Tangled Multi-Walled Carbon Nanotubes

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In standard in vitro assays (Ames test according to OECD TG 471, mouse lymphoma TK locus assay according to OECD TG 476 and chromosome aberration test on human lymphocytes according to OECD TG 473), Graphistrength C100 was not mutagenic nor clastogenic.

 

Gene mutations

The potential of Graphistrength™ C100 to induce gene mutations has been evaluated in bacteria and mammalian cells performed following the OECD TG 471 and 476, respectively.

 

In bacteria

In a key study, the potential of Graphistrength™ C100 (micronized, the mean diameter of the agglomerates was reduced to 30 µm instead 400 µm to increase the bioavailability) to induce reverse mutation was evaluated in Salmonella typhimurium strains (Sire, 2009a). A preliminary toxicity test was performed to define the dose-levels of Graphistrength™ C100 micronized to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item Graphistrength™ C100 micronized was suspended in ethanol. The test item was found not soluble in the vehicles usually used for this type of assay. Consequently, a suspension was selected for the treatments. A homogenous suspension (to the naked eye) was obtained in ethanol at the concentration of 10 mg/mL. Since the test item was not soluble and non-toxic in the preliminary test, the highest dose-level was selected on the basis of the precipitate observed in the Petri plates, according to the criteria specified in the international guidelines. The selected treatment-levels were 15.6, 31.3, 62.5, 125, 250 and 500µg/plate, for both mutagenicity experiments with and without S9 mix. A moderate to marked precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. In the first experiment without S9 mix, a marked toxicity was noted at the dose-level of 500µg/plate in the five strains used. The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Graphistrength™ C100 micronized did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium. 

In mammalian cells

In a key study, the potential of Graphistrength™ C100 (micronized, the mean diameter of the agglomerates was reduced to 30 µm instead 400 µm to increase the bioavailability) to induce mutations at the TK (Thymidine Kinase) locus was evaluated in L5178Y mouse lymphoma cells (Sire, 2009b). After a preliminary toxicity test, Graphistrength™ C100 micronized was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 0.5 x 106 (3-hour treatment) or 0.15 x 106 (24-hour treatment) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. Cytotoxicity was measured by assessment of adjusted relative total growth (Adj. RTG) and relative suspension growth (Adj. RSG) as well as cloning efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was checked after the expression of the mutant phenotype. The test item was suspended in ethanol and the positive controls were methylmethane sulfonate (without S9 mix) and Cyclophosphamide (with S9 mix). In the culture medium, the concentration of 50µg/mL showed a moderate precipitate. At this dose-level, the pH and the osmolality values were comparable to those of the vehicle control culture. The cloning efficiencies CE2 and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. Since the test item was non-toxic but poorly soluble, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines. Using a treatment volume of 100µL/20 mL, the selected concentrations were 0.625, 1.25, 2.5, 5, 10 and 20µg/mL for both experiments, with and without S9 mix. At the end of the treatment periods (3- or 24-hour treatments), a slight to marked precipitate was noted in the culture medium, at concentrations = 0.625µg/mL. Following the 3-hour treatment either with or without S9 mix as well as the 24-hour treatment without S9 mix, no toxicity was induced at any of the tested dose-levels as shown by the absence of any noteworthy decrease in the Adj. RTG. Following the 3-hour treatment either with or without S9 mix as well as the 24-hour treatment without S9 mix, no noteworthy increase in the mutation frequency, in comparison to the vehicle control was noted. Graphistrength™ C100 micronized did not show any mutagenic activity in the mouse lymphoma assay.

 

In the frame of the Nanogenotox program, the potential of Graphistrength™ C100 (named NM 402 in the study report) to induce mutations at the TK (Thymidine Kinase) locus was evaluated in L5178Y mouse lymphoma cells (Norppa et al., 2013). The test was conducted at concentrations of 0, 16, 32, 64 and 128 µg/ml without metabolic activation. No mutagenic activity was observed.

 

In conclusion, Graphistrength™ C100 is not mutagenic in bacteria and mammalian cells.

 

Chromosomal aberration assay

The in vitro clastogenic potential of Graphistrength™ C100 was evaluated in a chromosome aberration test on human lymphocytes following the OECD TG 473, the cytokinesis-block micronucleus assay following the OECD TG 487 on human lymphocytes, A549, BEAS 2B, 16 HBE and Caco-2 cells.

 

In a key study, the potential of the test item Graphistrength™ C100 (micronized, the mean diameter of the agglomerates was reduced to 30 µm instead 400 µm to increase the bioavailability) to induce chromosome aberrations was evaluated in cultured human lymphocytes (Sire, 2009c). The test item was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. Treatment of cells with the test substance in the presence or absence of S9 resulted in similar numbers of aberrations compared to negative control (ethanol), while cell cultures treated with positive control substances resulted in significant elevation of aberrations. In the culture medium, the concentration of 50µg/mL showed a marked precipitate. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture. Since the test item was non-toxic but poorly soluble, the choice of the highest concentration was based on the level of precipitate, according to the criteria specified in the international guidelines. With a treatment volume of 27.5µL/5.5 mL culture medium, the treatment-concentrations were 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50µg/mL, for the first experiment, both with and without S9 mix, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50µg/mL, for the second experiment, both with and without S9 mix. A slight to marked precipitate was observed at the end of the treatment period, generally at concentrations = 12.5µg/mL. Without S9 mix, following the 3-hour treatment, only a slight decrease in mitotic index was noted, without any clear evidence of a concentration relationship (up to 25% decrease). Following the 20-hour treatment, a slight decrease in mitotic index was noted at concentrations = 25µg/mL (26-29% decrease). Following the 44-hour treatment, a slight decrease in mitotic index was noted at 50µg/mL (24% decrease). The concentrations selected for metaphase analysis without S9 were 3.13, 6.25 and 12.5µg/mL for the 3-hour treatment, the latter showing precipitate in the culture medium at the end of the treatment period, 3.13, 6.25 and 12.5µg/mL for the 20-hour treatments, the latter showing precipitate in the culture medium at the end of the treatment period, and 25µg/mL for the 44-hour treatment, this dose-level showing precipitate in the culture medium at the end of the treatment period. No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3-, 20- as well as 44-hour treatments. With S9, no noteworthy decrease in mitotic index was noted at the 20-hour harvest time in the first experiment. At the 20-hour harvest time in the second experiment, slight decreases in mitotic index were noted at concentrations = 12.5µg/mL (26-31% decrease). No noteworthy decrease in mitotic index was noted at the 44-hour harvest time. The concentrations selected for metaphase analysis with S9 were 3.13, 6.25 and 12.5µg/mL for the 20-hour harvest time in both experiments, the latter showing precipitate in the culture medium at the end of the treatment period, 12.5µg/mL for the 44-hour harvest time, this dose-level showing precipitate in the culture medium at the end of the treatment period. No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiments and at either harvest times. The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered valid. Graphistrength™ C100 micronized did not induce chromosome aberrations in cultured human lymphocytes.

