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Description of key information

Not irritating on rabbit skin (OECD 404)

Not irritating in rabbit eyes (OECD 405, OECD 437, OECD 492)

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
(no informations if untreated skin served as control).
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
(adopted May 12, 1981)
Deviations:
yes
Remarks:
(no informations if untreated skin was used as control)
GLP compliance:
yes
Specific details on test material used for the study:
commercial grade
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, Fuellinsdorf / Switzerland
- Age at study initiation: 12 - 14 weeks
- Weight at study initiation: 2410-2530 g
- Housing: Individually
- Diet: Rabbit food, NAFAG, No. 814 Tox, NAFAG AG, Gossau, SG (Switzerland), ad libitum
- Water: ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
other: moistened with water before application
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g
Duration of treatment / exposure:
4 h
Observation period:
7 days
Number of animals:
3 female animals
Details on study design:
TEST SITE
- Area of exposure: at least 6 cm²
- Type of wrap if used: The gauze patch with the test substance was applied to the skin and loosely held in place by adhesive but non irritating tape (ISOPLAST AG, Brugg / Switzerland).

REMOVAL OF TEST SUBSTANCE
- Washing: no washing conducted

SCORING SYSTEM: according to OECD 404
Irritation parameter:
erythema score
Basis:
mean
Remarks:
(animal 1 - 3)
Time point:
24/48/72 h
Score:
0.4
Max. score:
4
Reversibility:
fully reversible within: 48 - 72 h
Irritation parameter:
edema score
Basis:
mean
Remarks:
(animal 1 - 3)
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no effects observed
Irritant / corrosive response data:
Due to intensive staining by the test compound scoring after 1 h was not possible. Erythema (score 1) were detected 24 h after treatment in 2 animals. One animal recovered within the next 24 h, whereas the other animal still showed erythema (score 2). This animal recovered 72 h after treatment. No edema were observed.
Other effects:
The body weight development was not affected by the application of the test article.

Table 1: Scoring of erythema and edema formation

Exposition:  4 h, semiocclusive
Animal  Reading  Erythema Edema
1 1 h na 0
2 1 h na 0
3 1 h na 0
1 24 h 1 0
2 24 h 0 0
3 24 h 1 0
1 48 h 0 0
2 48 h 0 0
3 48 h 2 0
1 72 h 0 0
2 72 h 0 0
3 72 h 0 0
1 7 d 0 0
2 7 d 0 0
3 7 d 0 0
1 24/48/72 hrs 0.3 0
2 24/48/72 hrs 0 0
3 24/48/72 hrs 1 0
mean 24 - 72 h 0.44 0.00

na = not assessable due to staining produced by the test item.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present experiment the test substance was found to be non-irritant and not corrosive when applied to the rabbit skin.
Executive summary:

In a primary dermal irritation study (OECD 404, Ciba-Geigy Ltd., 1983), 3 female New Zealand White rabbits were dermally exposed to 0.5 g of the test substance for 4 hours under semiocclusive conditions. Animals then were observed for 7 days. Irritation was scored by the method of Draize as described in the OECD guideline 404. The mean score for the 24, 48 and 72 h reading for erythema was 0.44 and for edema was 0. The skin reactions observed were fully reversible within 48 hours. Under the conditions of the present experiment the test substance was found to be non-irritant and not corrosive when applied to the rabbit skin.

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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
(adopted May 12, 1981)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
commercial grade
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, Fuellinsdorf / Switzerland
- Age at study initiation: 12 - 14 weeks
- Weight at study initiation: 2410 - 2660 g
- Housing: individually
- Diet: Rabbit food, NAFAG, No. 814 Tox, NAFAG AG, Gossau / Switzerland; ad libitum
- Water: ad libitum
- Acclimation period: 5 days (23 June, 1983 - 27 June, 1983)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

Duration of treatment / exposure:
see observation period (no washing conducted)
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
3 males
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: not done

SCORING SYSTEM: according to OECD 405

Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
(animal 1 -3)
Time point:
24/48/72 h
Score:
0.1
Max. score:
4
Reversibility:
fully reversible within: 24 - 48 h
Irritation parameter:
iris score
Basis:
mean
Remarks:
(animal 1 - 3)
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: no effects observed
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
(animal 1 - 3)
Time point:
24/48/72 h
Score:
0.1
Max. score:
3
Reversibility:
fully reversible within: 24 - 48 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
(animal 1 - 3)
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 - 72 h
Other effects:
The body weight development was not affected by the application of the test article. No other reactions to treatment were recorded during the whole observation period.

