Non-graphitic carbon fibre

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity potential of the test item (milled carbonised PAN based fibre) has been evaluated in three in vitro studies (in vitro mammalian cell gene mutation test (OECD 476), bacterial reverse mutation test (OECD 471), and in vitro mammalian chromosome aberration test (OECD 473)). In all three tests, the test substance was negative for any genotoxic potential. Thus, in accordance to the CLP regulation 1272/2008 no classification is warranted.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-09-17 to 2012-12-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: overlay agar (for S. typhimurium strains consisting of 0.6% agar, 0.5% NaCl and 10% 0.5 mM histidine/biotin solution)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not reported
- Periodically checked for karyotype stability: The test strains were checked at regular intervals for their genetic markers, according to the methods by MARON and AMES (1983):
- Amino acid requirement (histidine for S. typhimurium strains)
- rfa mutation where appropriate: crystal violet sensitivity
- uvrB/uvrA mutation: sensibility to ultraviolet (UV) radiation
- Presence of R-factor plasmids where appropriate: ampicilline resistance of strains TA98, TA100
- Periodically "cleansed" against high spontaneous background: no

Additional strain / cell type characteristics:

other: see section "any other information on materials and methods incl. tables"

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

- Type and identity of media: overlay agar (for E.coli strain consisting of 0.6% agar, 0.5% NaCl and 10% 0.25 mM tryptophane solution)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not known
- Periodically checked for karyotype stability:
The test strains were checked at regular intervals for their genetic markers, according to the methods by MARON and AMES (1983):
- amino acid requirement (tryptophan for E.coli WP2 strain)
- uvrB/uvrA mutation: sensibility to ultraviolet (UV) radiation
- Presence of R-factor plasmids where appropriate: ampicilline resistance of E.coli WP2 uvrA (pKM101)
- Periodically "cleansed" against high spontaneous background: no

Additional strain / cell type characteristics:

other: see section "any other information on materials and methods incl. tables"

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (S9 fraction from phenobarbital / ß-naphthoflavone induced rat liver + co-factors)

Test concentrations with justification for top dose:

5.0 μg/plate, 50 μg/plate, 150 μg/plate, 500 μg/plate, 1500 μg/plate, 2500 μg/plate, 5000 μg/plate (with and without S9 mix)

Vehicle / solvent:

DMSO or H20 (for strain specific control experiments) served as negative (vehicle) controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO or H20

True negative controls:

yes

Remarks:

Phosphate buffer (pH 7.4)

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-Amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: 12 hrs
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: not applicable

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: the cytotoxicity of a substance is shown by reduction in the number of revertant colonies and/or inhibition (clearing or diminution) of the background lawn or the degree of survival of treated cultures

Evaluation criteria:

- the study is considered as valid if the background growth of the test organisms in the negative control plates is close and the mean colony counts of the control values of the strains (spontaneous reversion rates of the control plates without S9 mix) are within the historical control data range
- positive reference items should induce a distinct enhancement of revertant rates over the control plates
- slight toxicity or precipitates in the treatment groups will not invalidate the experiments or the doses at which they are found
-a test item is considered to show a positive response in this test system if there is a reproducible demonstration in two independent assays of a
dose-effect relation with a strain dependent increase in the mean number of revertants per plate compared to the vehicle control in at least one
strain with or without metabolic activation
- in the strains TA98, TA100 and WP2uvra the increase should be at least 2-fold, for tester strains TA1535 and TA 1537 this increase should be at least 3-fold
- a test item is considered to show a negative response if no dose-related increase in the mean number of revertant colonies is observed nor a
reproducible positive response at any of the test points in at least two independent experiments
- if there is a clear positive response the test is not repeated
- in case of a negative or equivocal result the test is repeated using different exposure conditions (concentration spacing and/or amount of S9 mix used)

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Table 2a.: Results of the follow-up experiments with different concentrations of the milled PAN based carbon fibre (trade name: Sigrafil C30 M150 UNS) in Salmonella TA 98