 

In the frame of the Nanogenotox program, the potential cyto- and genotoxic effects of Graphistrength™ C100 (named NM 402 in the study report) was investigated in the in vitro micronucleus test (OECD TG 487) in bronchial epithelial BEAS 2B and 16 HBE cells, adenocarcinomic human alveolar basal epithelial A549 cells, epithelial colorectal adenocarcinoma Caco-2 cells and in a primary culture of Human lymphocytes (Tavares et al. 2014; Norppa et al, 2013; Louro et al., 2012).

Dispersions of NM 402 were freshly prepared and cultures were exposed without metabolic activation to concentrations of 32, 64, 128 and 256 µg/ml, 2, 4 and 8 µg/ml, 0.52-52.08 µg/cm², 9.5, 28, 85, 128 and 256 µg/ml and 2.5, 5.0, 15, 45, 125, and 256 µg/ml in BEAS 2B, 16 HBE, A549, Caco-2 cells and human lymphocytes, respectively. The clonogenic assay was used to determine in situ cell survival (8-days exposure) and the cytokinesis-block micronucleus assay was carried out (30-52h-exposure) to evaluate genotoxicity. Concurrent control cultures were also analysed: vehicle control, positive control (mitomycin C, MMC) and reference NM (ZnO-NM110).

In A549 cells (Louro et al., 2016; Norppa et al, 2013; Louro et al., 2012), the results of the clonogenic assay showed that Graphistrength™ C100 induced a concentration-dependent reduction of the cell survival with IC50 of 41.33 µg/cm². The highest concentrations, 26.04 and 52.08 µg/cm², induced a 2-fold significant increase in micronucleated binucleate cells (MNBCs) compared with the vehicle controls (P=0.006 and 0.019, respectively). Regression analysis indicated a concentration-response relationship that was best fitted to a linear-quadratic model (R²= 0.861). The cytokinesis-block proliferation index (CBPI) remained unaltered following A549 cells exposure to Graphistrength™ C100. The positive controls, MMC and ZnO significantly increased MNBCs frequency and concomitantly decreased CBPI.

In human lymphocytes (Tavares et al. 2014; Norppa et al, 2013), a significant increases in MNBC frequencies were detected after exposure to one dose of Graphistrength™ C100 (15 µg/ml, p = 0.015). However, the increase is only 1.5-fold compared to the control, in the range of the historical control and no dose-effect relationship was found by regression analysis.

In BEAS 2B cells (Louro et al., 2016; Norppa et al, 2013), no significant increase in MNBC frequencies was detected after exposure to all doses of Graphistrength™ C100.

In 16 HBE and Caco-2 cells, Graphistrength™ C100 was negative for the induction of MNBC (Norppa et al., 2013).

 

Primary DNA damage assay

The potential of Graphistrength™ C100 to induce primary DNA damage was evaluated in in vitro comet assays performed on human hepatoblastoma C3A, human hepatocellular carcinoma (HepG2), human renal proximal tubule epithelial (HK-2), human bronchial epithelial BEAS 2B and 16 HBE, adenocarcinomic human alveolar basal epithelial A549, epithelial colorectal adenocarcinoma Caco-2 cells and FE1-Muta(TM) Mouse lung epithelial cells.

 

In the frame of the ENPRA program (Kermanizadeh et al., 2012), intracellular levels of glutathione were measured in human hepatoblastoma C3A cell line after the exposure to Graphistrength™ C100 (named NM 402 in the publication) as well as intracellular ROS using the DCFH-DA assay. Antioxidants were used to investigate the role of ROS in the responses observed. The short term genotoxic property was investigated utilising the comet assay. The fpg (formamidopyrimidine [fapy] – DNA glycosylase) modified comet assay was used to measure DNA strand breaks and specific oxidative DNA damage (such as 7, 8-dihydro-8-oxoguanine, 8-oxoadenine, fapy-guanine etc.). C3A cells were exposed to the Graphistrength™ C100 for 4hr at concentrations up to 20 µg/ml. A small but significant increase in percentage tail DNA was observed following exposure to Graphistrength™ C100. Addition of the fpg enzyme to the samples resulted in increased percentage of tail DNA following treatment with Graphistrength™ C100. This indicates that the damage witnessed is partially due to oxidative DNA damage. In conclusion, Graphistrength™ C100 induced a low cytotoxicity, generated intracellular ROS, induced oxidative stress (GSH depletion), and an oxidative mechanism was involved in both the induction of IL8 protein production and genotoxicity according to the Comet assay.

In the frame of the ENPRA program (Kermanizadeh et al., 2013), the toxicological impact of Graphistrength™ C100 (named NM 402 in the publication) was assessed on HK-2 cells - WST-1 cytotoxicity assay, FACS Array, HE oxidation and the FpG(formamidopyrimidine-DNA-glycoslyase) modified Comet assay were used. Exposure of the HK-2 cells to Graphistrength™ C100 resulted in a dose dependant increase in both IL6 and IL8 following a 24 hr exposure. The HE oxidation assay showed no intracellular ROS following exposure. Finally, a short term (4 hr) exposure of the HK-2 cells to Graphistrength™ C100 at sub-lethal concentrations resulted in weak DNA damages.

 

In the frame of the Nanogenotox program (Louro et al., 2016; Norppa et al., 2013), Graphistrength™ C100 (named NM 402) was tested in the comet assay using various human cell lines: pulmonary (bronchial epithelial BEAS 2B and 16 HBE; adenocarcinomic human alveolar basal epithelial A549) and intestinal (epithelial colorectal adenocarcinoma Caco-2). In A549, BEAS 2B and Caco-2cells, Graphistrength™ C100 was also studied using the FpG-modified comet capable of improved detection of oxidative DNA damage. The comet assay was negative for all cell lines with or without FpG.

