Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1)

Endpoint summary

Description of key information

Skin:

In an in vitro skin irritation study according to OECD Guideline 439 (BASF Colors&Effects, 2017), a cell viability of 109.6 % was determined in reconstructed human epithelial cells.

In a Draize test according to Fed. Reg. 38, No. 187, § 1500.41, the test substance showed no skin irritating properties, when tested as 50 % aequous preperation (BASF SE, 1982).

Eye:

In an in vivo eye irritation study in rabbits according to Fed. Reg. 38, No 187, Paragraph 1500.42 (Draize test) (BASF SE, 1981), the test substance showed no eye irritating properties.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-04-07 to 2016-07-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: 002-141407, Test-substance No.: 16/0046-1

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

OTHER SPECIFICS: Solid / orange, pH ~ 5 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue lot number: 23328
- Date of initiation of testing: 2016-04-12

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature, 37 °C, incubator
- Temperature of post-treatment incubation: 37 °C, incubator

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with sterile PBS to remove residual test substance.
- Observable damage in the tissue due to washing: Due to mechanical damage of tissue 1 during the washing procedure, only two tissues of the negative control could be evaluated. Since the OD570 values of tissue 2 and 3 are well within the historical control data, this deviation is not considered to adversely affect the result of this study.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm, without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
MTT QC assay, 4 hours.
Acceptance criteria OD (540 - 570 nm) [1.0 - 3.0]
Result:1.744 ± 0.201
- Barrier function:
ET-50 assay, 100 µL 1 % Triton X-100. 4 timepoints, n = 3, MTT assay
Acceptance criteria: ET-50 (4.77 - 8.72 h)
Result: 5.81 h
- Contamination: Longterm antibiotic and antimycotic free culture, no contamination observed

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean relative tissue viability with a test substance is less than or equal to 45 %.
- The test substance is considered to be non-irritant to skin if the mean relative tissue viability with a test substance is higher than 55 %.
- The test substance is considered to be borderline, if the mean relative tissue viability with a test substance is lesser than or equal to 55 % and higher then 45 %.

The „borderline"-evaluation (50 ± 5 %) was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is confirming the borderline range provided in OECD Guideline 439.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 µL bulk volume of the solid test substance


NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 %

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

42 ± 4 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

109.6

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Yes, tissue 1
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance identification		tissue 1	tissue 2	tissue 3	mean	SD	CV [%]
NC	mean OD570	*	1.953	2.198	2.075
	viability [% of NC]	*	94.1	105.9	100.0	8.3	8.3
16/0046-1	mean OD570	2.311	2.104	2.412	2.275
	viability [% of NC]	111.3	101.4	116.2	109.6	7.6	6.9
PC	mean OD570	0.087	0.088	0.075	0.084
	viability [% of NC]	4.2	4.3	3.6	4.0	0.3	8.7

 

* Due to mechanical damage of tissue 1 during the washing procedure, only two tissues of the negative control could be evaluated. Since the OD570 values of tissue 2 and 3 are well within the historical control data, this deviation is not considered to adversely affect the result of this study.

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: Fed. Reg. 38, No. 187, § 1500.41

Version / remarks:

1973

Principles of method if other than guideline:

Draize test

GLP compliance:

no

Remarks:

prior to GLP implementation

Species:

rabbit

Strain:

Vienna White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 3.1 - 3.33 kg
- Diet: SSNIFF (INTERMAST)

Type of coverage:

occlusive

Preparation of test site:

other: intact and abraded skin

Vehicle:

water

Controls:

other: untreated skin of the same animal

Amount / concentration applied:

ca. 0.5 g of an 50 % aqueous test substance preparation

Duration of treatment / exposure:

24 hours

Observation period:

8 days

Number of animals:

6

Details on study design:

Application area: 2.5 x 2.5 cm

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 24 h and 72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 24 h and 72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 24 h and 72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #4

Time point:

other: 24 h and 72 h

Score:

0.5

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #5

Time point:

other: 24 h and 72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #6

Time point:

other: 24 h and 72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24 h and 72 h

Max. score:

4

Remarks on result:

not determinable

Remarks:

The test substance coloured the skin

Irritant / corrosive response data:

Due to test substance-coloured skin, determination of the erythema score was not possible (intact and abraded skin).
Slight formation of oedema was observed in one animal (#4) only in the abranded skin area. Oedema were reversible within 8 days.

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

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: Fed. Reg. 38, No. 187, § 1500.42

Version / remarks:

1973-09-27

Principles of method if other than guideline:

Draize test

GLP compliance:

no

Remarks:

prior to GLP implmentation

Species:

rabbit

Strain:

Vienna White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 3.03 - 3.04 kg

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated eye of the same animal

Amount / concentration applied:

11 mg (= 0.1 mL bulk volume)

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

6

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: no

SCORING SYSTEM: according to Draize (1959)

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Irritation parameter:

conjunctivae score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Slght redness of the conjuntiva was observed in four animals, which was reversible within 2 days. One animal showed slight opacity of the cornea after 72 h.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The objective was to assess the skin irritation and corrosion potential of the test substance. Using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential of a substance. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT) according to OECD Guideline 439. However, in the current case, the results derived with SIT alone were sufficient for a final assessment. Therefore the SCT was waived.

The potential of the test substance to cause dermal irritation was assessed by a single topical application of 25 µL (irritation test) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-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 was compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. The following results were obtained in the EpiDerm™ skin irritation test:

After an exposure period of 1 hour with about 42 h post-incubation, the mean viability of the test-substance treated tissueswasdetermined at 109.6 % (values for single tissues: 111.3 %, 101.4 % and 116.2 %). All acceptance criteria were met.

Based on this and applying the evaluation criteria it was concluded, that the test substance did not show a skin irritation potential in the EpiDerm™in vitro skin irritation test. For this reason, corrosion to skin could be excluded and the respective test was waived.

This finding is supported by the results of a Draize test according to Fed. Reg. 38, No. 187, § 1500.41 (BASF SE, 1982). The test substance (0.5 g, 50 % aequous preparation) was applied to the intact skin area of six vienna white rabbits under occlusive conditions for 24 h. Readings were performed after 24 and 72 h followed by an observation period of 8 days. A determination of the erythema score was not possible due to test substance-coloured skin. Mean edema score of all animals was 0.0, except for one animal (edema score 0.5). According to these results, the test substance did not show skin irritating properties.

Eye irritation:

In a Draize study (BASF SE, 1981) of acceptable quality (validation criteria 2), a sample of 11 mg (0.1 mL) test substance was tested in a group of 6 vienna white rabbits. The eyes were not washed after application. Irritation was scored at 24, 48, and 72 h after application. No further observation period was indicated.

A slight conjunctival redness was observed but was reversible within 2 days. No chemosis or iritis was observed. According to the test results, the test substance was not irritating to the eyes.

Following individual irritation mean scores (24, 48, and 72 h) were determined:

Cornea Opacity Score/Iris Score/Conjunctivae Score/Chemosis Score

Animal 1: 0.33/0/0.67/0

Animal 2: 0/0/0/0

Animal 3: 0/0/0.67/0

Animal 4: 0/0/0/0

Animal 5: 0/0/0.67/0

Animal 6: 0/0/0.33/0

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

No Eye and skin irritating properties were documented. As a result the substance should not be classified as eye irritating or skin irritating under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.