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Genetic toxicity: in vitro

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Administrative data

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:
2013-11-30 to 2013-10-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to the respective OECD guideline and under GLP conditions.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
The objective of this bacterial reverse mutation assay was to evaluate the potential of Charcoal- High VOCs sample, to induce point mutations which revert mutations present in several tester strains of Salmonella typhimurium, and restore the functional capability of bacteria to synthesize an essential amino acid, viz. histidine. The study would provide information on genotoxic hazards likely to arise from the exposure to the test article.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Name of the Test Article Charcoal- High VOCs sample
CAS No. 16291-96-6
Physical Appearance Solid, black, odourless
Batch No. K2555
Purity- Total Carbon 83.11% w/w (C-Fix = 75.72%)
Date of Manufacture 10-12-2012
Date of Expiry 10-12-2030
Storage condition (at INTOX)
Test Article was stored in its original container, as supplied by the Sponsor at Room Temperature (27 ± 9 °C) in the Test Article Store of INTOX. The stability of the test article. in storage will be the responsibility of the Sponsor.

Method

Target gene:
Tester strains of Salmonella typhimurium were employed for this study as detailed below.
Tester Bacteria: Salmonella typhimurium
Tester Strains Used: TA1535, TA97a, TA98, TA100, TA102
Rationale for selection of the tester strains: These strains have been recommended by the OECD Guideline
Source: Tester Strains were procured from Molecular Toxicology, Inc. (MOLTOX®), PO Box 1189, Boone, NC 28607, USA.
At INTOX Pvt. Ltd., these were maintained at the In-vitro Toxicity and Mutagenicity Section of the test facility
Storage: -196 °C, in liquid nitrogen cylinders (cryocan)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA 97a
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: Mutations in his D6610, uvrB, pKM 101 and rfa
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 / 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 / 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 / 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:
Based on the results of above preliminary tests, since the test article did not exhibit any cytotoxicity at 5000 IJg/plate, for the definitive (main) study the highest test dose, both in presence and absence of metabolic activation system, was selected as 5000 IJg/plate. Four lower test concentrations were 1500 IJg, 500 IJg, 150 IJg and 50 IJg/plate.
Vehicle / solvent:
DMSO was used as vehicle in this study. Plates containing the reaction mixture comprising of DMSO but not Charcoal - High VOCs sample, and overlay agar supplemented with histidine, served as a vehicle control.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone / n-hexane 50:50 (%vlv) in ethanol: all strains with and without metabolic activation (0.5 mL solvent in 10 mL ethanol)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR 191
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone / n-hexane 50:50 (%vlv) in ethanol: all strains with and without metabolic activation (0.5 mL solvent in 10 mL ethanol)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone / n-hexane 50:50 (%vlv) in ethanol: all strains with and without metabolic activation (0.5 mL solvent in 10 mL ethanol)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Amino-anthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone / n-hexane 50:50 (%vlv) in ethanol: all strains with and without metabolic activation (0.5 mL solvent in 10 mL ethanol)
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
The bacterial strains were cultured in Oxoid nutrient broth No. 2. Minimal glucose agar contained agar, Vogel-Bonner minimal medium E and 40% glucose. The overlay agar contained 0.6% agar,
0.5% NaCI and 0.05 mM histidine- biotin (Maron and Ames, 1983).
The cofactor supplemented post-mitochondrial fraction (S9) was employed as the metabolic activation system.
Rat liver S9 fraction preparation was carried out at INTOX laboratories, using phenobarbitone and
-naphthoflavone as inducing agents.
The S9 fraction was tested for protein content, sterility and its metabolic activation before preserving in cryocans at about -196 °C. For this experiment, fresh vials were removed from the cryocan, thawed and used in the preparation of S9 mix. Each batch of S9 prepared as above is validated for its metabolic activation by verifying its activity.
The S9 mix was prepared in cold condition at - 4 °C, immediately prior to its use in the experiment. The microsomal enzyme reaction mixture contained following components for 10 mi.
Evaluation criteria:
The mean number of histidine revertant colonies for all the treatment groups was compared with the number of revertants in the respective vehicle control group. The mutagenic activity of the test article was assessed by applying the following criteria:
Criteria for Evaluation

1. Criteria for a Positive Response

A test article is considered to be positive (mutagenic), if it induces a concentration dependent increase and/or an increase at one or more concentrations, in revertant frequency, which is at least
2-fold (3-fold for TA1535) of that observed in the corresponding concurrent vehicle control.

