[1,1':3',1''-Terphenyl]-2'-ol, 5',5''''-(2,2,2-trifluoro-1-methylethylidene)bis-

Endpoint summary

Description of key information

- no skin corrosion or irritation potential in vitro (acc. to OECD 431 and OECD 439, respectively)
- no eye corrosion or irritation potential (HET-CAM and EpiOcular(TM) test, respectively)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study according to guideline and GLP

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EpiDerm(TM)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE

Species:

other: not applicable (in vitro test)

Strain:

other: not applicable (in vitro test)

Type of coverage:

other: not applicable (in vitro test)

Preparation of test site:

other: not applicable (in vitro test)

Vehicle:

physiological saline

Controls:

other: not applicable (in vitro test)

Amount / concentration applied:

negative control tissues: 30 ul PBS
positive control tissues: 30 ul 5% SDS
test tissues: 25 ul PBS + 25 ul solid test material

Duration of treatment / exposure:

1 hour (25 min at room temperature + 35 min at 37°C), then tissues were washed with sterile PBS, transferred into fresh medium and dried with a sterile cotton swab

Observation period:

42 hours (2nd medium change at 24 +/- 2 hours after exposure)

Number of animals:

not applicable (in vitro test)
For negative control, positive control and test substance, three tissues each were employed. Additionally, one killed tissue each was used for the test substance and negative control in order to detect direct MTT reduction

Details on study design:

The EpiDerm(TM) Skin Irritation Test is based on the finding that chmical-induced skin irritation is the result of a cascade of events that is initiated by penetration of the chemicals through the stratum corneum where they may damage the underlying layers of keratinocytes and other skin cells. The test method used here measures the initiating event in the cascade, i.e. cell / tissue damage. This is accomplished via the means of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Living cells convert MTT enzymatically into blue formazan salt which is quantitatively measured after extraction from tissues.
EpiDerm(TM) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. Based on the results of a validation study, it was concluded by EVCAM that the EpiDerm(TM) human epidermis model is suitable to be used for distinguishing between irritant and non-irritant chemicals.

To determine whether Ligand TFME-DPP constitutes a skin irritant, EpiDerm tissues were preincubated for 16-22 hours in 0.9 mL medium and subsequently treated with the test substance, the positive control or the negative control. For test substance treatment, 25 ul of PBS were added first. Thereafter, a bulk volume of 25 ul of the solid test material (ca. 12 mg) was applied with a sharp spoon and homogeneously distributed together with the fluid. Control tissues were concurrently applied with 30 ul of sterile PBS (negative control, NC) or with 30 ul of 5 % SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface of the NC and PC controls afterwards.
Following incubation for 25 min at room temperature and 35 min at 37°C, the tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted onto sterile absorbent paper and transferred into new 6-well-plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. The tissues were then incubated at 37°C for 24 +/- 2 hours, subsequently transferred into new 6-well plates pre-filled with 0.9 mL fresh medium and incubated at 37°C for additional 18 +/- 2 hours.
The medium was then replaced by 0.3 mL MTT solution and the tissues were incubated at 37°C for 3 hours. They were then washed with PBS to stop the MTT-incubation. The formazan salt was extracted with isopropanol from the tissues and the optical density was determined spectrophotometrically. The quotient of test tissue absorption divided by negative control absorption was used to calculate tissue viability.

Preliminary findings indicated that the test substance can reduce MTT directly. Therefore, killed control tissues treated as negative control were performed in parallel. However, the result of the killed control did not indicate an increased MTT reduction, therefore the killed control was not used for viability calculation.

The EpiDerm(TM) in vitro skin irritation test showed no reduction in cell viability after treatment with test substance compared to negative control (115% and 100%, respectively). In contrast, mean viability of the positive control was 4%.

		tissue 1	tissue 2	tissue 3	mean	SD
Negative control (NC)	mean OD(570)	2.160	2.206	2.317	2.228
	viability (% of NC)	97.0	99.0	104.0	100	3.62
Test substance	mean OD(570)	2.608	2.645	2.420	2.558
	viability (% of NC)	117.1	118.7	108.7	115	5.4
Positive control	mean OD(570)	0.082	0.081	0.072	0.079
	viability (% of NC)	3.7	3.7	3.2	4	0.25