Study: 16G12025 Experiment: Follow-Up Experiment Test strain: TA 98
		revertants/plate				revertants/plate
Plate lD	dose/plate	-S9	Mean	± SD	Q	+S9	Mean	± SD	Q
		25				45
Spontaneous rate		41	35	9		46	44	3
		38				41
		24				38
DMSO	100 µL/plate	28	28	5	0,8	48	40	7	0,9
		33				34
		32				51
Sigrafil	50 µg/plate	33	34	3	1	57	49	10	1,1
		37				38
		52				40
Sigrafil	150 µg/plate	30	37	13	1,1	40	40	1	0,9
		30				41
		53				49
Sigrafil	500 µg/plate	39	41	11	1,2	46	41	12	0,9
		32				27
		26				41
Sigrafil	1500 µg/plate	26	26	1	0,7	67	48	17	1,1
		27				36
		35				51
Sigrafil	5000 µg/plate	43	42	6	1,2	26	47	19	1,1
		47				64
		1012
2-NF	5 µg/plate	809	824	180	29,4	*
		652
		56				1864
2-AA	1 µg/plate	56	56	1	2,0	1488	1654	192	41,4
		57				1611

Table 2b: Results of the follow-up experiments with different concentrations of the milled PAN based carbon fibre (trade name: Sigrafil

C30 M150 UNS) in Salmonella TA 100

Study: 16G12025 Experiment: Follow-Up Experiment Test strain: TA 100
		revertants/plate				revertants/plate
Plate lD	dose/plate	-S9	Mean	± SD	Q	+S9	Mean	± SD	Q
		143				129
Spontaneous rate		150	151	9		139	139	11
		160				150
		193				173
DMSO	100 µL/plate	178	182	9	1,2	130	160	26	1,2
		176				176
		151				171
Sigrafil	50 µg/plate	177	158	17	1,0	184	189	21	1,4
		145				212
		185				175
Sigrafil	150 µg/plate	194	185	10	1,2	163	175	12	1,3
		175				186
		189				209
Sigrafil	500 µg/plate	175	189	14	1,3	150	180	30	1,3
		202				182
		176				149
Sigrafil	1500 µg/plate	187	179	7	1,2	156	162	17	1,2
		175				181
		192				175
Sigrafil	5000 µg/plate	173	178	13	1,2	208	194	17	1,4
		168				198
		1249
2-NF	5 µg/plate	1512	1331	157	7,3	*
		1231
		177				2946
2-AA	1 µg/plate	199	196	17	1,1	2883	2876	74	18,0
		211				2799

Table 2c: Results of the follow-up experiments with different concentrations of the milled PAN based carbon fibre (trade name: Sigrafil C30 M150 UNS) in Salmonella TA 1535

Study: 16G12025 Experiment: Follow-Up Experiment Test strain: TA 1535
		revertants/plate				revertants/plate
Plate lD	dose/plate	-S9	Mean	± SD	Q	+S9	Mean	± SD	Q
		12				21
Spontaneous rate		18	18	6		20	19	2
		23				17
		19				15
DMSO	100 µL/plate	15	20	6	1,1	11	15	4	0,8
		26				19
		16				16
H2O	100 µg/plate	12	25	19	1,4	10	12	3	0,6
		46				11
		14				18
Sigrafil	50 µg/plate	20	17	3	0,9	21	16	6	0,8
		17				10
		16				18
Sigrafil	150 µg/plate	15	19	6	1,1	12	20	9	1,1
		25				30
		24				26
Sigrafil	500 µg/plate	14	16	7	0,9	25	24	3	1,3
		10				21
		15				23
Sigrafil	1500 µg/plate	18	17	2	0,9	19	18	6	0,9
		17				11
		14				22
Sigrafil	5000 µg/plate	22	16	5	0,9	22	23	2	1,2
		13				26
		386
NaN3	1 µg/plate	543	463	79	18,5	*
		459
		26				305
2-AA	1 µg/plate	10	15	10	0,8	603	402	174	26,8
		9				298

Table 2d: Results of the follow-up experiments with different concentrations of the milled PAN based carbon fibre (trade name: Sigrafil C30 M150 UNS) in Salmonella TA 1537

