Non-graphitic carbon fibre

Genetic toxicity: in vitro

Administrative data

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

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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

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"

Method

Species / strain

Species / strain 1

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 2

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

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)

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.