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP, test method is under validation

Qualifier:

according to guideline

Guideline:

other: OECD (2014a) Draft Proposal for a New Test Guideline: Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Principles of method if other than guideline:

The study method is based on the assumption that chemicals inducing serious eye damage or eye irritation will induce cytotoxicity in the corneal epithelium. 3D models of human corneal epithelium are treated with test substance, followed by a recovery period and measurement of tissue viability. Relative viability (test substance vs. negative control) is used to predict whether a substance requires classification or not.
Study guideline is available at: http://www.oecd.org/chemicalsafety/testing/RhCE-Test-Method-for-Identifying-Chemicals-Not-Requiring%20Classification-Labelling-for-Eye-Irritation-Draft-New-TG-2014-07-25.pdf

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab

Species:

other: not applicable (in vitro)

Strain:

other: not applicable (in vitro)

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

- treated tissues: bulk volume of 50 µl of test material (about 16 mg)
- negative control: 50 µl sterile de-ionized water
- positive control: 50 µl methyl acetate (98+%)

Duration of treatment / exposure:

After application of test substances or controls, the tissues were incubated for 6 hours at 37°C. They were subsequently washed with sterile PBS and immersed in pre-warmed medium to remove residual chemical.

Observation period (in vivo):

After washing, the tissues were returned into the incubator for 18 hours prior to MTT testing.

Number of animals or in vitro replicates:

not applicable (in vitro)

Due to the ability of the test substance to directly reduce MTT in pre-tests, killed controls were applied in parallel. However, the result of the killed control did not indicate an increased MTT reduction, thus it was not used for viability calculation.

The data obtained is conformant with the above-mentioned acceptance criteria.

Findings:

		tissue 1	tissue 2	mean KC	mean	inter-tissue variability (%)
NC	mean OD(570)	1.775	1.652	0.043	1.714
	viability (% of NC)	103.6	96.4		100	7.2
08/0260-2	mean OD(570)	1.832	1.753	0.034	1.793
	viability (% of NC)	106 .9	102.3		105	4.6
PC	mean OD(570)	0.398	0.363		0.381
	viability (% of NC)	23.2	21.2		22	2.0

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irriation / corrosion:

An EpiDerm(TM) Skin Corrosivity Test was performed according to OECD Guideline 431 (BASF 2008). The tissues were treated with the test substance (25 µl bulk volume, corresponding to ca. 8 mg), incubated for 3 minutes or 1 hour with the test chemical and cytotoxicity was measured. The results showed that the test substance was not able to reduce MTT directly. The viability of the tissue was 121% and 107% after 3 minutes and 1 hour, respectively.

An EpiDerm(TM) Skin Irritation Test was performed according to OECD Guideline 439 (BASF 2014). The test substance (25 µl bulk volume, ca. 12 mg) was applied once undiluted onto the tissue and incubated for 1 hour, followed by intensive rinsing of the tissue and a 42-hours post-incubation period. Tissue viability was tested using MTT and the results showed a mean tissue viability of 115% (> 50% = non-irritant).

Eye irritation / corrosion:

A HET-CAM test was performed to determine whether Ligand TFME-DPP was corrosive (BASF 2008). Therefore, 25 µl (ca. 8 mg) of Ligand TFME-DPP were added onto the chorion allantoid membrane (CAM) of fertilized white longbeak eggs under SPF conditions. The CAM was observed for >100 seconds for the presence of hemorrhagia and coagulation. During this period, no hemorrhagia or coagulation could be observed.

An EpiOcular(TM) test was used to assess eye irritation potential of Ligand TFME-DPP (BASF 2014). Reconstructed human Cornea Epithelium was treated once with 50 µl bulk volume (about 16 mg) test substance for 6 hours, followed by a 18 hours post-incubation period and determination of tissue viability with MTT. Mean tissue viability was 105% after test substance incubation and post-exposure incubation (> 60% viability = non-irritant).

Justification for selection of skin irritation / corrosion endpoint:
study according to guideline and GLP

Justification for selection of eye irritation endpoint:
study according to guideline and GLP

Justification for classification or non-classification

Skin irritation / corrosion:

The present data on skin irritation do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a non-classification is warranted.

Eye irritation / corrosion:

The present data on eye irritation do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a non-classification is warranted.