5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid

Endpoint summary

Description of key information

There are reliable in vivo studies available to assess the potential of the test substance for skin and eye irritation.

skin irritation
key study, in vivo, rabbit: not irritant (GLP, OECD 404, Clariant, 2008)
key study, in vivo, rabbit: not irritant (non-GLP, similar to OECD 404; BASF SE, 1973)
eye irritation
key study, in vivo, rabbit: not irritant (GLP, OECD 405, BASF SE, 2005)
key study, in vivo, rabbit: not irritant (non-GLP, similar to OECD 405; BASF SE, 1973)
key study, in vivo, rabbit: not irritant (GLP, OECD 405; Clariant, 2010)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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:

other: Comparable to to guideline study with acceptable restrictions (incomplete documentation, 20 h occlusive treatment, 2 animals)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

(occlusive treatment, 20 h treatment duration, 2 animals)

Principles of method if other than guideline:

standardized internal test method (BASF test)

GLP compliance:

no

Species:

rabbit

Strain:

Vienna White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Source: Breeder
- Weight at study initiation: females: 3.21 and 3.22 kg

ENVIRONMENTAL CONDITIONS: not reported

Type of coverage:

occlusive

Preparation of test site:

other: clipped

Vehicle:

water

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 50 % aqueous solution

Duration of treatment / exposure:

20 h

Observation period:

7 days

Number of animals:

2

Details on study design:

Since irritation data are recommended for a 4 h skin exposition, results of the available 20 h exposition will serve as approximation.

TEST SITE
- Area of exposure: 2.5 x 2.5 cm (dorsal)

SCORING SYSTEM: Descriptive scores of the raw data have been converted to Draize numerical scores

Irritation parameter:

edema score

Basis:

mean

Remarks:

(2 animals)

Time point:

other: 24 - 48 - 72 h

Score:

0

Max. score:

4

Reversibility:

other: no effects

Irritation parameter:

erythema score

Basis:

mean

Remarks:

(2 animals)

Time point:

other: 24 - 48 - 72 h

Score:

0

Max. score:

4

Reversibility:

other: no effects

Other effects:

orange substance residues (lasting 20 hrs)

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD testing guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV, NL-5961 NM Horst
- Age at study initiation: 13 weeks
- Weight at study initiation: 2292 g – 2451 g
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2008-07-30 To: 2008-08-18

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

not required

Amount / concentration applied:

0.5g

Duration of treatment / exposure:

4h

Observation period:

14 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm ( 10 cm x 10 cm shaved , left flank)
- Type of wrap if used: gauze patch, covered with a semi-occlusive dressing

REMOVAL OF TEST SUBSTANCE
- Washing: lukewarm tap water
- Time after start of exposure: 4h

SCORING SYSTEM: Draize

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24, 48 and 72h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

other: At the 1h reading, yellow staining by the test item interfered with erythema scoring.

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24, 48 and 72h

Score:

0

Max. score:

0

Reversibility:

other: Not applicable

Irritant / corrosive response data:

The test item caused marked yellow staining of the treated skin at the 1-hour reading in all animals which persisted as light yellow staining up to 72 hours after treatment or test day 10. Due to the marked staining the skin of all the rabbits was not assessable at the 1-hour reading. When assessable, from the 24- hour observation except the staining no skin reactions were observed in all animals up to day 14 post treatment, the end of observation time.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

(2002)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Physical state: Solid; ground / dark yellow
- Analytical purity: 92 %
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Centre Lago S.A., 01540 Vonnas, France
- Age at study initiation: about 6 months
- Weight at study initiation: 3.60 kg - 3.95 kg
- Housing: single
- Diet (e.g. ad libitum): Kliba-Labordiaet (Kaninchen & Meerschweinchenhaltung "GLP"), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland (about 130 g per animal per day)
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12h/12h

Check for dead or moribund animals: Twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.