 

FE1-Muta(TM) Mouse lung epithelial cells were exposed for 24 hr to Graphistrength™C100 (named NM 402 in the publication) dispersed in MilliQ filtered Nanopure water with 2% serum from C57BL/6N mice (Jackson et al., 2015). The levels of DNA strand breaks (SB) were evaluated using the comet assay. Exposure to Graphistrength™C100 (12.5–200 µg/ml) did not induce significant cytotoxicity (viability above 96%). Cell proliferation was reduced to 47% at 200 µg/ml and was associated with generation of reactive oxygen species and high surface area. No increased level of DNA SB was observed.

 

Unaltered levels of DNA strand breaks was observed in human alveolar adenocarcinoma (A549), human hepatocellular carcinoma (HepG2) and human renal proximal tubule epithelial (HK-2) cell lines after 4 h exposure to concentrations up to 40 µg/cm² (128 µg/ml) (Thongkam et al., 2016).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

march 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD 471)

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

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Arochlor1254-induced liver rats

Test concentrations with justification for top dose:

1, 10, 50, 100, 250 and 500 µg/plate for the preliminary toxicity test.
15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for both mutagenicity experiments

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:ethanol
- Justification for choice of solvent/vehicle:
The test item was found not soluble in the vehicles usually used for this type of assay.
Consequently, a suspension was selected for the treatment. An homogenous suspension (to the naked eye) was obtained in ethanol at the concentration of 10 mg/mL.

For positive control : DMSO was used as solvent (except for Mitomycin C which was dissolved in distilled water)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: azide de sodium, 9-Aminoacridine, 2-Nitrofluorène, Mitomycin C, 2-Anthramine, Benzo(a)pyrène

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

DURATION
- Preincubation period: During the preincubation method, test item solution, S9 mix and the bacterial suspension were incubated for 60 minutes at 37°C, under shaking.
- Exposure duration: 48 to 72 hours of incubation at 37°C

NUMBER OF REPLICATIONS: Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level).

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains.

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as
a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

no

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the dose-level of 500 µg/plate in the five strains used without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the dose-level of 500 µg/plate in the five strains used without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the dose-level of 500 µg/plate in the five strains used without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the dose-level of 500 µg/plate in the five strains used without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the dose-level of 500 µg/plate in the five strains used without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

See tables 1, 2.

Table 1: First experiment

              Direct plate incorporation method

Strain	Compound	Dose level per plate (µg)	S9 mix	Mean revertant colony counts	SD	Ration treated / solvent
TA 1535	ETHANOL		-	16	5
	TEST ITEM	15.6	-	12	5	0.8
		31.3	-	21	7	1.3
		62.5	-	21	8	1.4
		125	-	11	3	0.7
		250	-	15	6	1.0
		500	-	0	1	0.0
	NAN3	1	-	610	39	38.9
	ETHANOL		+	19	3
	TEST ITEM	15.6	+	12	2	0.6
		31.3	+	16	3	0.8
		62.5	+	16	2	0.8
		125	+	20	7	1.1
		250	+	16	8	0.8
		500	+	16	6	0.8
	2AM	2	+	286	16	14.8
TA 1537	ETHANOL		-	6	1
	TEST ITEM	15.6	-	9	8	1.6
		31.3	-	9	3	1.4
		62.5	-	12	3	2.1
		125	-	9	3	1.4
		250	-	10	6	1.6
		500	-	0	0	0.0
	9AA	50	-	1168	324	194.7
	ETHANOL		+	8	2
	TEST ITEM	15.6	+	10	4	1.2
		31.3	+	9	5	1.1
		62.5	+	9	4	1.0
		125	+	10	2	1.2
		250	+	8	5	1.0
		500	+	5	5	0.6
	2AM	2	+	144	15	17.3
TA 98	ETHANOL		-	30	4
	TEST ITEM	15.6	-	36	6	1.2
		31.3	-	28	5	0.9
		62.5	-	27	8	0.9
		125	-	31	7	1.0
		250	-	30	1	1.0
		500	-	1	2	0.0
	2NF	0.5	-	248	16	8.3
	ETHANOL		+	35	6
	TEST ITEM	15.6	+	38	4	1.1
		31.3	+	40	11	1.1
		62.5	+	36	2	1.0
		125	+	43	5	1.2
		250	+	40	4	1.1
		500	+	35	7	1.0
	2AM	2	+	1462	112	41.4
TA 100	ETHANOL		-	95	19
	TEST ITEM	15.6	-	125	22	1.3
		31.3	-	120	10	1.3
		62.5	-	112	30	1.2
		125	-	132	14	1.4
		250	-	138	1	1.5
		500	-	36	52	0.4
	NAN3	1	-	744	82	7.8
	ETHANOL		+	106	16
	TEST ITEM	15.6	+	117	8	1.1
		31.3	+	118	22	1.1
		62.5	+	119	8	1.1
		125	+	117	10	1.1
		250	+	122	6	1.2
		500	+	129	15	1.2
	BAP	5	+	957	28	9.0
TA 102	ETHANOL		-	290	52
	TEST ITEM	15.6	-	349	51	1.2
		31.3	-	241	34	0.8
		62.5	-	344	53	1.2
		125	-	345	16	1.2
		250	-	403	53	1.4
		500	-	12	5	0.0
	MMC	0.5	-	2764	159	9.5
	ETHANOL		+	382	41
	TEST ITEM	15.6	+	433	10	1.1
		31.3	+	433	22	1.1
		62.5	+	407	54	1.1
		125	+	327	27	0.9
		250	+	469	41	1.2
		500	+	505	22	1.3
	2AM	10	+	2778	162	7.3

Conclusions:

Interpretation of results (migrated information):
negative

GRAPHISTRENGTH C100 MICRONISED did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

The potential of the test item GRAPHISTRENGTH C100 MICRONISED to induce reverse mutation in Salmonella typhimurium was evaluated. The study was performed according to the international guidelines OECD 471 and Good Laboratory Practices.

A preliminary toxicity test was performed to define the dose-levels of GRAPHISTRENGTH C100 MICRONISED to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item GRAPHISTRENGTH C100 MICRONISED was suspended in ethanol.

The test item was found not soluble in the vehicles usually used for this type of assay. Consequently, a suspension was selected for the treatments. An homogenous suspension (to the naked eye) was obtained in ethanol at the concentration of 10 mg/mL. Since the test item was not soluble and non-toxic in the preliminary test, the highest dose-level was selected on the basis of the precipitate observed in the Petri plates, according to the criteria specified in the international guidelines. The selected treatment-levels were 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate, for both mutagenicity experiments with and without S9 mix.