Study: 16G12025 Experiment: Follow-Up Experiment Test strain: TA 1537
		revertants/plate				revertants/plate
Plate lD	dose/plate	-S9	Mean	± SD	Q	+S9	Mean	± SD	Q
		10				4
Spontaneous rate		5	8	3		15	9	6
		10				9
		7				13
DMSO	100 µL/plate	9	8	1	1,0	5	9	4	1,0
		7				8
		26				12
Sigrafil	50 µg/plate	12	17	8	2,1	5	11	5	1,2
		13				15
		22				26
Sigrafil	150 µg/plate	12	15	6	1,9	16	19	6	2,1
		12				16
		17				12
Sigrafil	500 µg/plate	11	13	3	1,6	17	12	6	1,3
		12				6
		15				15
Sigrafil	1500 µg/plate	11	12	2	1,5	14	17	4	1,9
		11				21
		29				11
Sigrafil	5000 µg/plate	14	19	8	2,4	11	15	7	1,7
		15				23
		526
9 -Aminoacridine	50 µg/plate	1204	763	383	95,4	*
		558
		22				129
2-AA	1 µg/plate	13	15	7	1,9	131	129	2	14,3
		9				127

Table 2e: Results of the follow-up experiments with different concentrations of the milled PAN based carbon fibre (trade name: Sigrafil C30 M150 UNS) in Ecoli WP2 uvrA pKM101

Study: 16G12025 Experiment: Follow-Up Experiment Test strain: Ecoli WP2 uvrA pKM101
		revertants/plate				revertants/plate
Plate lD	dose/plate	-S9	Mean	± SD	Q	+S9	Mean	± SD	Q
		193				222
Spontaneous rate		172	183	11		252	234	16
		184				227
		162				220
DMSO	100 µL/plate	153	155	6	0,8	226	226	6	1,0
		150				232
		175				251
Sigrafil	50 µg/plate	172	180	12	1,0	251	238	23	1,0
		194				211
		173				239
Sigrafil	150 µg/plate	163	165	7	0,9	254	247	8	1,1
		160				247
		180				285
Sigrafil	500 µg/plate	170	172	8	0,9	239	258	24	1,1
		165				251
		207				257
Sigrafil	1500 µg/plate	160	169	34	0,9	217	238	20	1,0
		140				241
		173				226
Sigrafil	5000 µg/plate	156	166	9	0,9	212	227	15	1,0
		169				242
		3269
MMS	2 µL/plate	3300	3344	104	21,6	*
		3462
		179				942
2-AA	1 µg/plate	143	170	24	1,1	886	925	34	4,1
		188				948

± SD: Standard Deviation

Q: Quotient of mean revertants (test item) / mean revertants (concurrent negative reference item)

-S9: without S9 mix

+S9: with S9 mix

DMSO: dimethyl sulfoxide

H₂O: water

2-NF: 2-nitrofluorene

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

MMS: methyl methanesulfonate

NaN3: sodium azide

* not tested

Conclusions:

The study is regarded as valid because there were no deviations from the test protocols, the study has been performed according to the principles of Good Laboratory Practice, and all validity criteria are fulfilled. Under the test conditions (both with and without metabolic activation) and with the bacterial strains used the test item (milled carbonised PAN based fibre) is considered not to induce a mutagenic effect (induction of gene mutation).

Executive summary:

At a maximum concentration of 5000 μg test substance/plate the test substance (milled carbonised PAN based fibre) did not show any cytotoxic effects in the preliminary experiment. This concentration was therefore chosen as the maximum test substance concentration for the mutagenicity experiments (initial and follow-up experiments). All experiments are regarded as valid. The mean numbers of the revertant colonies of the negative controls were within the acceptable ranges. The mean number of revertants induced by the positive control items was adequately enhanced in all bacterial strains compared to the spontaneous reversion rate. In all experiments the test item (milled carbonised PAN based fibre) did not induce any responses, neither a positive nor ar dose-related response.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-08-08 to 2013-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

type and identity of media: RPMI-1640 medium with 25 mM HEPES, 2 mM L-glutamine and 1 mM pyruvate. Ten percent heat-inactivated horse serum and penicillin/streptomycin (10000 U/10000 μg/mL, 1%) were added directly before use
- Properly maintained: yes
- checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not applicable
- "cleansed" against high spontaneous background: yes

Thymidine kinase (TK) proficient mouse lymphoma L5178Y/TK+/- cells are sensitive to the cytotoxic effects of the pyrimidine analogue trifluorothmidine (TFT), which causes inhibition of cellular metabolism and stops further cell divisions. Cells, deficient in TK due to the forward mutation TK+/- → TK-/- are resistant to TFT. Thus, mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain TK, are not. The enzyme TK is not essential for surviving of mutants as thymidine nucleotides can be synthesized de novo via an alternative pathway.

