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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from publication.

Data source

Reference
Reference Type:
publication
Title:
Gene mutation toxicity study of the test chemical
Author:
Seifried et al
Year:
2006
Bibliographic source:
Chem. Res. Toxicol.

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
as per Prival and Mitchell (1982)
Principles of method if other than guideline:
Salmonella/Mammalian-Microsome Mutagenicity Assay was conducted by using the given test chemical.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate
Cas Number:
1934-21-0
Molecular formula:
C16H12N4O9-S2.3Na
IUPAC Name:
Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate
Details on test material:
IUPAC name: Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate
Commen name:Tartrazine
Molecular weight :534.3681 g/mol
Molecular formula:C16H12N4O9-S2.3Na
Smiles:n1(c2ccc(cc2)S(=O)(=O)[O-])c(c(c(n1)C(=O)[O-])/N=N/c1ccc(cc1)S(=O)(=O)[O-])O.[Na+].[Na+].[Na+]
InChI:1S/C16H12N4O9S2.3Na/c21-15-13(18-17-9-1-5-11(6-2-9)30(24,25)26)14(16(22)23)19-20(15)10-3-7-12(8-4-10)31(27,28)29;;;/h1-8,13H,(H,22,23)(H,24,25,26)(H,27,28,29);;;/q;3*+1/p-3/b18-17+;;
substance type: organic

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA98 and TA100
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking.
- source of S9 : male uninduced hamster liver S9
- method of preparation of S9 mix: No data
- concentration or volume of S9 mix and S9 in the final culture medium: 30% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) - Nitrogen was blown over the preincubation tube to keep the atmosphere reduced.
Test concentrations with justification for top dose:
0, 333, 1000, 3333, 6666, 10000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Test chemical was soluble in water.
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
congo red
Details on test system and experimental conditions:
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): (1-2) x10^9 cells/mL
- Test substance added - preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 min
- Exposure duration/duration of treatment: 48 h
- Harvest time after the end of treatment (sampling/recovery times): No data

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: Preincubation Method - All plates were counted with an Artek automated colony counter (Artek 880, DynaTech, Chantilly, VA) or Minicount colony counter (Imaging Products International, Inc., Chantilly, VA), which was calibrated prior to use.
Evaluation criteria:
All plates were observed for increase in mutant frequencies.
Statistics:
No data

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES (if applicable): The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

Table

Test Chemical

Dose

TA98

TA100

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

Prival Modification

Negative

 

 

 

 

 

 

 

 

 

H2O

---

---

50 ± 5

---

---

217 ± 18

 

 

333ug

---

---

53 ± 2

---

---

187 ± 28

 

 

1000ug

---

---

55 ± 10

---

---

215 ± 14

 

 

3333ug

---

---

52 ± 5

---

---

189 ± 6

 

 

6666ug

---

---

59 ± 1

---

---

211 ± 50

 

 

10000ug

---

---

57 ± 11

---

---

208 ± 20

 

 

Positive 1

---

---

349 ± 33

---

---

785 ± 151

 

 

Positive 2

---

---

397 ± 41

---

---

1316 ± 98

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
The given test chemical failed to induce mutation in Salmonella typhimurium TA98 and TA100 in the presence and absence of S9 metabolic activation and hence it is not likely to be mutagenic by in-vitro test.
Executive summary:

Bacterial Reverse Mutation Assay was conducted by using the given test chemical as per OECD Guideline 471 (Bacterial Reverse Mutation Assay) and Prival modification by Prival and Mitchell (1982). For tests using the FMN-modified assay, strains TA98 and TA100 were used at doses 0, 333, 1000, 3333, 6666, 10000 ug/plate dissolved in water. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical was added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by Ames et al. The plates were then incubated at 37 °C for 48 h. The positive control in FMN experiment was Congo red. All plates were counted with an Artek automated colony counter or Minicount Colony counter, which was calibrated prior to use. The given test chemical failed to induce mutation in Salmonella typhimurium TA98 and TA100 in the presence and absence of S9 metabolic activation and hence it is not likely to be mutagenic by in-vitro test.