A moderate to marked precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. In the first experiment without S9 mix, a marked toxicity was noted at the dose-level of 500 µg/plate in the five strains used. The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

GRAPHISTRENGTH C100 MICRONISED did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

december 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD 476)

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

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL).
This medium (RPMI 0) was supplemented by heat inactivated horse serum at 10%, v/v (RPMI 10) or 20%, v/v (RPMI 20).
RPMI 10 was diluted with RPMI 0 (1:1, v/v) in order to obtain RPMI 5 which was used for treatment.

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from a liver microsomal fraction of rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

0.625, 1.25, 2.5, 5, 10 and 20 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

- Justification for choice of solvent/vehicle:
The test item was found not soluble in the vehicles usually used for this type of assay.
Consequently, a suspension was selected for the treatment.
The test item was suspended in the vehicle at concentrations of:
. 10 mg/mL for the preliminary toxicity test,
. 4 mg/mL for both experiments.
The preparations were made immediately before use.

Concerning the positive controls: distilled water was used as solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethane sulfonate (without S9 mix), Cyclophosphamide (with S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in suspension

DURATION
- Exposure duration:
Preliminary toxicity test: A treatment of 3 hours (with and without S9 mix) and 24 hours (without S9 mix) was performed.
Mutagenicity experiments: concerning the first experiment, a treatment of 3 hours (with and without S9 mix) was performed; concerning the second experiment, a treatment of 24 hours was performed without S9 mix and a treatment of 3 hours was led with S9 mix.

- Expression time (cells in growth medium):48 hours
- Selection time (if incubation with a selection agent):11-12 days

SELECTION AGENT (mutation assays):Trifluorothymidine

NUMBER OF REPLICATIONS:
Preliminary test: at least six dose-levels (one culture/concentration) were tested both with and without S9 mix.
Mutagenicity experiments: In two independent experiments, at least four dose-levels of the test item (two cultures/concentration) were tested both with and without metabolic activation.

NUMBER OF CELLS EVALUATED:
-Viability plates:
An average of 1.6 cells/well (two 96-well plates/culture = four plates/dose-level) to define the number of viable cells (CE2).
-Mutant plates:
2000 cells/well (four 96-well plates/culture = eight plates/dose-level) to select the TFTR (trifluorothymidine resistant) mutant cells (for the determination of CEmutant).
The clones were counted, differentiating small and large colonies:
. size of small colonies: < 25% of the diameter of the well,
. size of large colonies: > 25% of the diameter of the well.
For scoring of colonies in mutant plates, the following parameters were considered:
. well containing mutant colony (small or large),
. well not containing mutant colony,
. when both small and large colonies are present in the same well both mutant colonies were counted (one small and one large).

DETERMINATION OF CYTOTOXICITY
- Method:
Cytotoxicity was measured by assessment of adjusted relative total growth (Adj. RTG) and relative suspension growth (Adj. RSG) as well as cloning efficiency following the expression time (CE2).
The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype.

Evaluation criteria:

IWGT recommendations (d) were followed for the determination of a positive result which should fulfill the following criteria:
. at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control equals or exceeds the global evaluation factor (126 x 10-6 for the microtiter method),
. and a dose-related trend is demonstrated by a statistically significant trend test.

Statistics:

no

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH, Effects of osmolality: At 50µg/mL, the pH was approximately 7.6 (as for the vehicle control) and the osmolality was equal to 411 mOsm/kg H2O (402 mOsm/kg H2O for the vehicle control).

- Water solubility:insoluble

- Precipitation:
Preliminary toxicity test: Using a treatment volume of 100 µL/20 mL of culture medium, the final concentration of 50 µg/mL showed a moderate precipitate.
Mutagenicity experiments: At the end of the treatment periods (3- or 24-hour treatments), a slight to marked precipitate was noted in the culture medium, at concentrations = 0.625 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
The dose-levels used for treatment (cytotoxicity study) were 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL.
At the end of the treatment periods (3- or 24-hour treatments), a slight to marked precipitate was noted in the culture medium, generally at dose-levels = 1.56 µg/mL.
Following the 3-hour and the 24-hour treatments without S9 mix as well as the 3-hour treatment with S9 mix, no toxicity was induced at any of the dose-levels tested as shown by the absence of any noteworthy decrease in the adjusted relative total growth (Adj. RTG).

COMPARISON WITH HISTORICAL CONTROL DATA:yes

Conclusions:

Interpretation of results (migrated information):
negative

GRAPHISTRENGTH C100 micronised did not show any mutagenic activity in the mouse lymphoma assay.

Executive summary:

The potential of the test item GRAPHISTRENGTH C100 micronised to induce mutations at the TK (Thymidine Kinase) locus in L5178Y mouse lymphoma cells was evaluated. The study was performed according to the international guidelines OECD No. 476 and Good Laboratory Practice.

After a preliminary toxicity test, GRAPHISTRENGTH C100 micronised was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 0.5 x 106 (3-hour treatment) or 0.15 x 106 (24-hour treatment) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. Cytotoxicity was measured by assessment of adjusted relative total growth (Adj. RTG) and relative suspension growth (Adj. RSG) as well as cloning efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype.

The test item was suspended in ethanol and the positive controls were methylmethane sulfonate (without S9 mix) and Cyclophosphamide (with S9 mix). In the culture medium, the concentration of 50 µg/mL showed a moderate precipitate. At this dose-level, the pH and the osmolality values were comparable to those of the vehicle control culture.

The cloning efficiencies CE2 and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. Since the test item was non-toxic but poorly soluble, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines. Using a treatment volume of 100 µL/20 mL, the selected concentrations were 0.625, 1.25, 2.5, 5, 10 and 20 µg/mL for both experiments, with and without S9 mix. At the end of the treatment periods (3- or 24-hour treatments), a slight to marked precipitate was noted in the culture medium, at concentrations = 0.625 µg/mL.

Cytotoxicity: Following the 3-hour treatment either with or without S9 mix as well as the 24-hour treatment without S9 mix, no toxicity was induced at any of the tested dose-levels as shown by the absence of any noteworthy decrease in the Adj. RTG. Mutagenicity: Following the 3-hour treatment either with or without S9 mix as well as the 24-hour treatment without S9 mix, no noteworthy increase in the mutation frequency, in comparison to the vehicle control was noted.