Additional strain / cell type characteristics:

other: thymidine kinase (TK) proficient mouse lymphoma cells L5178Y/TK+/-

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, homogenate was prepared from 8-10 weeks old male Wistar rats which received triple treatments of 80 mg/kg bw of both phenobarbital intraperitoneally and ß-naphthoflavone orally on consecutive days

Test concentrations with justification for top dose:

-S9 mix: 3 μg/mL, 10 μg/mL, 30 μg/mL, 100 μg/mL, 300 μg/mL, 900 μg/mL, 2700 μg/mL, 5000 μg/mL
+S9 mix: 3 μg/mL, 10 μg/mL, 30 μg/mL, 100 μg/mL, 300 μg/mL, 900 μg/mL, 2700 μg/mL, 5000 μg/mL

Vehicle / solvent:

none

Untreated negative controls:

yes

Remarks:

RPMI-1640 + 5% horse serum

Negative solvent / vehicle controls:

no

True negative controls:

yes

Remarks:

RPMI-1640 + 5% horse serum

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h in the presence of thymidine, hypoxanthine, methotrexate and glycine to reduce the number of spontaneously occuring TK-/- cells
- Exposure duration: 4 h at 37 ± 2°C
- Expression time (cells in growth medium): 2 days for mutant phenotype, 6 days for post-treatment cytotoxicity
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable
SELECTION AGENT (mutation assays): 5-trifluorothymidine
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: not applicable

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency (PE) and relative survival (RS) were calculated

Evaluation criteria:

- quantification of viable colonies of survivor I and II microscopically 6 days after plating
- TFT resistant colonies were evaluated microscopically after 14 days of plating
- viability was also judged by cell counting immediately after treatment and over the two-day expression period
- suspension growth (SG) and relative growth (RTG) were subsequently calculated
- referring to the OECD guideline:
- if the test item is positive/mutagenic colony sizing has to be performed on at least one of the test item concentrations (the highest positive
concentration) and on the negative/vehicle and positive controls using the criteria of small and large colonies
- if the test item is negative colony sizing has only to be done on negative and positive controls
- in this study, colony sizing was performed for all treatment groups
- colonies were counted as "large colonies" if they covered more than 1/4 of the well surface and exhibited a broad-faced growth
- the thickness of large colonies is generally not more than 1 or 2 cells
- colonies were counted as "small colonies" if they covered less than 1/4 of the well surface, exhibited a compact growth, and had a thickness of
more than 2 cells

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: see endpoint "water solubility"
- Precipitation: no
- Other confounding effects: no

RANGE FINDING/SCREENING STUDIES: for dose range finding, cytotoxicity of the test item was determined with and without metabolic activation with the following concentrations: 0 μg/L, 0.5 μg/L, 1.0 μg/L, 3.0 μg/L, 10 μg/L, 30 μg/L, 100 μg/L, 300 μg/L, 900 μg/L, 2700 μg/L, and 5000 μg/L

COMPARISON WITH HISTORICAL CONTROL DATA: All values were within the historical range for negative controls (-S9 mix: 54.0 to 192.1; +S9 mix: 49.7 to 196.4) of Fraunhofer ITEM and within the range proposed by Moore et al. (2003) for negative and vehicle controls (50-200 mutants/106
viable cells)

Table 1: Main tests, 4 h treatment with the test substance without S9 mix: One culture per treatment group and two cultures for the negative control.

