Non-graphitic carbon fibre

Endpoint summary

Description of key information

The skin irritant effect of the test substance was investigated in two tests, in the in vivo skin irritation study conducted according to OECD Guideline 404 and the Council Regulation (EC) No. 440/2008 part B.5 and in the in vitro skin irritating test conducted in accordance with the MatTek protocol EPI-200-SIT, the OECD guideline 439 as well as the draft updated guideline 439.

In both tests, the test substance showed no irritating potential and thus no classification for skin irritation is warranted according to CLP regulation 1272/2008.

To determine the eye irritating potential of the test substance two studies were conducted. In an study conducted in accordance to OECD 405 it was technically not possible in a pre-test to apply the material to the eyes of the animals due to the fibre dusty appearance, which made it impossible to apply a sufficient amount of the test item. In an in vitro eye irritation study (EpiOcular) the test substance showed no irritating effects on the tissues and thus the substance can be considered to be non-irritant to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-09 to 2013-01-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 761/2009, Annex III, B.46

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: reconstructed human epidermal skin model

Details on test animals or test system and environmental conditions:

not applicable

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Amount / concentration applied:

approx. 25 mg of test item per well

Duration of treatment / exposure:

60 +/- 1 min

Observation period:

MTT assay performed 42 hrs (in total) after incubation

Irritation / corrosion parameter:

% tissue viability

Value:

106.4

Negative controls validity:

valid

Positive controls validity:

valid

Table 1: Individual measured values of the optical density (OD) and percentages of the viability derived from these during the MTT assay of the main investigation as well as the additional investigation

Test substance	Tissue n	Measured OD values		Blank-corrected OD		Ø of the aliquots	Viability [%]
		Aliquot 1	Aliquot 2	Aliquot 1	Aliquot 2
Negative control	1	2,612	2,600	2,581	2,569	2,575	97,9
	2	2,642	2,616	2,611	2,585	2,598	98,8
	3	2,700	2,796	2,669	2,765	2,717	103,3
Positive control	1	0,101	0,100	0,070	0,069	0,070	2,6
	2	0,095	0,094	0,064	0,063	0,064	2,4
	3	0,104	0,103	0,073	0,072	0,073	2,8
MILLED CARBONISED PAN BASED FIBRE; SIGRAFIL C30 M150 UNS	1	2,735	2,695	2,704	2,664	2,684	102,1
	2	2,953	2,873	2,922	2,842	2,882	109,6
	3	2,895	2,825	2,864	2,794	2,829	107,6
MILLED CARBONISED PAN BASED FIBRE; SIGRAFIL C30 M150 UNS	1^step2	0,035	0,034	0,004	0,003	0,004
	2^step2	0,033	0,033	0,002	0,002	0,002
	3^step2	0,033	0,033	0,002	0,002	0,002

OD = optical density, Ø = mean,^step2 without MTT and incubated in medium only (step 2)

Table 2: Means of the optical density (OD) and viabilities during the MTT assay of the main investigation as well as the additional investigation (step 2)

Test substance	Ø of the OD	σ of the OD	Ø of the viability [%]	σ of the viability	CV % [%]
Negative control	2,630	0,076	100,0	2,90	2,90
Positive control	0,069	0,005	2,6	0,17	6,69
MILLED CARBONISED PAN BASED FIBRE; SIGRAFIL C30 M150 UNS	2,798	0,103	106,4	3,90	3,66
MILLED CARBONISED PAN BASED FIBRE; SIGRAFIL C30 M150 UNS^step2	0,003	0,001	0,1 % ≈ relative ODTI= 100 % x 0,003/2,630

OD = optical density, Ø = mean, σ = standard deviation, CV = coefficient of variation, ^step2 without MTT and incubated in medium only (step 2), TI = test item

Interpretation of results:

GHS criteria not met

Conclusions:

No deviations from the test protocols are reported and GLP criteria are fullfilled. Therefore, the study and the results are regarded as valid.
Based on the results described and according to the criteria of the Comission Regulation (EC) No.761/2009 the test item (milled carbonised PAN based fibre) shall not be classified into one of the GHS categories.