GRAPHISTRENGTH C100 micronised did not show any mutagenic activity in the mouse lymphoma assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

april 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD 473)

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:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media:0.5 mL of heparinized whole blood was added to 5 mL of RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL).

phytohemagglutinin: a mitogen to stimulate lymphocytes division

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from arochlor 1254 induced-rats

Test concentrations with justification for top dose:

· 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL (1st experiment)
· 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL (2nd experiment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:The test item was found not soluble in the vehicles usually used for this type of assay.
Consequently, a suspension was selected for the treatment. An homogenous suspension (to the naked eye) was obtained in ethanol at the concentrati on of 10 mg/mL.
Concerning positive controls, distilled water was used as solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C (without S9 mix), Cyclophosphamide (with S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration:
In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.
The second experiment was performed as follows:
· without S9 mix, cells were exposed continuously to the test or control items until harvest,
· with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.
Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.

- Fixation time (start of exposure up to fixation or harvest of cells):3 hours, 20 hours and 44 hours (for the second experiment with S9 mix). The cells were fixed in a methanol/acetic acid mixture (3/1; v/v), one and a half hours before harvest.

SPINDLE INHIBITOR (cytogenetic assays):a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis.

STAIN (for cytogenetic assays):Giemsa.

NUMBER OF REPLICATIONS: two independent experiments, using duplicate cultures, the cells were tested, both with and without S9 mix, using at least five concentrations of the test item. One male and one female donor.

NUMBER OF CELLS EVALUATED:Analysis of 200 metaphases/dose-level (with 44 to 46 chromosomes) was made, with 100 metaphases/culture, whenever possible. Only 50 metaphases/culture were analyzed when at least 10% cells with structural chromosome aberration were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (number of cells in mitosis/1000 cells examined), which indicates whether an item induces mitotic inhibition. Mitotic index was determined without blind scoring.
The following structural aberrations were recorded for each metaphase (c, d): gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverizations)

OTHER EXAMINATIONS:
- Determination of polyploidy:yes
- Determination of endoreplication:yes

Evaluation criteria:

A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the results were compared using the ¿2 test, in which p = 0.05 was used as the lowest level of significance.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH, Effects of osmolality:. At 50 µg/mL , the pH was approximately 7.6 (as for the vehicle control) and the osmolality equal to 411 mOsm/kg H2O (402 for the vehicle control).

- Evaporation from medium: no

- Water solubility:The test item was found not soluble in the vehicles usually used for this type of assay.

- Precipitation:In the culture medium, the concentration of 50 µg/mL showed a marked precipitate.
A slight to marked precipitate was observed in the culture medium at the end of the treatment period, generally at concentrations = 12.5 µg/mL.

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Conclusions:

Interpretation of results (migrated information):
negative

GRAPHISTRENGTH C100 micronised did not induce chromosome aberrations in cultured human lymphocytes.

Executive summary:

The potential of the test item GRAPHISTRENGTH C100 micronised to induce chromosome aberrations in cultured human lymphocytes was evaluated. The study was performed according to the international guidelines OECD 473 and Good Laboratory Practice Regulations. The test item was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. Treatment of cells with the test subtance in the presence or absence of S9 resulted in similar numbers of aberrations compared to negative control (ethanol), while cell cultures treated with positive control substances resulted in significant elevation of aberrations. In the culture medium, the concentration of 50 µg/mL showed a marked precipitate. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture. Since the test item was non-toxic but poorly soluble, the choice of the highest concentration was based on the level of precipitate, according to the criteria specified in the international guidelines. With a treatment volume of 27.5 µL/5.5 mL culture medium, the treatment-concentrations were as follows:

· 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL, for the first experiment, both with and without S9 mix,

· 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL, for the second experiment, both with and without S9 mix.

A slight to marked precipitate was observed at the end of the treatment period, generally at concentrations = 12.5 µg/mL. Experiments without S9 mix Cytotoxicity: Following the 3-hour treatment, only a slight decrease in mitotic index was noted, without any clear evidence of a concentration relationship (up to 25% decrease). Following the 20-hour treatment, a slight decrease in mitotic index was noted at concentrations = 25 µg/mL (26-29% decrease). Following the 44-hour treatment, a slight decrease in mitotic index was noted at 50 µg/mL (24% decrease).

Metaphase analysis:

The concentrations selected for metaphase analysis were as follows:

· 3.13, 6.25 and 12.5 µg/mL for the 3-hour treatment, the latter showing precipitate in the culture medium at the end of the treatment period,

· 3.13, 6.25 and 12.5 µg/mL for the 20-hour treatments, the latter showing precipitate in the culture medium at the end of the treatment period,

· 25 µg/mL for the 44-hour treatment, this dose-level showing precipitate in the culture medium at the end of the treatment period. No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3-, 20- as well as 44-hour treatments. Experiments with S9 mix Cytotoxicity: No noteworthy decrease in mitotic index was noted at the 20-hour harvest time in the first experiment. At the 20-hour harvest time in the second experiment, slight decreases in mitotic index were noted at concentrations = 12.5 µg/mL (26-31% decrease). No noteworthy decrease in mitotic index was noted at the 44-hour harvest time. Metaphase analysis:

The concentrations selected for metaphase analysis were as follows:

· 3.13, 6.25 and 12.5 µg/mL for the 20-hour harvest time in both experiments, the latter showing precipitate in the culture medium at the end of the treatment period,

· 12.5 µg/mL for the 44-hour harvest time, this dose-level showing precipitate in the culture medium at the end of the treatment period.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiments and at either harvest times. The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered valid.

GRAPHISTRENGTH C100 micronised did not induce chromosome aberrations in cultured human lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In standard in vivo assays, no DNA damage was observed by the comet assay (OECD TG 489) in lung, liver and kidney cells, and no clastogenic activity was observed by the micronucleus test (OECD TG 474) in the bone marrow cells of rats exposed by inhalation for 13 weeks to Graphistrength C100.

Chromosomal aberration assay

The in vivo clastogenic potential of Graphistrength™ C100 was evaluated in rat bone marrow micronucleus tests performed following the OECD TG 474 after a 13-week inhalation exposure, a 3-day intratracheal instillation and a 3-day oral gavage. A micronucleus assay was also performed on intestinal (colon) cells after a 3-day oral gavage.