17G12022	4 h without S9 mix
Treatment	SG	RTG	RS ( Survivor II ) [%]	MF
Negative control 1	18.33	1.00A	100.0A	83.6
Negative control 2	18.62			62.3
Test item: 3 μg/mL	19.86	1.16	108.1	88.2
Test item: 10 μg/mL	19.50	1.04	98.5	64.9
Test item: 30 μg/mL	18.13	0.97	98.5	83.2
Test item: 100 μg/mL	18.72	0.93	91.4	80.6
Test item: 300 μg/mL	17.19	0.85	91.4	89.6
Test item: 900 μg/mL	17.25	0.92	98.5	73.1
Test item: 2700 μg/mL	18.03	0.85	87.4	89.9
Test item: 5000 μg/mL	19.48	0.79	74.7	81.2
MMS: 10 μg/mL	10.82	0.47	80.2	713.9*

Table 2: Main tests, 4 h treatment with the test substance with S9 mix: One culture per treatment group and two cultures for the negative control.

17G12022	4 h with S9 mix
Treatment	SG	RTG	RS ( Survivor II ) [%]	MF
Negative control 1	13.68	1.00 A	100.0 A	86.9
Negative control 2	14.17			81.0
Test item: 3 μg/mL	13.71	1.05	106.6	93.0
Test item: 10 μg/mL	13.06	1.01	108.2	93.1
Test item: 30 μg/mL	13.48	1.06	109.9	68.9
Test item: 100 μg/mL	11.82	0.92	108.2	80.6
Test item: 300 μg/mL	14.03	1.11	109.9	87.0
Test item: 900 μg/mL	15.94	1.18	103.1	111.0
Test item: 2700 μg/mL	14.39	1.03	100.0	74.1
Test item: 5000 μg/mL	17.89	1.19	92.7	87.0
CP: 2.5 μg/mL	16.51	1.24	104.8	247.9*

SGmean for negative controls: 13.93

SG = Suspension Growth: ( Cell Countsday 1/ Cell Setupday 0) x ( Cell Countsday 2/ Cell Setupday 1)

RTG = Relative Total Growth: ( SGtest item/ SGnegative control) x ( PEtest item/ PEnegative control)

RS = Relative Survival: ( PEtest item/ PEnegative control) x 100

MF = Mutant Frequency (total colonies): ( PEmutant/ PEviable) x 106

PEmutant = Plating Efficiency TFT Selection Plates: -ln (Number of Empty Wells / Number of Total Wells Plated ) / 2000

PEviable = Plating Efficiency Survivor II: -ln ( Number of Empty Wells / Number of Total Wells Plated ) / 1.6

*Relevant increase based on the "Global Evaluation Factor" concept (Moore et al., 2003)

A Means of the two negative controls were set to 100%; mean SG and mean PE (Survivor II) of the two negative controls were used to calculate RTG and RS (Survivor II) values.

Conclusions:

The study is regarded as valid because all validity criteria are fulfilled. The test item (milled carbonised PAN based fibre), both with and without metabolic activation (S9 mix), did not induce a relevant increase in the frequency of trifluorothymidine (TFT) resistant mutants (mutant frequency) in the concentration range and under the test conditions used. Thus, under the restrictions of this assay, the test item did not show evidence of substance-specific induction of gene mutations in mouse lymphoma L5178Y/TK+/- cells and is thus judged to be non-mutagenic in mammalian cells.

Executive summary:

