Charcoal, coconut shell

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2010-6-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed according to the respective OECD guideline and under GLP conditions. "Charcoal" and "Coconut shell charcoal" are almost identical substances, but require two separate registrations, due to different CAS and EC numbers. The difference between both substances is a different origin (coconut shell/wooden origin), but after the carbonization the final products are identical and “Coconut shell charcoal” fits into the chemical sameness description of “Charcoal”. Both substances not only show a structural similarity, but are structural identical.

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

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)

Principles of method if other than guideline:

The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity of chemicals. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain. Since many compounds do not exert a mutagenic effect until they have been metabolized by enzyme systems not available in the bacterial cell, the test compound and test organisms are also incubated in the presence of a liver microsomal fraction.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Charcoal (Probe 1: C-Fix = 73.3%)

IUPAC Name:

Charcoal (Probe 1: C-Fix = 73.3%)

Constituent 2

Reference substance name:

Charcoal

EC Number:

240-383-3

EC Name:

Charcoal

Cas Number:

16291-96-6

IUPAC Name:

methane

Details on test material:

Name: charcoal (Probe 1: C-Fix = 73.3%)
Batch No.: 19062009 1PP
Appearance: charcoal typic
CAS-No.: 16291-96-6
EINECS-No.: 240-383-3
Purity: C-Fix = 73.3%
Date of expiry: Dec. 2030
Storage conditions: room temperature 20 ± 5 °C, keep away from humidity

Method

Target gene:

Tester strains TA98 and TA 97a is reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Strain TA 102 is sensitive to oxidizing DNA damage based on AT base pairs at the site of the mutation.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: Mutations in hisD6610, uvrB, pKM 101, and rfa

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: Mutations in hisD3052, uvrB, pKM 101, and rfa

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Mutations in hisG46, uvrB, pKM 101, and rfa

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Mutations in hisG428, pKM 101, and rfa

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: Mutations in hisG46, uvrB, and rfa

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate (S9 mix) obtained from male Sprague-Dawley rats pretreated with Aroclor 1254 (500 mg/kg body weight)

Test concentrations with justification for top dose:

plate incorporation method: 50, 150, 500, 1,500, and 5,000 µg/plate
pre-incubation method: 313, 625, ,1250, 2,500, and 5,000 µg/plate

Vehicle / solvent:

Ethanol

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine

Remarks:

concentration: 20 µg/plate; strains: TA 97a, TA 98 and TA 102 in the absence of metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide

Remarks:

concentration: 1 µg/plate; strains: TA 100 and TA 1535 in the absence of metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Amino-anthracene

Remarks:

concentration per plate: 1 µg; strains: TA 97a, TA 100, TA 102 and TA 1535 in the presence of metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

concentration: 20 µg; strain: TA 98; in the presence of metabolic activation

Details on test system and experimental conditions:

An extract was prepared from 250 g of the test item by extraction with 1 L acetone:n-hexane 50:50 (v/v) for 1 h at 50°C in an ultrasonic bath. The sample was washed with an additional 250 mL of the solvent. The combined extracts were reduced at 50°C in a rotary evaporator to approximately 150 mL and filled quantitatively in a 250 mL volumetric flask. At the start of the experiment, a stock solution containing 50 g/L of the test item in Ethanol was prepared and used for the dilutions.

Evaluation criteria:

The colonies were counted visually, the numbers were recorded.
The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants minus mean spontaneous revertants) were calculated

Statistics:

The use of statistics was limited to the calculation of means and standard deviations.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 97

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 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 3

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

Test results 4

Species / strain:

S. typhimurium TA 102

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 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

Additional information on results:

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration related increase over the range tested can also be taken as a sign of mutagenic activity.

Remarks on result:

other: other: plate incorporation method and pre-incubation method

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mean revertants incorporation method
Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	99	122	9	13	78	98	185	186	11	5
	sd	2.2	3.1	2.2	1.9	3.4	11.6	10.8	5.9	1.7	1.0
DMSO	Mean	113	128	9	9	108	112	196	192	6	14
	sd	18.2	4.4	5.0	3.4	12.4	7.0	19.5	9.3	1.7	3.1
Ethanol	Mean	123	106	9	16	113	108	211	184	6	14
	sd	10.2	13.4	6.6	1.0	6.6	8.8	11.5	7.5	2.4	0.8
Solvent control	Mean	97	122	7	9	104	90	212	189	8	14
	sd	1.5	14.1	1.6	2.5	5.4	12.5	14.1	10.0	3.0	3.7
Positive Controls	Mean	950	958	868	603	642	898	556	968	645	554
	sd	120	147	258	73	232	149	206	151	141	107
	f(I)	8.41	7.48	96.44	67.00	8.23	8.02	2.84	5.04	58.64	39.57
5000 µg/pl.	Mean	116	122	13	13	101	73	187	196	8	14
	sd	14	18	1	5	9	7	4	1	2	1
	f(I)	1.20	1.00	1.86	1.44	0.97	0.81	0.88	1.04	1.00	1.00
1500 µg/pl.	Mean	110	98	10	10	84	77	166	178	9	11
	sd	13	2	2	3	6	14	18	4	1	3
	f(I)	1.13	0.80	1.43	1.11	0.81	0.86	0.78	0.94	1.13	0.79
500 µg/pl.	Mean	116	109	6	9	103	99	183	190	7	6
	sd	18	9	1	3	10	7	11	8	1	1
	f(I)	1.20	0.89	0.86	1.00	0.99	1.10	0.86	1.01	0.88	0.43
150 µg/pl.	Mean	109	120	6	17	110	81	183	170	10	14
	sd	16	7	2	2	7	17	10	4	2	2
	f(I)	1.12	0.98	0.86	1.89	1.06	0.90	0.86	0.90	1.25	1.00
50 µg/pl.	Mean	98	113	8	17	103	103	203	199	11	7
	sd	4	12	1	2	14	10	2	3	1	2
	f(I)	1.01	0.93	1.14	1.89	0.99	1.14	0.96	1.05	1.38	0.50
Mean revertants preincubation method
Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	104	122	12	13	104	142	145	147	13	12
	sd	17.2	6.4	2.6	5.6	16.1	33.5	16.0	32.7	4.2	2.4
DMSO	Mean	114	104	14	16	120	96	128	213	10	13
	sd	5.0	7.2	2.2	2.9	28.0	10.2	12.4	35.9	0.0	1.0
Ethanol	Mean	114	111	15	16	116	113	150	232	11	13
	sd	6.4	5.2	3.7	2.2	13.1	27.3	16.7	20.3	4.1	2.1
Solvent control	Mean	124	129	11	10	137	143	172	212	8	11
	sd	6.2	36.3	1.0	4.7	26.1	7.1	30.4	31.9	3.9	1.5
Positive Controls	Mean	486	423	677	148	734	569	729	653	111	491
	sd	46	28	33	43	50	95	104	135	4	114
	f(I)	4.26	4.07	48.36	9.25	7.06	5.93	5.70	3.07	8.54	37.77
5000 µg/pl.	Mean	111	127	10	12	129	129	130	201	6	9
	sd	11	24	2	5	15	14	9	96	1	5
	f(I)	0.90	0.98	0.91	1.20	0.94	0.90	0.76	0.95	0.75	0.82
2500 µg/pl.	Mean	105	102	12	12	109	102	132	165	14	8
	sd	12	13	1	3	16	13	16	40	1	2
	f(I)	0.85	0.79	1.09	1.20	0.80	0.71	0.77	0.78	1.75	0.73
1250 µg/pl.	Mean	111	114	5	13	129	119	155	216	10	10
	sd	4	4	2	2	9	11	16	17	2	5
	f(I)	0.90	0.88	0.45	1.30	0.94	0.83	0.90	1.02	1.25	0.91
625 µg/pl.	Mean	118	126	11	9	120	163	194	232	9	13
	sd	10	9	2	4	9	5	31	58	1	3
	f(I)	0.95	0.98	1.00	0.90	0.88	1.14	1.13	1.09	1.13	1.18
313 µg/pl.	Mean	111	111	9	10	115	138	179	261	8	13
	sd	7	5	1	3	4	20	20	25	1	2
	f(I)	0.90	0.86	0.82	1.00	0.84	0.97	1.04	1.23	1.00	1.18

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with an without metabolic activation

It can be stated, that under the test conditions, the test item charcoal (Probe 1: C-Fix = 73.3%) is not mutagenic in the bacterial reverse mutation test using Salmonella typhimurium, strains TA97a, TA98, TA100, TA102, and TA1535.

Executive summary:

The test item charcoal (Probe 1: C-Fix = 73.3%) was tested in the bacterial reverse mutation assay (Ames test) using the Salmonella typhimurium tester strains TA97, TA 98, TA100, TA102, and TA1535 in the presence and absence of metabolic activation by rat liver S9 mix. The assay was performed using both the plate incorporation and the preincubation method. Under the conditions of this study, charcoal (Probe 1: C-Fix = 73.3%) was concluded to be negative in all tester strains in the presence and absence of metabolic activation.