 

In a key study, the genotoxic potential of Graphistrength™ C100 was investigated in the in vivo micronucleus assay performed on the bone marrow cells of 5 male and 5 female Rcc HanTM: WIST(SPF) rats per groups around 24 hours following a nose-only, flow-past 13-week inhalation exposure (6 hours/day, 5 days/week) to 0, 0.05, 0.25 and 5.0 mg/m3 air (Broich, 2016). Six animals/sex were treated with the positive control item cyclophosphamide. Per animal, 6000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the respective Air Control thus indicating that Graphistrength™ C100 did not exert any cytotoxic effects in the bone marrow in neither male nor female animals. In comparison to the respective Air Control values there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any sex and dose level after administration of the test item. Furthermore, no dose response was observed. In conclusion, Graphistrength™ C100 did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the rat.

 

In the frame of the Nanogenotox program, (Fessard, 2013), rats were treated for 3 days at 24-hour interval with Graphistrength™ C100 (named NM 402 in the study report) by intratracheal (i. t.) route at dose levels of 0.4, 0.8 and 1.6 mg/kg/d or by oral route (p. o.) at dose levels of 12.8, 25.6 and 51.2 mg/kg/d. Three to six hours after the third treatment, the rats were deeply euthanized and bone marrow cells from the i. t. and p. o. treated rats and the colon from the p. o. treated rats were isolated for the micronucleus test. No medullar toxicity and clastogenic activity was observed.

 

In conclusion, Graphistrength™ C100 is not clastogenic in the rats by inhalation, intratracheal and oral routes of exposure.

 

Primary DNA damage assay

An in vivo hOGG1-modified comet assay was performed on lung, liver and kidney cells of rats exposed by inhalation to Graphistrength™ C100 for 13 weeks (Broich, 2016).

 

In a key study, the genotoxic potential of Graphistrength™ C100 was investigated in the in vivo comet assay performed under alkaline conditions, i. e. pH > 13 (Alkaline Single Cell Gel Electrophoresis) followed by one expression time of around 24 hours after the last treatment in isolated lung, kidney and liver cells of male Rcc HanTM: WIST(SPF) rat, exposed by nose-only, flow-past inhalation at 3 dose levels (5.0, 0.25 and 0.05 mg/m3air), 6 hours/day, 5 days/week for a period of 13 weeks (Simar, 2014; Broich, 2016). The enzyme hOGG1 was also used in order to demonstrate eventual oxidative damage. Clear signs of inflammatory reaction were observed in the lungs of animals exposed to the high concentration. They were characterized by black particles deposition, changes of the cytology, biochemistry and cytokines levels in the brochoalveolar lavage fluid and histological signs of alveolar and bronchiolar inflammation. No statistically significant increases in tail intensity median were observed in isolated lung, liver and kidney cells in absence and presence of hOGG1, at the 3 tested concentrations. Graphistrength™ C100 was thus considered as not genotoxic in rat lung, kidney or liver cells. Indeed, no primary DNA damage was detected, either in absence or in presence of hOGG1 that demonstrates the lack of potential of Graphistrength™ C100 to induce oxidative damage. Graphistrength™ C100 did not induce primary DNA damage in the rat cells by inhalation exposure.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

other: RccHanTM: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

See Section 7.5.2.: Broich (2016) GRAPHISTRENGTH C100: 13-Week Inhalation Toxicity Study with Recovery Periods and Micronucleus and Comet Assays in the Wistar Rat. Study D48447, Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland

Route of administration:

inhalation: aerosol

Vehicle:

Clean air

Details on exposure:

See Section 7.5.2.: Broich (2016) GRAPHISTRENGTH C100: 13-Week Inhalation Toxicity Study with Recovery Periods and Micronucleus and Comet Assays in the Wistar Rat. Study D48447, Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6-hours daily, 5 days/week at approximately 24-hour intervals

Post exposure period:

Approximately 24 hours after the last treatment

Remarks:

(groups 2, 3 and 4, respectively)

Dose / conc.:

0.05 other: mg/m³ air (target)

Dose / conc.:

0.25 other: mg/m³ air (target)

Dose / conc.:

5 other: mg/m³ air (target)

No. of animals per sex per dose:

5

Control animals:

other: yes, sham-exposed (group 1)

Positive control(s):

- Cyclophosphamide (CPA)
- Justification for choice of positive control(s): recommended by the guideline
- Route of administration: oral
- Doses / concentrations: single administreation of 20 mg/kg bw 24 hours before sacrifice

Tissues and cell types examined:

Bone marrow cells (polychromatic erythrocytes)

Details of tissue and slide preparation:

Preparation of the Bone Marrow Smears
The femora were removed, the epiphyses cut off and the marrow will be flushed out with fetal calf serum, using a syringe (3 mL per femur). The nucleated cells were separated from the erythrocytes using the method of Romagna. Briefly, the cell suspensions were passed through a column consisting of a-Cellulose (Sigma) and Cellulose (Sigmacell type 50) (1:1 mixture). The columns were washed with Hank´s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. The pellet was resuspended in a small drop of FCS and spread on slides. The smears were air-dried, fixed in methanol and transported to Harlan CCR in Germany.
The slides were stained with May-Grünwald/Giemsa. For this at first the slides were incubated 1 minute in a 5% May-Grünwald solution followed by 1 minute in a 1 : 1 mixture of May-Grünwald : phosphate buffer (pH 7.4). After 20 minutes in 14% Giemsa solution the slides were washed twice in phosphate buffer and 10 seconds in deionised water. Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). Two slides were made from each bone marrow sample. One slide was shipped to Harlan CCR, the other was kept at Harlan Laboratories Ltd, Itingen. The slides were coded using a computer generated coding list.

Analysis of Cells
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test item producing neither a dose-related increase in the number of micronucleated polychro-matic erythrocytes nor a statistically significant positive response at any of the test points is con-sidered non-mutagenic in this system.

Statistics:

Nonparametric Mann-Whitney test, if necessary.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

Bone marrow

Toxicity:

yes

Remarks:

inflammatory reactions in the lungs. No effect on the bone marrow

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity
See Section 7.5.2.: Broich (2016) GRAPHISTRENGTH C100: 13-Week Inhalation Toxicity Study with Recovery Periods and Micronucleus and Comet Assays in the Wistar Rat. Study D48447, Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland

Genotoxicity
No increase of the frequency of micronucleated polychromatic erythrocytes (PCE) and signs of medullar toxicity were observed in the rats after 13 weeks of exposure to Graphistrength™ C100 (Table). A clear positive response was observed with the positive control.