The test item (milled carbonised PAN based fibre) at the limit concentration of 5000 μg/mL did not lead to significant changes in both pH and osmolality as compared to the negative control media. In the main experiments, the test item, both with and without metabolic activation (S9 mix), did not mediate marked acute cytotoxicity up to the limit concentration of 5000 μg/mL as judged by suspension growth (SG) and relative total growth (RTG). In addition, plating efficiency (PE) and relative survival (RS) of survivor II plates did not reflect significant cytotoxicity. RS (Survivor II) was maximally reduced to about 74.7 and 87.4% (without S9 mix, 5000 and 2700 μg/mL) and to 92.9% (with S9 mix, 5000 μg/mL) of the respective negative controls. The negative controls exhibited acceptable mean mutant frequencies (MF) which were within the normal range (50 -200 mutants/106 viable cells; Moore et al., 2003) for L5178Y/TK+/- cells at the TK+/- locus. The positive controls methylmethanesulfonate (MMS, -S9 mix) and cyclophosphamide (CP, +S9 mix) induced marked and relevant increases in MF, indicating satisfactory performance of the test and sufficient activity of the metabolising system. Based on the concept of the "Global Evaluation Factor" (Moore et al., 2003) the test item, both with and without metabolic activation (S9 mix), did not induce relevant increases in MF at the TK+/- locus of L5178Y/TK+/- cells. All values were within the historical range for negative controls (-S9 mix: 54.0 to 192.1; +S9 mix: 49.7 to 196.4) and within the range proposed by Moore et al. (2003) for negative and vehicle controls (50-200 mutants/106 viable cells).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-17 to 2013-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- type and identity of media: Dulbecco's minimum essential medium (D-MEM) with high glucose (4.5 g/L), GlutaMaxTM, sodium pyruvate (110 mg/L) concentration, supplemented with 10% fetal bovine serum (experiments without S9 mix) or D-MEM with 2 instead of 10% fetal bovine serum (experiments with S9 mix)
- properly maintained: yes
- checked for Mycoplasma contamination: yes
- periodically checked for karyotype stability: not reported
- periodically "cleansed" against high spontaneous background: not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, from rat liver induced by phenobarbital / ß-naphthoflavone, co-factors: KCl, MgCl2, NADP, glucose-6-phosphate

Test concentrations with justification for top dose:

4 h without S9 mix: 0 µg/mL, 60 µg/mL, 180 µg/mL, 540 µg/mL
24 h without S9 mix: 0 µg/mL, 60 µg/mL, 180 µg/mL, 540 µg/mL
4 h with S9-mix: 0 µg/mL, 3.5 µg/mL, 10 µg/mL, 30 µg/mL, 90 µg/mL, 270 µg/mL

True negative controls:

yes

Remarks:

growth medium (experiments without S9 mix) and growth medium with 2% instead of 10% FBS (experiments with S9 mix)

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- preincubation period: not applicable
- exposure duration: short term treatment: 3-6 h, continous treatment: 24 h
- expression time (cells in growth medium): not applicable
- selection time (if incubation with a selection agent): not applicable
- fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colcemid 2-3 h before harvesting
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: not applicable

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- method: mitotic index

OTHER EXAMINATIONS:
- determination of polyploidy: an increase in polyploid cells may indicate that the test item has the potential to inhibit mitotic processes and to
induce numerical chromosome aberrations

Evaluation criteria:

100 metaphases from each culture will be selected and evaluated for chromosome aberrations on the following criteria:
- Cells with 22 (or 22 +/- 2) chromosomes
- No centromere splitting
- No extensive overlap of chromosome
- Good chromosome spreading
- Good fixation and acceptable staining
- Only clear aberrations will be classified
- Metaphases showing structural aberrations and less than 20 chromosomes will be documented but not evaluated
- Numerical aberrations will be documented
- Metaphases showing complex structural aberrations and chromosomes not clearly identifiable will be evaluated as aberrant

- The test item will be considered to induce structural chromosome aberrations in cultured mammalian somatic cells (positive result), if the number of
aberrant cells falls within the range of concurrent positive controls or if there is a dose-related increase in the percentages of aberrant cells in
comparison to the concurrent negative controls
- The test item will also be considered to be positive if a reproducible increase in the number of cells with structural chromosome aberrations occurs for at least one test condition
- An increase in polyploid cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations
- The test item is considered not to induce structural chromosome aberrations in cultured mammalian somatic cells (negative result), if there is no
increase at any dose in the percentage of aberrant cells in comparison to concurrent negative/vehicle controls

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1: Mitotic indices (M.I.), main experiments, 4 and 24 h treatment, without S9 mix

Test item:                                                milled carbonised PAN based fibre (trade name: Sigrafil C30 M 150 UNS)

Replicate No.:                                         A and B

Exposure to the test item:                    4 h and 24 h without S9 mix

Fixation time:                                          24 h after treatment start