Table 1: Evaluation of the eye reactions

Animal  Reading  Cornea Opacity Iris Conjunctiva
Redness Chemosis
1 1 h 0 0 0 0
2 1 h 1 0 1 0
3 1 h 1 0 2 1
1 24 hrs 0 0 0 1
2 24 hrs 0 0 0 0
3 24 hrs 1 0 1 1
1 48 hrs 0 0 0 0
2 48 hrs 0 0 0 0
3 48 hrs 0 0 0 1
1 72 hrs 0 0 0 0
2 72 hrs 0 0 0 0
3 72 hrs 0 0 0 0
1 7 days 0 0 0 0
2 7 days 0 0 0 0
3 7 days 0 0 0 0
1 24/48/72 hrs 0 0 0 0.3
2 24/48/72 hrs 0 0 0 0
3 24/48/72 hrs 0.3 0 0.3 0.7
mean animal 1-3 24/48/72 hrs 0.1 0.0 0.1 0.3
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present experiment the test substance was found to be slightly irritant and not corrosive when applied to the rabbit eye mucosa.
Executive summary:

An eye irritation test was performed with three male New Zealand White rabbits acc. to OECD 405 (Ciba-Geigy Ltd., 1983). Eyes were left unwashed after application of 0.1 g of test substance and the animals were observed 7 days. The mean scores for the 24, 48 and 72 h reading for conjunctivae redness, iritis, cornea opacity and conjunctivae chemosis were 0.1, 0, 0.1, and 0, respectively. After 48 hours all reactions were reversed. Under the conditions of the present experiment the test substance was found to be slightly irritant and not corrosive when applied to the rabbit eye mucosa. There was a clear tendency of recovery towards the end of the observation period and no classification according to EU- or EU-GHS-requirements is necessary.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
commercial grade
Puriity>99%
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 µL bulk volume (about 9 mg) of the test substance
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used: Two EpiOcular™ tissue samples were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes. Thereafter the tissues were treated with ca. 50 μL bulk volume (about 9 mg) of 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 (MTI) 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.
- Positive and negative control means and acceptance ranges based on historical data: Yes
Irritation parameter:
other: Mean tissue viability
Run / experiment:
tissue 1
Value:
87
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: for details see table 2 in section "Any other information on results incl. tables"
Irritation parameter:
other: Mean tissue viability [% of NC]
Run / experiment:
tissue 2
Value:
95.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: for details see table 2 in section "Any other information on results incl. tables"

Table 2: Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Test substance

 

Tissue 1

Tissue 2

Mean

Inter-tissue variability [%]

NC

Mean OD570

1.774

1.678

1.726

 

Viability

[% of NC]

102.8

97.2

100.0

5.6

CAS 5280-80-8

Mean OD570

1.502

1.647

1.574

 

Viability

[% of NC]

87.0

95.4

91.2

8.4

PC

Mean OD570

0.379

0.414

0.396

 

Viability

[% of NC]

21.9

24.0

22.9

2.0

NC = Negative Control

PC = Positive Control

OD570 = Optical Density [wavelength 570 nm]

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the observed results and applying the evaluation criteria it was concluded that the test substance does not show an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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:
July 2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
commercial grade
purity > 99%


Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: A bulk volume of about 45 mg of the solid test substance was applied
Duration of treatment / exposure:
4 hours
Number of animals or in vitro replicates:
3
Details on study design:
METHOD:
The test method consists of a topical exposure of the test substance to the epithelial surface of isolated corneas from the eyes of freshly slaughtered cattle. Corneal opacity is measured quantitatively as the amount of light transmission through the cornea. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements are used to calculate an In Vitro lrritancy Score (IVIS) of the test substance, which is used for the prediction of serious eye damage.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes, De-ionized water

POSITIVE CONTROL USED: 20% imidazole in de-ionized water

APPLICATION DOSE AND EXPOSURE TIME: Three corneas were treated with the test substance for an exposure period of 4 hours. Because it was not possible to prepare a homogeneous 20% test substance preparation in water, the test substance was applied undiluted. For covering the whole surface of the cornea, a bulk volume of about 45 mg of the solid test substance was applied.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured quantitatively as the amount of light transmission through the cornea.
- Corneal permeability: Measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Decision criteria as indicated in the OECD guideline were used (see table 1 in section "Any other information on materials and methods incl. tables")
Irritation parameter:
in vitro irritation score
Remarks:
(IVIS, mean)
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Table 2: Opacity score of the test substance, NC and PC

Test substance

Cornea- No.

Initial opacity

Final opacity

Opacity Change

Corrected Opacity Change*

Mean

SD

CAS 5280-80-8 

13

14

15

2.7

2.5

2.2

3.9

1.9

3.3

1.2

-0.6

1.1

0.0

0.0

0.0

0.0

0.0

NC

1

2

3

2.5

2.8

3.7

14.2

5.3

13.9

11.7

2.5

10.2

NA

NA

NA

8.1

4.9

PC

4

5

6

2.8

1.8

2.2

101.2

98.2

80.5

98.5

96.4

78.3

90.4

88.3

70.2

82.9

11.1

*Negative values are set to zero for further calculation

 

Table 3: Permeability score of the test substance, NC and PC

Test substance

Cornea-No.