2. Criteria for a Negative Response

A test article, for which the results of two subsequent repeat tests do not meet the above criteria, is considered non-mutagenic in this test.

3. Criteria for an Equivocal Response

Occasionally, a test article cannot be judged to be positive or negative (e.g., concentration­ dependent increases that fail to reach 2-fold control values, or ;;.::: 2-fold increases that do not appear to be concentration dependent). In these rare instances, the results may be classified as equivocal. Equivocal or weak positive results may indicate the need to repeat the test, possibly with a modified protocol such as appropriate spacing of dose levels.
Statistics:
The use of statistics was limited to the calculation of means and standard deviations.

Results and discussion

Test resultsopen allclose all
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
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
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
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
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

 

 

 

 

Charcoal-HighVOCssample

(IJg/plate)

59

Mean

!S.D.

Mean

+S.D.

Mean

+S.D.

Mean

+S.D.

Mean

+S.D.

5000

 

15.33

0.58

130.67

5.03

25.33

11.15

126.00

9.17

318.67

12.22

1500

 

19.67

2.08

141.33

13.32

21.33

2.52

128.00

5.29

297.33

12.22

500

 

16.67

6.03

116.00

5.29

21.67

3.06

134.00

5.29

298.67

12.86

150

 

15.33

1.53

135.33

8.33

19.67

2.52

138.00

8.00

285.33

30.02

50

 

13.33

0.58

120.00

10.00

23.00

6.08

136.00

30.79

312.00

10.58

Vehicle Control (DMSO)

100f.JI

 

15.00

1.00

135.33

12.22

29.00

4.36

131.33

22.48

324.00

4.00

 

 

 

5000

 

 

 

18.33

 

 

3.21

 

 

120.00

 

 

6.00

 

 

21.00

 

 

1.00

 

 

138.67

 

 

28.94

 

 

298.67

 

 

16.65

Charcoal-High VOCs sample

1500

 

22.00

9.00

126.00

8.72

26.00

7.94

155.33

16.77

294.67

37.17

500

+

21.67

4.04

108.00

2.00

28.00

6.08

139.33

11.37

278.67

42.77

 

150

 

15.67

1.53

128.67

8.33

29.00

2.65

141.33

6.43

293.33

26.63

 

50

 

18.33

5.86

144.67

11.72

29.00

3.46

136.00

21.63

290.67

2.31

Vehicle Control (DMSO)

100f.JI

 

26.00

5.00

126.00

17.44

29.67

3.79

142.00

13.11

304.00

30.20

 

 

Positive Controls

 

 

 

 

 

 

 

 

 

 

 

 

Sodiumazide

2

 

1024.00

99.52

 

 

 

 

 

 

 

 

ICR191

1

 

 

 

938.67

41.05

 

 

 

 

 

 

4-Nitroquinoline-N-oxide

0.5

 

-

 

-

 

989.33

120.09

 

 

 

 

3-MethylmethaneSulphonate

1f.JI

 

 

 

-

 

 

 

1973.33

421.83

2117.33

108.91

2-Aminoanthracene

10

 

404.00

52.00

 

 

 

 

 

 

 

 

2-Aminoftuorene

20

+

 

 

1456.00

78.79

901.33

72.59

1477.33

502.56

 

 

 

Danthron

30

 

 

 

-

 

 

 

 

 

1616.00

125.73

 

 

 

 

 

 

S9:

 

WithoutS9

 

+

WithS9

 

 

 

 

 

 

Charcoal-HighVOCs sample

(JJg/plate)

59

Mean

!

S.D.

Mean

+S.D.

Mean

!

S.D.

Mean

!S.D.

Mean

+S.D.