Evaluated cells:                                     1000 per culture

17G12033	4 hours				24 hours
	A		B		A		B
Treatment	M.I.	%	M.I.	%	M.I.	%	M.I.	%
Negative control	11.0	100	10.7	100	10.1	100	10.3	100
Positive controlEMS: 400µg/mL	-	-	-	-	8.8	87	6.8	66
Positive controlEMS: 600µg/mL	6.3	57	6.7	63	-	-	-	-
Test item: 60µg/mL	8.1	74	9.0	84	8.6	85	8.9	86
Test item: 180µg/mL	7.3	66	7.8	73	8.5	84	6.2	60
Test item: 540µg/mL	3.5	32	4.2	39	4.9	49	3.6	35

M.I.     =   (number of metaphases/number of cells counted) x 100
%        =   percent of negative control

A         =   replicate A

B       =  replicate B
-        =  not determined

Table 2: Mitotic index (M.I.), main experiment, 4 h treatment, with S9 mix

Test item:                                                 milled carbonised PAN based fibre (trade name: Sigrafil C30 M 150 UNS)

Replicate No.:                                         A and B

Exposure to the test item:                  4 h with S9 mix

Fixation time:                                          24 h after treatment start

Evaluated cells:                                      1000 per culture

17G12033	4 hours
	A		B
Treatment	M.I.	%	M.I.	%
Negative control	9.3	100	8.6	100
Positive control CP: 2.5 µg/mL	6.3	68	5.1	59
Test item: 3.5 µg/mL	8.3	89	7.4	86
Test item: 10 µg/mL	7.6	82	6.9	80
Test item: 30 µg/mL	6.0	65	6.7	78
Test item: 90 µg/mL	5.4	58	5.2	60
Test item: 270 µg/mL	3.0	32	3.1	36

M.I.      =   (number of metaphases/number of cells counted) x 100
%         =   percent of control

A          =   replicate A 

B          =   replicate B

Table 3:  In vitro mammalian chromosome aberration test with V79 cells, main experiment, 4 h treatment, without S9 mix

Test item:                                                       milled carbonised PAN based fibre (trade name: Sigrafil C30 M 150 UNS)

Metabolic activation:                                     without S9 mix

Replicate No.:                                              A and B

Exposure to the test item:                       4 h

Fixation time:                                               24 h after treatment start

Number of analysed metaphases:         100 per replicate (A and B), 200 per treatment

Treatment	Rep.	Aberrations							Total number of aberrations		Cells with aberrations [%]
		g	G	b	B	ex	EX	Others
									+gaps	-gaps	+gaps	-gaps
Negative control	A B =	1 1 2	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	1 1 2	0 0 0	1 1 1.0	0 0 0.0
Positive control, 600 µg/mL EMS	A B =	10 8 18	0 1 1	3 2 5	4 5 9	5 3 8	0 0 0	0 2 py 2	22 19 41	12 10 22	18 16 17.0	11 7 9.0
Test item: 60 µg/mL	A B =	2 2 4	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 2py 2	2 2 4	0 0 0	2 2 2.0	0 0 0.0
Test item: 180 µg/mL	A B =	4 3 7	0 0 0	0 0 0	2 1 3	0 0 0	0 0 0	0 1py, 1ma 2	6 5 11	2 2 4	5 5 5.0	1 2 1.5
Test item: 540 µg/mL	A B =	7 2 9	0 1 1	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	7 3 10	0 0 0	7 2 4.5	0 0 0.0

Rep.: Replicate No.; = : Sum of aberrations for replicates A and B; “py”: As numerical aberration, polyploidy was recorded but not included in the total aberration frequency. 

For further tables/results, see section "overall remarks, attachments"

Conclusions:

The study is regarded as valid because there were no deviations from the test protocols, the study has been performed according to the principles of Good Laboratory Practice, and all validity criteria are fulfilled. The aberration frequencies in the treated carbon fibre containing cultures did not exceed 5% as the lower limit for positive control substances and were mostly in the range of the historical negative/vehicle controls. Therefore, the test substance (milled carbonised PAN based fibre) is considered to be negative in cultured mammalian somatic cells under the test conditions used (with and without metabolic activation).