Executive summary:

The skin irritating potential of the test item (milled carbonised PAN based fibre) was investigated in accordance with the methods of the Commission Regulation (EC) No. 761/2009 Annex III B.46., the MatTek protocol EPI-200-SIT, the OECD guideline 439 as well as the draft updated guideline 439.

An amount of approx. 25 mg of the test item was applied on reconstructed human epidermis EpiDerm moistened with 25 µL DBPS. The reference items [negative control = Dulbecco's phosphate buffered saline (DPBS), positive control = 5 % Sodium Dodecyl Sulfate (SDS)] were applied on non-moistened EpiDerm tissues in an amount of 30 µL. After the termination of the 60 minute exposure time the test substances were removed from the skin surface by washing with DPBS. Subsequently, the treated tissue models were post-incubated in medium for 42 hours. Afterwards, the viability of the treated EpiDerm tissues was determined using the MTT assay (MTT = 3 -(4,5 -dimethylthiazol-2 -yl)-2,5 -diphenyltetrazolium bromide).

A MTT viabilty of 106,4% was determined for the test item.

In an additional investigation, the coloured test item was tested on three more viable EpiDerm tissues which were not incubated in MTT solution but in medium only. Thereby, it was revealed that the test item was alomst completely removable from the tissues after the 60 minute exposure time and had no appreciable influence of the MTT endpoint determination. The relative optical density caused by the coloured test item was approx. 0.1% only and a correction calculation of the MTT viability not necessary.

Based on the results described and according to the criteria of the Comission Regulation (EC) No.761/2009 the test item shall not be classified into one of the GHS categories.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-23 to 2013-01-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

other: Crl:CHBB(HM) russian rabbit

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, D-88353 Kißlegg, Germany
- Age at study initiation: 7 months
- Weight at study initiation: 2.2 to 3.3 kg
- Housing: individually caged in PRO cages (floor area: 2576 cm2) with perforated floor
- Diet (e.g. ad libitum): ad libitum, pelleted complete rabbit diet "Altromin 2123", supplied by Altromin, D-32791 Lage, Germany
- Water (e.g. ad libitum): ad libitum, bottles with domestic qualitiy drinking water which is acidified with hydrochloric acid to pH 2.5 to prevent microbial growth
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 3 °C
- Humidity: 30% - 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped by electric clipper

Vehicle:

other: corn oil

Controls:

no

Amount / concentration applied:

500 mg / 2.5 x 2.5 cm

Duration of treatment / exposure:

Initial test: 3 min, 1 hr, 4 hrs

Observation period:

0, 1 hr, 24 hrs, 48 hrs, 72 hrs
The observation period can be extended up to 14 days to evaluate reversibility of observed effects.

Number of animals:

Initial test: 1 animal
Confirmatory test because no corrosive effects were observed in the initial test: 2 animals

Details on study design:

TEST SITE
- Area of exposure: back, 2.5 cm x 2.5 cm
- Type of wrap if used: 16-layer gauze patch is used as a cover. The patch is secured semi-occlusively with adhesive Gothaplast tape (2.5 cm)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): mild soap and lukewarm water
- Time after start of exposure: 3 min, 60 min, 4 hr

SCORING SYSTEM:
- According to "Grading of Skin Reactions" as found in OECD guidance 404 (adopted 2002)

ASSESSMENT:
- According to Annex 1, Part 3 of Regulation (EC) No 1272/208 (CLP)

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: no clinical signs observed

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: no clinical signs observed

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: no clinical signs observed

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 3 min,1hr

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 3 min, 1 hr

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 3 min, 1 hr

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: no clinical signs observed

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: no clinical signs observed

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: no clinical signs observed

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 3 min, 1hr

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 3 min, 1 hr

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 3 min, 1 hr

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Table 1: Assessment of the formation of erythema and oedema during the first 72 hrs from which the individual means were calculated

