Non-graphitic carbon fibre

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-18 to 2012-11-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: method by Stern et al.

Version / remarks:

M. Stern, M. Klausner, R. Alvarado, K. Renskers, M. Dickens (1998). Evaluation of the EpiOcular Tissue Model as an Alternative to the Draize Eye Irritation Test. Toxicology In Vitro 12, 455-461.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: protocol of the supplier of the tissue models

Version / remarks:

MatTek Corporation, 200 Homer Avenue, Ashland MA 01721 (2001). Ocular Irritation Protocol: Neat Method (MTT ET-50) For Use with EpiOcular Tissue Model (OCL-200). Rev. 1/1/01, 1-6.

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

carbon

EC Number:

701-026-1

Cas Number:

7440-44-0

Molecular formula:

C up to (C80N5H16O)n

IUPAC Name:

carbon

Test material form:

solid: fibres

Details on test material:

- milled carbonised PAN based fibre (Sigrafil C30 M150 UNS)
- for further details on the test material, see section 1.4, endpoint "Non-graphitic carbon fibre (carbonised PAN based fibre, milled)", attached document "Analytics_milled_carbonised_PAN_based_fibre.pdf"

Test animals / tissue source

Species:

other: corneal model consisting of human-derived epidermal keratinocytes

Strain:

other: not applicable

Test system

Vehicle:

water

Amount / concentration applied:

approximately 200 mg of the test substance in a 1:1 mixture with water

Duration of treatment / exposure:

test item: 3 min, 30 min, 60 min
negative control: 60 min
positive control: 4 min, 15 min, 45 min

Number of animals or in vitro replicates:

not applicable

Details on study design:

see description in "any other information on materials and methods inc. tables"

Results and discussion

In vitro

Results

Irritation parameter:

other: irritation class according to Stern et al.

Remarks:

- irritation class is derived from the ET50 value (effective time of exposure to reduce tissue viability to 50%) of the test item - the ET50 is determined in human-derived EpiOcularTM corneal tissue models via a colorimetric viability test (MTT assay)

Run / experiment:

- the ET50 is calculated from the optical density (OD) as the percentage of viability referred to the negative control (= 100%) - the OD is measured during the MTT viability test (MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

Value:

> 60

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

- the ET50 value of > 60 min corresponds to a classification as non/minimally irritant according to Stern et al.

Any other information on results incl. tables

Table 2: Individual values of the optical density (OD) being corrected about the blank as well as the means and percentages of the tissue viability calculated from these and derived ET₅₀ values.

test substance	exposure time [min]	tissue n	optical density minus Ø Blank = 0,031					viability [% NC]	ET50 [min]
			aliquot 1	aliquot 2	aliquot 3	Ø	Ø tissue
negative control	60	1 2 3	1,688 1,725 1,739	1,642 1,735 1,650	1,708 1,730 1,698	1,680 1,730 1,696	1,702	100
MILLED CARBONISED PAN BASED FIBRE; SIGRAFIL C30 M150 UNS	3   30   60	1 2 1 2 1 2	1,624 1,580 1,596 1,377 1,696 1,650	1,667 1,542 1,609 1,347 1,628 1,584	1,678 1,628 1,648 1,364 1,689 1,614	1,657 1,584 1,618 1,363 1,671 1,616	1,620   1,491   1,644	95   88   97	> 60
positive control	4 15 45	1 1 1	1,799 1,017 0,314	1,791 1,006 0,304	1,788 1,018 0,315	1,793 1,014 0,311		105 60 18	approx. 19

NC = negative control, OD = optical density, Ø = mean

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Because no deviations from the study protocol are reported and the study was performed according to the Principles of Good Laboratory Practice it is regarded as valid. The only slight decrease in tissue viability after exposition to the test item (milled carbonised PAN based fibre) and the determined ET50 value of > 60 minutes indicates at most a minimal irritation potential of the test item. Based on these results the test item shall be categorised as non/minimally irritant.

Executive summary:

The eye irritant potential of the test item (milled carbonised PAN based fibre) was investigated according to the methods by STERN et al. (1998)1 and the protocol2 of the tissue models supplier, the MatTek Corporation. Approximately 200 mg of a 1:1 mixture of the test item and water was applied to human, three-dimensional corneal models EpiOcularTM. The reference items (negative control = water; positive control = 0,3% Triton X-100) were applied in amounts of 100 µL each. After the termination of the 3, 30, and 60-minute exposure period the test item was removed from the tissue surface by rinsing with phosphate buffer saline (DPBS), the tissues were submerged into medium for 10 minutes and then the cell viability was determined using the MTT assay. The exposition periods for the reference items were 4, 14 and 45 minutes for the positive control as well as 60 minutes for the negative control. Based on the obtained exposure time-related viability curve the time of exposure needed for the test item to reduce the viability of the treated tissue to 50% of control tissues (ET50) was determined. Based on the exposure time-related viability curve as result of the MTT assay the ET50 value of the test item was determined to be above 60 minutes. The positive control responded with a decrease of the tissue viability depending on the exposure time and meeting the expectations. The determined ET50 value was approx. 19 minutes and showed a moderate irritant effect of the positive control.

Under the experimental conditions described and based on the obtained results the test item shall be categorised as non/minimally irritant.

References

1) M. Stern, M. Klausner, R. Alvarado. K. Renskers, M. Dickens (1998). Evaluation of the EpiOcular(TM) Tissue Model as an Alternative to the Draize Eye Irritation Test. Toxicology In Vitro 12, 455 -461.

2) MatTek Corporation, 200 Homer Avenue, Ashland MA 01721 (2001). Ocular Irritation Protocol: Neat Method (MTT ET-50) For Use with EpiOcular Tissue Model (OCL-200). Rev. 1/1/01, 1-6.