Executive summary:

The test item (milled carbonised PAN based fibre) did not induce marked changes in osmolality and the pH value of the incubation media except for a very slight increase in the pH at a concentration of 1080 µg/mL, but the pH value was still in the physiological range. In the main experiments, the test item demonstrated a concentration-dependent cytotoxicity both with and without S9 mix as determined by the mitotic index (M.I.). Cytotoxicity was slightly more pronounced in the main experiment with S9 mix (4 h of incubation) than in the experiments without S9 mix (4 h of incubation). Negative/vehicle controls demonstrated complete absence of spontaneous structural chromosome aberrations (only 5 chromatid gaps and 1 chromosome gap) and were within the range of historical negative controls. Positive controls (ethyl methanesulfonate (EMS) without S9 mix and cyclophosphamide (CP) with S9 mix) clearly increased the frequency of aberrant cells, with aberration frequencies exceeding 5% as the defined lower limit for positive control substances. Test performance and activity of the metabolising system were thus satisfactory. Irrespective of its cytotoxic potential, the test item, without S9 mix (both after 4 and 24 h of incubation), did not markedly increase the frequency of aberrant cells as compared to the respective negative/vehicle controls. Besides gaps which were also evident in the vehicle control cultures a sum of 7 chromosome breaks, 1 exchange, and 1 ma were observed in the two main experiments (= 6 aberrant metaphases) in a spotted way without clear concentration-dependency. In the main experiment with S9 mix some more structural chromosome aberrations were noted, amounting to 8 chromatid and 7 chromosome breaks, 7 chromatid and 2 chromosome exchanges, and 1 multi-aberrant metaphase within in total 19 aberrant metaphases per 1000 analysed cells. Nevertheless, the resulting aberration frequencies (without gaps) in no case exceeded 5% as defined lower limit for positive control substances and all aberration frequencies fell within the range of the historical negative controls (except for a test item concentration of 270 µg/mL with S9 mix: 2.5%, historical negative control range: 0.2%).

Due to the aberration frequencies in the treated carbon fibre containing cultures which did not exceed 5% as the lower limit for positive control substances and which were mostly in the range of the historical negative/vehicle controls the test item (milled carbonised PAN based fibre) is considered to be negative in cultured mammalian somatic cells under the test conditions used (with and without metabolic activation).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

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. The obtained test results and the resulting classification are therefore regarded to also cover all other qualities of the substance non-graphitic carbon fibre within the scope of this registration.

The genetic toxicity potential of the test item (milled carbonised PAN based fibre) has been evaluated in three in vitro studies (in vitro mammalian cell gene mutation test (OECD 476), bacterial reverse mutation test (OECD 471), and in vitro mammalian chromosome aberration test (OECD 473)):

- In vitro mammalian cell gene mutation test (OECD 476): For the applied concentration range and under the test conditions the test substance, both with and without metabolic activation (S9 mix), did not induce a relevant increase in the frequency of trifluorothymidine (TFT) resistant mutants (mutation frequency). Thus, under the restrictions of this assay, the test substance did not show evidence of a substance-specific induction of gene mutations in mouse lymphoma L5178Y/TK+/- cells and is therefore regarded to be non-mutagenic in mammalian cells.

- Bacterial reverse mutation test (OECD 471): Under the test conditions applied the test substance is considered not to induce a mutagenic effect (induction of gene mutation) in the examined bacterial strains (Salmonella typhimurium TA 98, TA 100, TA 1535, and TA 1537; E. coli WP2 uvr A pKM 101).

- In vitro mammalian chromosome aberration test (OECD 473): The aberration frequencies in the treated carbon fibre containing cultures did not exceed 5% as the lower limit for positive control substances and were mostly in the range of the historical negative/vehicle controls. Therefore, the test substance (milled carbonised PAN based fibre) is considered to be negative in cultured mammalian somatic cells (Chinese hamster lung fibroblasts, V79) under the applied test conditions (with and without metabolic activation).

In conclusion, no adverse effects (negative) were observed in the three studies. All studies are regarded as valid because there were no deviations from the test protocols, the studies have been performed according to the principles of Good Laboratory Practice, and all validity criteria were fulfilled.

Justification for classification or non-classification

The results of all three OECD compliant studies assessing genotoxicity showed a clearly negative outcome. Hence, there is no evidence for any genotoxic potential of the test substance (milled carbonised PAN based fibre). According to the classification criteria within the Regulation (EC) No 1272/2008 (CLP Regulation), the test substance is regarded as not genotoxic.