Table: Results of the micronucleus assay in the bone marrow of male and female rats exposed nose-only to respirable aerosols of Graphistrength™ C100 for 13 weeks

Test group (n=5)	Concentration mg/m³ air	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythrocytes
Males
Air control	0	24	0.340	2 -17	1099
Graphistrength™ C100	0.05	24	0.440	5 -11	1073
	0.25	24	0.280	2 -10	1094
	5.00	24	0.210	3 -8	1081
Positive control^1	20	24	0.833	10 -26	806
Females
Vehicle	0	24	0.290	3 -9	1155
Graphistrength™ C100	0.05	24	0.210	1 -8	1223
	0.25	24	0.190	1 -5	1147
	5.00	24	0.220	2 -9	1091
Positive control^1	20	24	0.750	8 -28	813

¹cyclophosphamide, 20 mg/kg

Conclusions:

negative

Executive summary:

The genotoxic potential of Graphistrength C100 was investigated in thein vivomicronucleus assay performed on the bone marrow cells of 5 male and 5 female Rcc HanTM: WIST(SPF) rats per groups around 24 hours following a nose-only, flow-past 13-week inhalation exposure (6 hours/day, 5 days/week) to 0, 0.05, 0.25 and 5.0 mg/m³air. Six animals/sex were treated with the positive control item cyclophosphamide. Per animal, 6000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the respective Air Control thus indicating that GRAPHISTRENGTH C100 did not exert any cytotoxic effects in the bone marrow in neither male nor female animals. In comparison to the respective Air Control values there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any sex and dose level after administration of the test item.Furthermore, no dose response was observed.In conclusion, GRAPHISTRENGTH C100 did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the rat.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 489 (In Vivo Mammalian Alkaline Comet Assay)

Deviations:

yes

Remarks:

A sampling time 24-h after the last treatment was used instead of 2-6h as recommended by the guideline.

GLP compliance:

yes

Type of assay:

mammalian comet assay

Species:

rat

Strain:

other: RccHanTM: WIST(SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

See Section 7.5.2.: Broich (2016) GRAPHISTRENGTH C100: 13-Week Inhalation Toxicity Study with Recovery Periods and Micronucleus and Comet Assays in the Wistar Rat. Study D48447, Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland

Route of administration:

inhalation: aerosol

Vehicle:

Clean air

Details on exposure:

See Section 7.5.2.: Broich (2016) GRAPHISTRENGTH C100: 13-Week Inhalation Toxicity Study with Recovery Periods and Micronucleus and Comet Assays in the Wistar Rat. Study D48447, Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6-hours daily, 5 days/week at approximately 24-hour intervals

Post exposure period:

Approximately 24 hours after the last treatment

Remarks:

(groups 2, 3 and 4, respectively)

Dose / conc.:

0.05 other: mg/m³ air (target)

Dose / conc.:

0.25 other: mg/m³ air (target)

Dose / conc.:

5 other: mg/m³ air (target)

No. of animals per sex per dose:

5

Control animals:

other: yes, sham-exposed (group 1)

Positive control(s):

- methylmethanesulfonate
- Justification for choice of positive control(s): recommended by the guideline
- Route of administration: oral
- Doses / concentrations: twice at 24-hour interval at 100 mg/kg and once 3 to 6 hours before sacrifice with 70 mg/kg

Tissues and cell types examined:

Lung, kidneys and kiver cells

Details of tissue and slide preparation:

In this Test Site Phase, the Comet assay was performed under alkaline conditions essentially following the procedure of Singh et al. (1988), Tice et al. (2000), Hartmann et al. (2004), Burlinson et al. (2007).
The essential steps of the comet assay are successively: layering of cells mixed with low melting point agarose (over coated glass microscope slides), lysis (to lyse the cell and nuclear membranes and other proteins), unwinding of DNA, electrophoresis, neutralization, staining and scoring.
Approximately 24 hours after the last treatment, the rats were humanely killed and cells from the selected target organs were isolated in mincing solution.

Cell isolations
Single cell preparations were done within one hour after animal sacrifice.
A portion of (1) the lobe of the liver (2) the lobe of the lung or (3) one quarter of a kidney were removed and washed in the cold mincing buffer until as much blood as possible has been removed.
It is noteworthy that deposits of GRAPHISTRENGTH C100 were present on the lungs. Their levels increased with the doses.
The portion of each organs was minced with a pair of fine scissors to release the cells. Each cell suspension was stored on ice for 15-30 seconds to allow large clumps to settle, and the supernatant was used to prepare comet slides.

Slide preparation
Before use, a volume of 85 µL of 0.8% of NA was added on the microscope slide pre-layered with 1.5%-NA and then covered with a glass coverslip. Slides were placed at room temperature until the agarose layer hardens (3 to 5 minutes). Around 3 x 104 cells of the different doses tested was mixed with 75 µL of 0.5% of Low Melting Point Agarose (LMPA) kept at 37 °C and added on the microscope slide after gently sliding off the coverslip. The slides were then covered with a new glass coverslip, and were placed once again at room temperature for 3 to 5 minutes.
Three slides were prepared for the Comet assay. Concurrently, three other slides were prepared for the comet assay with the use of hOGG1.

Protocol for the Comet assay
Lysis
After the top layer of agarose has solidified, the glass coverslips were removed and the slides were immersed for one night at + 4 °C in the dark in the lysing solution.
Unwinding, electrophoresis
After this incubation period, the slides were then removed and placed on a horizontal gel electrophoresis unit and the unit filled with freshly prepared alkaline buffer to around 0.25 cm above the slides. In order to avoid excessive variation across the groups during each electrophoretic run, only one of the replicate slides were processed in each run for each animal (DNA – unwinding and electrophoresis). The cells were exposed to the alkali for 20 minutes to allow the DNA unwinding, and expression of single-strand breaks and alkali-labile sites. Next, electrophoresis was conducted for 20 minutes at 0-4°C by applying an electric current of 0.7 V / cm (25 V / 300 mA). All these steps were conducted protected from daylight to prevent the occurrence of additional DNA damage. After electrophoresis at pH >13, the slides were neutralized twice for 5 minutes and the DNA was exposed for 5 minutes to absolute ethanol in order to preserve all the Comet assay slides. Subsequently, the slides were air dried and then stored at room temperature until they were scored for DNA migration at IPL.
Protocol for the Comet assay with the use of hOGG1
For the hOGG1-modified comet assay, following lysis as described above, slides were washed two times 5 min with the hOGG1 incubation buffer at room temperature and then incubated for 35 min at 37°C with 0.12 x 10-3 U/slide of hOGG1 (Biolabs, ref 0031303; see § 9) in the hOGG1 incubation buffer. Slides were then processed as described above.

