Frits, chemicals and bismuth oxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Sep - 08 Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Frits, chemicals

EC Number:

266-047-6

EC Name:

Frits, chemicals

Cas Number:

65997-18-4

Molecular formula:

not applicable

IUPAC Name:

Frits, chemicals

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ SIT (Epi-200)

Justification for test system used:

The test was carried out with the reconstituted three-dimensional human skin model EpiDermTM (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multi layered, highly differentiated model of the human epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ SIT (EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue Lot number(s): 25840
- Date of initiation of testing: 31 Aug 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with DPBS (Dulbecco’s Phosphate-Buffered Sallines) to remove any residual test item; excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.504 ± 0.03 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 7.65 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3 each, for the test substance, positive and negative control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTI ON: single experiment.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after 60 minutes exposure and 42 h post-incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 60 minutes exposure and 42 h post-incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg test item + 25 µL DPBS

NEGATIVE CONTROL
- Amount applied: 30 μL DPBS

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

Duration of treatment / exposure:

60 ± 1 min

Duration of post-treatment incubation (if applicable):

42 ± 4 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 ± 1 min

Value:

87.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT (non-specific reduction of MTT) equaled 0%.
- Colour interference with MTT: The mixture of 25 mg of the test item per 300 µl aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC (non-specific colour) equaled 0%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean absolute OD570 of the three negative control tissues was >= 0.8 and <= 2.8.
- Acceptance criteria met for positive control: the mean relative tissue viability (% negative control) of the three positive control tissues was <= 20% (3.3 %).
- Acceptance criteria met for variability between replicate measurements: the standard deviation (SD) of viability of replicate tissues of all dose groups was <= 18% (1.0% - 4.3%).

Any other information on results incl. tables

Table 2: Result of the Test Item Frits

Name		Negative control				Positive control				Test item
Tissue		1	2	3		1	2	3		1	2	3
Absolute OD570		2.378	2.488	2.632		0.147	0.105	0.108		2.183	2.089	2.304
		2.329	2.472	2.491		0.155	0.107	0.110		2.126	2.069	2.231
OD570 (blank-corrected)		2.335	2.446	2.590		0.105	0.062	0.065		2.140	2.046	2.262
		2.286	2.429	2.448		0.112	0.065	0.067		2.083	2.027	2.188
Mean OD570 of the duplicates (blank-corrected)		2.311	2.437	2.519		0.108	0.063	0.066		2.112	2.037	2.225
Total mean OD570 of 3 replicate tissues (blank-corrected)		2.422*				0.079				2.125
SD OD570		0.105				0.025				0.095
relative tissue viability [%]		95.4	100.6	104.0		4.5	2.6	2.7		87.2	84.1	91.9
mean relative tissue viability [%]		100.0				3.3**				87.7
SD tissue viability [%]***		4.3				1.0				3.9
CV [% viabilities]		4.3				31.7				4.5

*      Blank-corrected mean OD570 nm of the negative control corresponds to 100% absolute tissue viability.

**    Mean relative tissue viability of the three positive control tissues is ≤ 20%.

***  Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

In this study under the given conditions the test item showed no irritant effects.