treatment, NC, PC)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 548 opacity units were discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Yes, 750 µL de-ionized water

POSITIVE CONTROL USED
Yes, 20% (w/v) solution of imidazole in de-ionized water

POST-INCUBATION PERIOD
no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: corneal opacity readings were taken for each cornea with an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a 96-well microtiter plate reader (OD490)

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS)
Evaluation of the test results based on to the criteria given in OECD 437 (July 2013).

Irritation parameter:

in vitro irritation score

Run / experiment:

BCOP Test

Value:

0.1

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

BCOP Test results:

Test substance identification	Mean Opacity Value	Mean Permeability Value	Mean In Vitro Irritancy Score
Test substance	0.0	0.004	0.1
NC	10.6	0.003	10.6
PC	84.5	2.259	118.4

Interpretation of results:

other: not severely irritating

Conclusions:

Based on the results for BCOP and EpiOcular Test and applying the evaluation criteria described, the test item shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen but is not severely irritating or corrosive.

Executive summary:

The potential of the test tem to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL of a 20% test-substance preparation to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. In addition to the test substance a negative control (NC; de-ionized water) and a positive control

(PC; 20% imidazole in de-ionized water) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.

Based on the results for BCOP and EpiOcular Test and applying the evaluation criteria described, the test item shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen but is not severely irritating or corrosive.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-06-22 to 2016-08-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Species:

human

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
The test substance is applied undiluted. 50 µL, ~11 mg test substance

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2

Details on study design:

- RhCE tissue construct used: EpiOcularTM model (OCL-200), Lot No. 23718
- Doses of test chemical and control substances used: Test substance 50 µL (~11 mg), PC 50 µL methyl acetate, NC 50 µL sterile de-ionized water
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Exposure: 6 h, 37 °C, Post-exposure incubation: 18 h, 37 °C
- Number of tissue replicates used per test chemical and controls: 2 replicates in each group (treatment, NC, PC)
- Description of the method used to quantify MTT formazan: After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
- Acceptance criteria:
Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 (min) value following exposure to 100 μL of 0.3% Triton X-100 for each EpiOcular™ EIT (OCL-200) batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD Guideline.
Lower acceptance limit: ET50 = 12.2 min
Upper acceptance limit: ET50 = 37.5 min
Acceptance criteria for the NC: The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is > 0.8. The mean OD570 of the NC should not exceed 2.5.
Acceptance criteria for the PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
Acceptance criteria for tissue variability: Two tissues were treated under the same conditions. A variability between the the two tissues is considered to be acceptable if the relative difference of the viability is < 20%.- Details of the test procedure used

Irritation parameter:

other: tissue viability [%]

Value:

38.4

Negative controls validity:

valid

Positive controls validity:

valid

		Tissue 1	Tissue 2	Mean	Inter-tissue variability [%]
NC	mean OD 570	1.769	1.867	1.818
	Viability [% of NC]	97.3	102.7	100.0	5.4
Test substance	mean OD 570	0.923	0.474	0.698
	Viability [% of NC]	50.8	26.1	38.4	24.7
PC	mean OD 570	0.380	0.481	0.430
	Viability [% of NC]	20.9	26.5	23.7	5.6

 

The value for inter-tissue variability of the test substance is about 24.7% and therefore out of the acceptance range (see above). Since all other quality criteria of the test were met and the viability values of both tissues are well below the cut off for eye irritation, this deviation is not considered to adversely affect the result of this study.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the results and applying the evaluation criteria the test item shows an eye irritation potential in the in vitro eye irritation test under the test conditions chosen.

Executive summary:

The potential of the test item to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (about 11 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissues were incubated with the test substance for 6 hours followed by an 18-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a

tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 38.4% (single values 50.8% and 26.1%). The value for inter-tissue variability of the test substance treated tissues is about 24.7% and therefore out of the acceptance range. Since all other quality criteria of the test were met and the viability values of both tissues are well below the cut off for eye irritation, this deviation is not considered to adversely affect the result of this study. Based on the results and applying the evaluation criteria the test item shows an eye irritation potential in the in vitro eye irritation test under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

In a primary dermal irritation study, adult Himalayan rabbits (3/sex) were dermally exposed to 0.5 g of disodium sebacate for 24 hours to 6.25 cm² skin. Animals then were observed for 7 days. Irritation was scored according to Draize et al. (1959). Minimal to moderate erythrema below the threshold of classification was observed in two out of six animals, being completely reversible after 4 days. No edema were obeserved in any of the animals. Disodium sebacate was minimally irritating to the skin based on the mean score.

Eye irritation

In an in vivo eye irritation study mild corneal and conjunctival reactions were reported. Since the results where reported in a format not allowing for an assessment according to GHS classification requirements, clarification was sought by performing in vitro studies. A single in vitro assay may not always be sufficient to cover the full range of eye irritating potential. Consequently, two in vitro assays were performed: The Bovine Corneal Opacity and Permeability Test (BCOP Test) according to OECD guideline 437 and the EpiOcular Eye Irritation Test according to OECD guideline 492.

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed in the BCOP assay by a single topical application of 50 μL of a 20 % (w/v) test-substance preparation to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. In addition to the test substance a negative control (NC; de-ionized water) and a positive control (PC; 20 % imidazole in de-ionized water) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. The mean IVIS of the test-substance treated corneas was 0.1, and based on this result, the test item is not regarded as corrosive or severely irritant.

The potential of the test substance to cause ocular irritation was assessed in the EpiOcular test by a single topical application of 50 μL bulk volume (~11 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test substance for 6 hours followed by a 18-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The mean viability of the test-substance treated tissues was 38.4 %.

Summary of the individual test results of the in vitro eye irritation turnkey testing strategy:

Test method	Test result	Test evaluation	Evaluation of the Test strategy
BCOP Test	the mean IVIS of the test substance treated corneas was 0.1	not identified as corrosive or severe irritant	irritant
EpiOcular	the mean viability of the test substance treated tissues was 38.4 %	irritant

Based on the results for BCOP and EpiOcular Test and considering the evaluation criteria, the test substance does show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

Eye and skin irritating properties were documented. As a result the substance should be classified as eye irritating (category 2, H319) under Regulation (EC) No. 1272/2008, as amended for the ninth time in Regulation (EC) No. 2016/1179. Classification for skin irritation is not warranted.