Quino[2,3-b]acridine-6,7,13,14(5H,12H)-tetrone

Endpoint summary

Description of key information

No indication of skin irritation was observed in-vitro (OECD 439). No indication of eye irritation were observed in an in-vitro assay (EpiOcular), in an animal study and via the inclusion and exclusion rules of the BfR.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented, according to OECD guideline and under GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Epi-200

Strain:

other: normal, human-derived epidermal keratinocytes , cultured to form a model of the human epidermis, consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum

Details on test animals or test system and environmental conditions:

MATERIAL and EQUIPMENT
EpiDerm TM 200 kit: MatTek ln Vitro Life Science Laboratories, Bratislava, Slovakia containing: 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm2 cultured in Millicells® 0 1 cm
Tissue for MTTreduction control: Epi-200 tissue that is killed by freezing at -20°C
Assay medium: Dulbecco's modified eagle's medium (DMEM); for the assay and for diluting MTT
Wash buffer: Dulbecco's phosphate buffered saline (P8S), w/o Ca2 + , Mg2 +
Extracting agent: lsopropanol p.a.
Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 1.0 mg I ml assay medium

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

other: not applicable

Controls:

other: not applicable

Amount / concentration applied:

25 μL de-ionized water was applied first. Thereafter, a bulk volume of 25 μL of the solid test
material was applied with a sharp spoon and homogeneously distributed with the water

Duration of treatment / exposure:

1h

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

Pretest for direct MTT reduction:
To determine whether the test substance is able to reduce MTT directly, the test substance will be incubated with the substrate. Approximately 50 ul is added to 0.9 ml of the MTT solution. The mixture is incubated in the dark at about 37 oc for 55 to 65 minutes. When the color of the mixture turns blue/purple, it is assumed that the test substance can directly reduce MTT.
Preincubation of the tissues:
Corrosion test: At least 1 hour but not more than 1.5 hours before test-substance application, tissues are transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37C°.
Irritation test: on the day of arrival in the laboratory, the tissues will be transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the pre-incubation medium is replaced with fresh medium and preconditioning continues for 18 ± 3 hours.
MTT incubation:
After the incubation / postincubation period, the assay medium is replaced by 0.3 ml MTT solution and the tissues are incubated in the incubator for 3 hours. After incubation, the tissues are washed with PBS to stop the MTT-incubation.
Detection of MTT metabolism:
The formazan that is metabolically produced by the tissues will be extracted by incubation of the tissues in 2 ml isopropanol at room temperature overnight (corrosion test) or for at least 2 hours on a plate shaker (ca. 120 rpm) (irritation test). After shaking the isopropanol extract and piercing the tissues, 2 aliquots of each extract per tissue will be transferred to a 96-well microtiter plate. The optical density will be determined
spectrophotometrically using a filter with a wavelength of 570 nm

Irritation / corrosion parameter:

other: other: Viability (%)

Value:

94

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1h. Max. score: 100.0. Reversibility: no data. (migrated information)

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

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:

weight of evidence

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Qualitative descripton of effects observed in a single animal. Little information on test item and animals.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

2 animals only, one with flushed eye 30 seconds after treatment. Qualitative description of effects.

GLP compliance:

no

Species:

rabbit

Strain:

other: albino, unspecified

Details on test animals or tissues and environmental conditions:

No data reported

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0,1 ml (17.76 mg)

Duration of treatment / exposure:

single treatment

Observation period (in vivo):

14 days

Number of animals or in vitro replicates:

1 with flushed eye 30 seconds after treatment
1 without flushing

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): flushing with tap water for one of the two animals
- Time after start of exposure: 30 seconds

SCORING SYSTEM: qualitative description

TOOL USED TO ASSESS SCORE: hand-slit lamp (1 and 4h, days 1,2,3,7,14)/ biomicroscope and fluorescein on the day after treatment

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

without flushing

Time point:

other: 24, 48 and 72 h

Remarks on result:

other: no findings on iris observed

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

without flushing

Time point:

other: 1h, 4h, days 1 and 2

Reversibility:

fully reversible within: 72h

Remarks on result:

other: scattered cloudiness

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

no flushing

Time point:

other: 1 and 4h

Reversibility:

fully reversible within: 1 day

Remarks on result:

other: mild redness

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

without flushing

Time point:

other: 1 and 4h

Reversibility:

fully reversible within: 1 day

Remarks on result:

other: slight swelling

Irritation parameter:

other: conjunctivae - discharge

Basis:

animal #1

Remarks:

wihtout flushing

Time point:

other: 1 - 4h, 2- 7 days

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: mild, haemastix positive on day 2 and 3

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

eye flushed

Time point:

other: 4h

Reversibility:

fully reversible within: 1 day

Remarks on result:

other: slightly injected iris

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: all

Remarks on result:

other: no chemosis observed

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

eye flushed

Time point:

other: 1 - 4h

Reversibility:

fully reversible within: 1 day

Remarks on result:

other: slight redness

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

eye flushed

Time point:

other: day 1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: general cloudiness

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Regarding skin irritation, no cytotoxicity was measured in cultived human keratinocytes (OECD 439). This assay has been validated to determine the skin irritation hazard category according to EU GHS and therefore the substance is considered to be non irritating without the need of further in-vivo testing.

The potential of Quino[2,3-b]acridine-6,7,13,14(5H,12H)-tetrone to cause dermal irritation was assessed by a single topical application of 25 μL bulk volume (about 4 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissue samples, 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 testsubstancetreated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The mean viability of the test-substance treated tissues determined after an exposure period

of 1 hour with about 42 hours post-incubation was 94%.

Regarding eye irritation, the in-vivo study in rabbits was performed prior to the introduction of GLP and OECD testing guidelines (BASF 1979). The study is reported in adequate detail and was performed with a test substance of high purity. However, the number of animals was so low that the study on its own does give sufficient information for classification and labelling. There were only 2 treated animals ,and for one, the treated eye was flushed 30 seconds after treatment. The effects were described qualitatively. Observed findings were reversible and mild.

Applying the inclusion and exclusion rules of the BfR (validated in Tsakovska 2005), the substance is predicted as non-irritating. The substance has no features that would include it in the category of irritants. In contrast, it has three excluding features (high molecular weight and melting point, low solubility). The substance was investigated in the EpiOcular in-vitro assay for eye irritation.

As the only OECD guideline for eye irritation available can only distinguish highly irritating substances from irritating substances, another in-vitro study had to be applied. The chosen assay uses a reconstructed three dimensional human cornea model (EpiOcular™). It has been validated at the performing laboratory to distinguish a non irritant from an irritant. Viability of the tissue is determined after a 90 min exposure plus an 18h recovery period. Any substance causing a reduction in viability of more than 50% is considered to be an irritant.

Since treatment of the human cornea cultures with the substance only marginally reduced viability, the substance was found to be non-irritating.

Overall, three independent building blocks support the weight-of-evidence" assessment as "non irritating to eyes".

Justification for selection of skin irritation / corrosion endpoint:
Only sudy available. Valid study

Justification for selection of eye irritation endpoint:
arbitrary, both studies needed for weight-of-evidence

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for skin or eye irritation under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

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. 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 fifth time in Directive EC 944/2013.