Vehicle:

unchanged (no vehicle)

Controls:

other: Untreated left eye

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL bulk volume (about 26 mg of the comminuted test substance)
- Concentration (if solution): undiluted

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

1, 24, 48, 72 h and 7 days after application

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
The treated eye of the animal(s) was rinsed with 3 to 6 mL of hand warm tap water for 1 to 2 minutes using a syringe with a blunt probe.
- Time after start of exposure:
About 1 hour after application of the solid test substance

SCORING SYSTEM: Draize test

TOOL USED TO ASSESS SCORE:
Daylight tubes "Lumilux" (L 58W/860 PLUS EGO 25X1, Osram, Germany)

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24 - 72 h

Score:

0

Max. score:

4

Reversibility:

other: no effects

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24 - 72 h

Score:

0

Max. score:

2

Reversibility:

other: no effects

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 24 - 72 h

Score:

1.22

Max. score:

3

Reversibility:

fully reversible within: 7 d

Remarks on result:

other: see table for details

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24 - 72 h

Score:

0.22

Max. score:

3

Reversibility:

fully reversible within: 24/48 h

Remarks on result:

other: fully reversible in 1/3 animals within 24 h, in 2/3 animals within 48 h

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: 24, 48 and 72h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

other: 24, 48 and 72h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

other: 24, 48 and 72h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: 24, 48 and 72h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

other: 24, 48 and 72h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

other: 24, 48 and 72h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: 24, 48 and 72h (mean)

Score:

1.3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

other: 24, 48 and 72h (mean)

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

other: 24, 48 and 72h (mean)

Score:

1.3

Max. score:

3

Reversibility:

fully reversible within: 72h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: 24, 48 and 72h (mean)

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: 24, 48 and 72h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 72h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

other: 24, 48 and 72h (mean)

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 72h

Other effects:

Discharge: mean 24 - 72 h: 0.22/max.score 3; reversible in 1/3 animals within 24 h; in 2/3 animals within 48 h.

Animal	Reading	Cornea		Iris	Conjunctiva			Additional findings
		Opacity	Cornea area		Redness	Chemosis	Discharge
1	1 h	0	0	0	2	1	1	Scleral vessels injected, circular
2	1 h	0	0	0	2	1	2	Scleral vessels injected, circular
3	1 h	0	0	0	2	1	2	Scleral vessels injected, circumscribed area
1	24 h	0	0	0	2	0	0	Scleral vessels injected, circumscribed area
2	24 h	0	0	0	2	1	1	Scleral vessels injected, circumscribed area
3	24 h	0	0	0	2	1	1	Scleral vessels injected, circumscribed area
1	48 h	0	0	0	1	0	0	Scleral vessels injected, circumscribed area
2	48 h	0	0	0	1	0	0	Scleral vessels injected, circumscribed area
3	48 h	0	0	0	2	0	0	Scleral vessels injected, circumscribed area
1	72 h	0	0	0	1	0	0	Scleral vessels injected, circumscribed area
2	72 h	0	0	0	0	0	0	Study discontinued because the animal was free of findings
3	72 h	0	0	0	0	0	0	Study discontinued because the animal was free of findings
1	7 d	0	0	0	0	0	0
mean	animal 1 24 - 72 h	0.00		0.00	1.33	0.00
mean	animal 2  24 - 72 h	0.00		0.00	1.00	0.33
mean	animal 3  24 - 72 h	0.00		0.00	1.33	0.33
mean	total 24 - 72 h	0.00		0.00	1.22	0.22

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

There are reliable studies available to assess the skin and eye irritation potential of the Pigment Yellow 139. The studies were performed in 1973 and 2008 when characterization of powders in regard to particles with a size of < 100 nm was not routinely performed. Retrospective analysis of the products indicates that one test material used for eye irritation testing was a nanomaterial wheras for the other materials, no conclusions could be drawn. Therefore, information on the whole range of organic pigments is taken into account.

Organic pigments, which are powders that often have a nanosize fraction, consistently show absence of a hazard for skin and eye irritation (Stratmann, et al. Indicators for lack of systemic availability of organic pigments, https://doi.org/10.1016/j.yrtph.2020.104719, Regulatory Toxicology and Pharmacology Volume 115, August 2020, 104719). The publication lists 106 and 107 pigments tested for skin ande eye irritation, respectively and for of them, the classification critieria as an irritant were not met.

The question on the impact of the particle size on the suitability of the in-vitro studies for eye irritation was adressed in the publication by Kolle et al in 20016 (Eye irritation testing of nanomaterials using the EpiOcular™ eye irritation test and the bovine corneal opacity and permeability assay, DOI 10.1186/s12989-016-0128-6, Particle and Fibre Toxicology (2016) 13:18). None of the dry-powder nanomaterials elicited eye irritation in either the EpiOcular™-EIT or the BCOP assay which was consistent with available in vivo data for the same test materials or for nanosized or bulk materials of the same

composition.

Therefore, the available experimental data is considered adequate to assess the hazard of the pigment both in the bulk and in the nano form.