Image analysis
Just prior to scoring, the DNA was stained using propidium iodide (25 µL/slide).
Slides were examined with a 200 x magnification, using a fluorescent microscope (Leica Microsystems SAS - DM 2000, Heerbrugg, Switzerland), equiped with an excitation filter of 515-560 nm and a barrier filter of 590 nm, connected through a gated monochrome CCD IEEE1394 FireWire video camera (Allied Vision Technologies), to Comet Assay IV Image Analysis System, version 4.11 with Windows XP Pro Software (Perceptive Instruments Ltd, Suffolk, UK).
For control and treated groups three slides were analysed with 50 nuclei per slide randomly scored. Five animals were retained per group, i.e. 15 slides per group, 750 analysed nuclei per group.
Recent publications focused on the interpretation of the results through the analysis of the median of the percentage of DNA in tail, with the animal as statistical unit (D. Lovell and T. Omori, 2008).
In fact, this parameter appears to be the most linearly related to dose (B. Burlinson et al., 2007).
The results obtained in the different treatments are presented in tabular form giving the median of the percentages of DNA in tail for 50 cells per slides, and the mean of the medians for 3 slides per animal (i.e. 150 cells per animal).
However, one slide from the liver of the animals Nos. 1 and 5 prepared in the absence of hOGG (i.e. Slides Nos. 394A and 398B), were lacking. Therefore 100 cells were analysed for these animals i.e. a total 700 cells for the 5 animals of this dose in this specific condition instead of 750 cells. However, it was considered that this slight deviation did not impact the current study, and the conclusions remain the same.

Evaluation criteria:

A study is accepted if the following criteria are fulfilled:
- In the positive control groups, the median of percentage of DNA in tail were statistically increased when compared to the control group.
- The values for the median of percentage of DNA in tail in positive control groups were consistent with historical values, contrarily to the ones of negative control.
- No toxicity was noted in the negative control group as assessed by the enumeration of ghost cells. The low frequencies of hedgehogs also demonstrate the good slides preparation.
The validity criteria for the test were fulfilled and the tests were validated.
The historical data for negative and positive controls obtained in the absence of hOGG1 and constituted with 3 (lung and liver) or 5 (kidney) assays after three treatments followed by one sampling time, 3 to 6 hours after the last treatment.

Statistics:

In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software Stat view®, version 5.
As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskall-Wallis test was performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups.
The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each group versus the vehicle control. This test was also used to compare vehicle control and positive control to determine acceptable criteria of a valid test.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

lungs, liver and kidneys

Toxicity:

yes

Remarks:

inflammatory reactions in the lungs

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity
See Section 7.5.2.: Broich (2016) GRAPHISTRENGTH C100: 13-Week Inhalation Toxicity Study with Recovery Periods and Micronucleus and Comet Assays in the Wistar Rat. Study D48447, Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland

Genotoxicity
No statistically significant increases in tail intensity median were observed in isolated lung, liver and kidney cells in absence and presence of hOGG1, at the 3 tested concentrations of 0.05, 0.25 and 5.0 mg/m3 air (Table).

Table: Results of the comet assay with and without hOGG1 in the lung; kidneys and liver of male rats exposed nose-only to respirable aerosols ofGraphistrength™ C100 for 13 weeks

	hOGG1	Air control	Graphistrength™ C100 (mg/m^3air)			Positive control^3
			0.05	0.25	5.0
n (males)		5	5	5	5	5
Lung
% of DNA in tail^1	- +	8.8 ± 5.5 20.2 ± 5.2	3.4 ± 1.5* 16.7 ± 5.7	8.2 ± 5.1 22.3 ± 11.8	4.7 ± 3.7 17.9 ± 5.9	48.7 ± 3.0** 79.1 ± 7.9**
Relative ratio of ghost cell^2	- +	- -	1.0 2.1***	1.2 2.0***	0.8 1.3	0.6** 1.1
Kidneys
% of DNA in tail^1	- +	7.4 ± 4.4 22.8 ± 7.4	7.5± 4.2 22.4 ± 7.3	8.2± 4.2 23.4 ± 8.4	9.7± 3.1 24.3 ± 8.0	72.5± 5.8** 74.0 ± 5.9**
Relative ratio of ghost cell^2	- +	- -	1.6 1.1	0.9 1.6*	1.5 1.7**	2.8*** 0.6*
Liver
% of DNA in tail^1	- +	3.8 ± 3.1 11.6 ± 6.4	3.4 ± 1.6 7.5 ± 1.5	1.8 ± 1.0 7.7 ± 1.1	5.5 ± 2.9 9.6 ± 3.0	71.2 ± 9.1** 78.6 ± 3.7**
Relative ratio of ghost cell^2	- +	- -	0.2 0.5*	0.5 0.2***	2.8** 1.1	4.9*** 1.7**

p Mann-Whitney: * p<0.05. ** p< 0.01. *** p< 0.001

¹Mean of Median ± sd

²Mean value in treated groups/mean control value

³MMS

Conclusions:

negative

Executive summary:

The genotoxic potential of Graphistrength C100 was investigated in the in vivo comet assay performed under alkaline conditions, i.e.pH > 13 (Alkaline Single Cell Gel Electrophoresis) followed by one expression time of around 24 hours after the last treatment in isolated lung, kidney and liver cells of male Rcc HanTM: WIST(SPF) rat, exposed by nose-only, flow-past inhalation at 3 dose levels (5.0, 0.25 and 0.05 mg/m³air), 6 hours/day, 5 days/week for a period of 13 weeks. The enzyme hOGG1 was also used in order to demonstrate eventual oxidative damage. Clear signs of inflammatory reaction were observed in the lungs of animals exposed to the high concentration.They were characterized by black particles deposition, changes of the cytology, biochemistry and cytokines levels in the brochoalveolar lavage fluid and histological signs of alveolar and bronchiolar inflammation. No statistically significant increases in tail intensity median were observed in isolated lung, liver and kidney cells in absence and presence of hOGG1, at the 3 tested concentrations. Graphistrength C100 was thus considered as not genotoxic in rat lung, kidney or liver cells. Indeed, no primary DNA damage was detected, either in absence or in presence of hOGG1, that demonstrates the lack of potential of graphistrength C100 to induce oxidative damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

According to the available data on Graphistrength C100 and Regulation (EC) No 1272/2008, no classification is required for germ cell mutagenicity.