Mean OD490

Dilution Factor

Mean Corrected OD490

Mean

SD

CAS 5280-80-8 

13

14

15

0.007

0.009

0.007

1

1

1

0.001

0.003

0.001

0.002

0.001

NC

1

2

3

0.004

0.007

0.007

1

1

1

NA

NA

NA

0.006

0.002

PC

4

5

6

1.408

1.086

1.428

1

1

1

1.402

1.080

1.422

1.301

0.192

 

Table 4: In Vitro score (IVIS) of the test substance, NC and PC

Test substance

Cornea-No.

Opacity per cornea

Permeability per cornea

IVIS

Per cornea

Per group

mean

SD

CAS 5280-80-8 

13

14

15

0.0

0.0

0.0

0.001

0.003

0.001

0.0

0.0

0.0

0.0

0.0

NC

1

2

3

11.7

2.5

10.2

0.004

0.007

0.007

11.7

2.6

10.3

8.2

4.9

PC

4

5

6

90.4

88.3

70.2

1.402

1.080

1.422

111.4

104.5

91.5

102.4

10.1

 NA = not applicable; SD = standard derivation; OD490= optical density at 490 nano meter

 

 Table 5: Histopathological findings of the test substance, NC and PC

Test substance

Cornea-No.

Histological evaluation

finding

score

CAS 5280-80-8

13

14

15

no findings

no findings

no findings

0

0

0

NC

1

2

3

no findings

no findings

no findings

0

0

0

PC

4

5

6

Severe

Severe

severe

IV

IV

IV

 

 

 

Interpretation of results:
GHS criteria not met
Conclusions:
Applying the citreria of the OECD 437 for the assessment of the BCOP test results, the IVIS score of the test substance of 0.0 does not support to classify the substance as causing eye irritation or serious eye damage.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin:
Reliable data from two studies on skin irritation are available for the test substance. These data reveal a very low skin irritation potential for the 'yellow disazo condensation pigment'.
In a primary dermal OECD guideline irritation study (OECD 404, Ciba-Geigy Ltd., 1983), 3 female New Zealand White rabbits were dermally exposed to 0.5 g of the test substance (CAS 5280-80-8) for 4 hours under semiocclusive conditions. Animals then were observed for 7 days. Irritation was scored by the method of Draize as described in the OECD guideline 404. The mean score for the 24, 48 and 72 h reading for erythema was 0.44 and for edema was 0. The skin reactions observed were fully reversible within 48 hours. Under the conditions of the present experiment the test substance was found to be non-irritant and not corrosive when applied to the rabbit skin (BASF, 1983).
In a supporting study 3 male and 3 female New Zealand White rabbits were dermally exposed to 0.5 g of the test substance for 24 h under occlusive conditions. After 24 and 72 hours the application sites were assessed for oedema and erythema. Under the conditions of this test, the pigment was also not considered to be irritating to the rabbit skin (Ciba-Geigy Ltd., 1975).
 
Eye:
Reliable data from two in vitro and two in vivo studies on eye irritation are available for the test substance.

For the test substance a Bovine Corneal Opacity and Permeability Test (BCOP Test) according to OECD guideline 437 were conducted (BASF, 2015). Three corneas were treated with the undiluted test substance (bulk volume of about 45 mg) for an exposure period of 4 hours. Based on the observed results (IVIS score = 0.0) it was concluded, that the test substance does not cause ocular corrosion or severe irritation in the Bovine Corneal Opacity and Permeability Test (BCOP Test). However, the Guideline also states, that due to limited accuracy of the BCOP test to correctly identify substances that do not require classification for eye irritation or serious eye damage, this test method should not be the first choice to initiate a bottom-up approach. Thus, the substance was also examined in an EpiOcular to exclude an eye irritation potential.
The conducted EpiOcular™ Eye irritation study with the test substance was performed according to OECD guideline 492 (BASF, 2015). The tissue was treated with 50 µL bulk volume (about 9 g) of the test substance for 6 hours followed by a 18-hours post-incubation period. The determined mean relative tissue viability compared to the negative control is 91.2 %. Since the mean tissue viability was >60 % of the negative control, the test substance does not show any eye irritation potential in the EpiOcular™ eye irritation test.

Further, two in vivo eye irritation test with New Zealand White rabbits according to OECD 405 are available. In one test (Ciba-Geigy Ltd., 1983) the eyes were left unwashed after application of 0.1 g of test substance and the animals were observed 7 days. The mean scores for the 24, 48 and 72 h reading for conjunctivae redness, iritis, cornea opacity and conjunctivae chemosis were 0.1, 0, 0.1, and 0, respectively. After 48 hours all reactions were reversed. In the other test (Ciba-Geigy Ltd., 1975) three animals eyes were left unwashed and three animals the eyes were washed out after 30 seconds after application of 0.1 g of test substance and the animals were observed 72 hours. The mean scores for the 24, 48 and 72 h reading for conjunctivae redness, iritis, cornea opacity and conjunctivae chemosis were 0 at any reading time point. Conjunctival redness was only observed after 1 hour after application.

Based on the results of the in vitro and in vivo tests with the test substance it is concluded that the pigment is not irritating to the eye.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin irritation/corrosion and eye irritation, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.