Skin irritation

A GLP conform in vivo study was performed to assess the acute dermal irritation or corrosion potential of the test substance in New Zealand White rabbits according to OECD guideline 404 (Clariant, 2008). 0.5 g test item was applied by topical semi-occlusive application to the intact left flank of each of three rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours, as well as 7, 10 and 14 days after removal of the dressing.

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. The test item did not elicit any skin reactions at the application site of any animal at any of the observation times (all scores 0).

The test item caused marked yellow staining of the treated skin at the 1-hour reading in all animals which persisted as light yellow staining up to 72 hours after treatment in two animals or test day 10 in the third animal.

Due to the marked yellow staining in all animals 1 hour after treatment, erythema was not assessable at this time point. When assessable, from the 24-hour observation (except the staining) no skin reactions were observed in all animals up to day 14 post treatment (the end of observation time).

 

An Industrial Hygiene Orientating Investigation study was performed to assess the primary skin irritation potential of the test substance similar to OECD guideline 404 with deviations in a Draize test (BASF SE, 1973). The undiluted test substance was applied to the dorsal skin of twoVienna White rabbits for 20 hours under occlusive dressing. No substance related findings were noted at 24 h and 8 days other than orange remainders of the test substance on the skin. The substance was therefore found to be not irritating to the skin of rabbits.

 

Eye irritation

In a GLP conform study the primary eye irritation potential of the test substance (purity: 92 weight-%) was assessed by a single ocular application of 0.1mL bulk volume (about 26 mg) of the test substance to one eye of three White New Zealand rabbits (stepwise procedure starting with one animal and supplementing two additional animals) (BASF SE, 2005). About one hour after application the eye was rinsed with tap water. The ocular reactions were assessed approximately 1, 24, 48 and 72 hours after application and on day 7. Considering the trade name given in the study report, the test material was likely to be a nanomaterial.

Slight or moderate conjunctival redness, slight conjunctival chemosis and slight to moderate discharge were observed in the animals during the course of the study. In addition injected scleral vessels in a circumscribed area or circular were noted in the animals during the observation period.

The ocular reactions were reversible in all animals within 7 days after application at latest. The average score (24 to72 hours) for irritation was calculated to be 0.0 for corneal opacity and for iris, 1.2 for conjunctival redness and 0.2 for chemosis. Mean scores for 24, 48 and 72h per animal for cornea were 1.3, 1.0 and 1.3. Mean scores for 24, 48 and 72h per animal for chemosis were 0, 0.3 and 03.

Considering the described ocular reactions as well as the average score for irritation and applying the CLP criteria, the test item is considered not to be irritating to the eyes.

 

Two additional studies support the results of the study described above.

One is an Industrial Hygiene Orientating Investigation study performed with two Vienna White rabbits in a test protocol similar to OECD guideline 405 with deviations (BASF SE, 1973). A 50 % aqueous suspension of the test substance was instilled into the eyes for one hour. Afterwards the test item was washed out and the eyes were observed for a period of 7 days.

Slight conjunctival redness was observed one hour and 24 hours after the beginning of the study. Slight edema was found only one hour after application. All ocular reactions were fully reversible within 8 days after application.

The second study was conducted according to OECD 405. 0.1 g test item was applied into the left eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours, as well as 7 days after test item instillation.

The mean score was calculated separately for each animal across three scoring times (24, 48 and 72 hours after instillation) for corneal opacity, iris light reflex, redness and chemosis of the conjunctivae, respectively. The individual mean scores for corneal opacity and iris light reflex were 0.0 for all three animals. The individual mean scores for the conjunctivae were 1.0, 0.33 and 0.33 for reddening, and 0.0, 0.0 and 0.0 for chemosis, respectively.

The instillation of the test item into the eye resulted in mild to moderate, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, ocular discharge and chemosis. These effects were reversible and were no longer evident 7 days after treatment, the end of the observation period for all animals. No abnormal findings were observed in the cornea or for the iris light reflex of any animals at any of the examinations. No staining of the treated eyes by the test item and no clinical signs were observed.

Thus, the test item did not induce significant or irreversible damage to the rabbit eye. Based upon the referred classification criteria (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), the test item does not have to be classified with respect to eye irritation in rabbits.

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 1272/2008. A GLP-compliant OECD 404 study is available for skin irritation.

A GLP-compliant OECD 405 study is available for eye irritation.

The scores for the test item treated tissues were below the thresholds for classification as an irritant. As a result, the substance is not considered to be classified for skin or eye irritation under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.