Animal No.	Weight in kg	Exposure time	Test field	Observation assessment of the formation of erythema after the termination of exposure					Ø*	Observation assessment of the formation of oedema after the termination of exposure					Ø*
				0 hrs	1 hr	24 hrs	48 hrs	72 hrs		0 hrs	1 hr	24 hrs	48 hrs	72 hrs
#1	3.3	3 min	anterior right	0	0	0	0	0		0	0	0	0	0
		1 h	middle right	0	0	0	0	0		0	0	0	0	0
		4 hrs	posterior right	0	0	0	0	0	0.0	0	0	0	0	0	0.0
#2	2.2	4 hrs	anterior right		0	0	0	0	0.0		0	0	0	0	0.0
#3	2.7	4 hrs	anterior right		0	0	0	0	0.0		0	0	0	0	0.0

Ø = mean value

* only the scores for the 4 hour exposure from the readings after 24 hrs, 48 hrs and 72 hrs are included in the calculation of the individual mean values

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. Based on the study results the test item (milled carbonised PAN based fibre) shall not be classified as skin irritant, category 1 or 2, according to the classification criteria of the Regulation (EC) No 1272/2008 (CLP Regulation).

Executive summary:

The skin irritant effect of the test item (milled carbonised PAN based fibre) was investigated according to the method recommended in the OECD Guideline 404 and the Council Regulation (EC) No. 440/2008, part B.5.

In an initial test with one female albino rabbit a sequential application of the test item (exposure time 3 min, 1 hr and 4 hrs) was carried out on one site of the right back area each. The skin was examined for signs of irritaion immediately as well as 1 hr, 24 hrs and 72 hrs after the termination of exposure. Since the animal did not show signs of skin irritation it was decided to carry out a confirmatory test with two additional animals in order to allow a categorisation of the test item based on the classification criteria.

Two female albino rabbits were exposed to the test item on one skin site of the back during the confirmatory test. After a 4 hour exposure time the test item was removed and the skin was examined 1 hr, 24 hrs, 48 hrs and 72 hrs after the termination of exposure. No signs of skin irritation were observed in these animals.

Under the experimental conditions described the mean scores after the 4 hour exposure were:

Animal No.       Erythema    Oedema

#1                   0.0               0.0

#2                   0.0               0.0   

#3                   0.0               0.0

Based on the results described the test item shall not be classified as skin irritant, category 1 or 2, according to the classification criteria of the Regulation (EC) No 1272/2008 (CLP Regulation).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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

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

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)

Species:

other: corneal model consisting of human-derived epidermal keratinocytes

Strain:

other: not applicable

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"

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test substance (milled carbonised PAN based fibre) falls within the typical range of the substance definition including the impurities (in total < 0.3%) which in principle consist of metal and mixed metal oxides (mainly Al, Ca, Fe, Na, and Si oxides). These substances are of negligible concern regarding human health and the environment.

The different manufacturing processes and precursor materials for non-graphitic carbon fibres within the scope of this registration lead to slightly different compositions. Accordingly, the choice of the test material is based on the substance with the highest content of nitrogen. Therefore, the obtained test results are regarded to also cover all other qualities of the substance non-graphitic carbon fibre within the scope of this registration.

The skin irritant effect of the test substance was investigated in two tests, in the in vivo skin irritation study conducted according to OECD Guideline 404 and the Council Regulation (EC) No. 440/2008 part B.5 and in the in vitro skin irritating test conducted in accordance with the MatTek protocol EPI-200-SIT, the OECD guideline 439 as well as the draft updated guideline 439. In both tests, the test substance showed no irritating potential and thus no classification for skin irritation is warranted according to CLP regulation 1272/2008.

To determine the eye irritating potential of the test substance two studies were conducted. In an study conducted in accordance to OECD 405 it was technically not possible in a pre-test to apply the material to the eyes of the animals due to the fibre dusty appearance, which made it impossible to apply a sufficient amount of the test item. In an in vitro eye irritation study (EpiOcular) the test substance showed no irritating effects on the tissues and thus the substance can be considered to be non-irritant to the eye.

Justification for classification or non-classification

Based on the results of the conducted studies, the test substance showed no potential to induce skin or eye irritation. In accordance with the CLP regulation 1272/2008 no classifcation is warranted.