5000

 

16.00

 

3.46

136.00

10.00

23.00

 

2.65

127.33

4.16

254.67

12.86

1500

500

 

14.00

15.33

 

2.65

1.15

118.67

126.67

9.02

11.02

29.00

24.33

 

3.46

1.15

128.67

120.67

11.37

3.06

238.67

269.33

22.03

24.11

 

150

-

 

14.00

 

 

4.58

 

123.33

 

12.22

 

27.33

 

 

2.52

 

120.67

 

11.37

 

314.00

 

21.63

50

 

15.00

 

5.29

146.67

10.26

33.33

 

4.04

126.00

12.17

278.67

38.44

Vehicle Control (DMSO)

100IJI

 

10.67

 

3.51

122.67

15.53

26.67

 

2.31

123.33

7.57

293.33

28.10

 

 

 

5000

 

 

 

18.67

 

 

 

1.53

 

 

132.67

 

 

8.33

 

 

38.67

 

 

 

2.89

 

 

136.00

 

 

8.72

 

 

270.67

 

 

18.48

Charcoal-High VOCs sample

1500

 

20.33

 

1.15

128.67

11.02

31.67

 

9.29

118.00

9.17

282.67

30.02

500

+

21.33

 

2.08

120.67

15.14

30.00

 

5.29

130.67

12.86

286.67

12.86

 

150

 

16.00

 

2.65

119.33

15.28

35.33

 

9.07

140.00

20.00

276.00

46.13

 

50

 

17.00

 

4.36

127.33

6.11

34.33

 

5.69

128.00

7.21

276.00

10.58

Vehicle Control (DMSO)

100 1-11

 

18.33

 

5.86

129.33

14.47

29.00

 

1.73

136.00

11.14

300.00

28.84

Applicant's summary and conclusion

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

Salmonella typhimurium Reverse Mutation Assay of Charcoal - High VOCs sample was carried out in compliance with the OECD Guidelines for Testing of Chemicals (Guideline No. 471, Section 4: Health Effects) on conduct of "Bacterial Reverse Mutation Test", adopted 21 July 1997 and 92/69/EEC: Method B.13/14, 'Bacterial Reverse Mutation Test'. European Commission Guidelines.
Under the conditions described for this study, it is concluded that, Charcoal - High VOCs sample is non-mutagenic in Salmonella typhimurium, Reverse Mutation Test (Ames test).
Executive summary:

Salmonella typhimurium Reverse Mutation Assay of Charcoal -High VOCs sample was carried out in compliance with the OECD Guidelines for Testing of Chemicals (No.471, Section 4: Health Effects)on conduct of "Bacterial Reverse Mutation Test", adopted 21 July 1997, 921691 EEC: Method B.13/14, 'Bacterial Reverse Mutation Test'. European Commission Guidelines and as per mutually agreed protocol.

Charcoal-HighVOCs sample was evaluated in the Ames I Salmonella Pre-incubation Assay to determine its ability to induce reverse mutation atselected histidine loci in five tester strains of Salmonella typhimurium viz. TA1535, TA97a, TA98, TA100 and TA102 in the presence and absence of metabolic activation system (S9).

Based up on the preliminary tests conducted to assess the solubility I precipitation and cytotoxicity of Charcoal -High VOCs sample, the tester strains were exposed to the test article in triplicate cultures at the doses of 5000 IJg,1500 IJg,500 IJg,150 IJg and50 IJg plate for with and without metabolic activation system (S9). Liver S9, induced in rats by phenobarbitone with [3-naphthoflavone, was used for this purpose. Dimethyl sulphoxide was used as a vehicle. The exposed bacteria were plated ontominimal glucose agarmedium supplemented withL-histidine. The plates were incubated at 37 ± 1°C for about 49 to 68 hours after which the histidine revertant colonies were counted and their frequency was compared with that invehicle control groups. Concurrent  positive control group was also included in the experiment,  as specified by the Guideline.

Results of the test indicated that the test article did not induce any cytotoxic effects intester strains TA 97a,TA 98,TA 100,TA 102 and TA 1535when tested both inpresence and absence of metabolic activation systems when compared with vehicle control groups according to the criteria of evaluation off indings of this Ames test.

The observed frequencies of histidine revertant colonies of Salmonella typhimurium tester strains TA1535, TA97a, TA98, TA100 and TA102 tested atand up to the concentration of 5000IJgl plate of Charcoal-HighVOCs sample, both in presence and absence of metabolic activation system, did not reveal any dose-dependentor 2-fold (3-fold for TA1535) increases as cornpared to the respective vehicle control groups according to the criteria of evaluation of findings of this Ames Test.

Plate counts for the spontaneous histidine revertant colonies inthe vehicle control groups were found to be comparable  with the range reported in the literature.  Concurrent positive controls demonstrated sensitivity of the assay with and without metabolic activation.

Under the conditions described for this study, it is concluded that,Charcoal-HighVOCs sample is

non-mutagenic in Salmonella typhimurium Reverse Mutation Test (Ames test).