[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 November, 1993 to 16 June, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

1992

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

1987

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-hydroxynaphthalene-1-sulphonato(3-)]chromium

EC Number:

271-352-2

EC Name:

[4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-hydroxynaphthalene-1-sulphonato(3-)]chromium

Cas Number:

68541-72-0

Molecular formula:

C20H13CrN4O5S

IUPAC Name:

[4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-hydroxynaphthalene-1-sulphonato(3-)]chromium

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Test material: FAT 20042/D (Neolan Rosa BE ZP feucht)
Batch No.: 276
Purity: Approx. 50 %
Appearance: Black-reddish humid pieces
Expiry date: October 1998
Storage: Room temperature
Material submitted by: CIBA-GEIGY Limited, Dyestuffs Division, Basle, Switzerland

Method

Target gene:

Histidine gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Original and confirmatory experiment- 123.46, 370.37, 1111.11, 3333.33 and 10000.00 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

other: same as solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: all strains (except TA 1535) with S9: 2-aminoanthracene; for TA 1535 with S9: cyclophosphamide; for TA 100 and 1535 without S9: sodium azide, for TA 102 without S9: mitomycin-C; for TA 98 without S9: 2 nitrofluorene, for TA1537 without S9: 9-aminoacridine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 per test substance concentration and controls


OTHER EXAMINATIONS:
- Colonies were counted electronically using an Artek Colony Counter (Fisher Scientific), or manually where minor agar damage or test chemical precipitates might have interfered with automating counting. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means for all mutagenicity assays were calculated and included in the Results section.

Rationale for test conditions:

None

Evaluation criteria:

Assay acceptance criteria: A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.

Statistics:

A statistical analysis of the test data was not performed.

Results and discussion

Test results

Test results 1

Key result

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:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

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:

not examined

Positive controls validity:

valid

Test results 4

Key result

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:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Range finding test
Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 10000 µg/plate with and without metabolic activation (since the purity of the test substance is about 50%, this corresponds to a concentration of 5000 µg/plate).

Mutagenicity test, original experiment
In the experiment performed without metabolic activation, treatment of strain TA 98 with FAT 20042/D (Neolan Rosa BE ZP feucht) led to a slight increase in the number of back-mutant colonies at the highest concentration. A marginal increase at two intermediate concentrations was also registered with strain TA 102 in both, the experiment with and without metabolic activation. No effects were observed with the other strains.

Mutagenicity test, confirmatory experiment
In the experiment performed without metabolic activation, again after treatment of strain TA 98 with FAT 20042/D (Neolan Rosa BE ZP feucht) a slight increase in the incidence of histidineprototrophic mutants was observed in comparison with the negative control. The slight increase in the number of revenants in the experiment without activation on strain TA 1535 at the concentration of 3333.3 µg/plate was not observed in the original experiment conducted on this strain and is attributed to spontaneously occurring back-mutants. No effects at all occurred and in the experiments with activation on strains TA 98 and TA 1535 and with the other strains.

The slight effect observed with strain TA 102 in the original experiment also is attributed to fluctuations in the incidence of spontaneously occurring back-mutant colonies.
In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were occasionally reduced at the upper concentrations.
There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.

Any other information on results incl. tables

SUMMARY OF THE MUTAGENICITY EXPERIMENTS

• Original experiments with metabolic activation

  Strain	Treatment	Mean counts
  TA 100	Negative control	124.00
  	123.46 µg/plate	110.33
  	370.37 µg/plate	111.00
  	1111.11 µg/plate	113.67
  	3333.33 µg/plate	131.67
  	10000.00 µg/plate	122.00
  	Positive control	2303.33

  Strain	Treatment	Mean counts
  TA 1535	Negative control	15.33
  	123.46 µg/plate	17.33
  	370.37 µg/plate	17.67
  	1111.11 µg/plate	16.00
  	3333.33 µg/plate	17.67
  	10000.00 µg/plate	14.00
  	Positive control	352.33

  Strain	Treatment	Mean counts
  TA 98	Negative control	28.33
  	123.46 µg/plate	32.00
  	370.37 µg/plate	35.67
  	1111.11 µg/plate	30.67
  	3333.33 µg/plate	44.33
  	10000.00 µg/plate	31.00
  	Positive control	2263.67

  Strain	Treatment	Mean counts
  TA 1537	Negative control	8.00
  	123.46 µg/plate	6.33
  	370.37 µg/plate	8.00
  	1111.11 µg/plate	6.67
  	3333.33 µg/plate	7.00
  	10000.00 µg/plate	8.33
  	Positive control	268.00

  Strain	Treatment	Mean counts
  TA 102	Negative control	254.33
  	123.46 µg/plate	323.67
  	370.37 µg/plate	363.33
  	1111.11 µg/plate	395.33
  	3333.33 µg/plate	376.33
  	10000.00 µg/plate	256.33
  	Positive control	2453.67

• Original experiments without metabolic activation

  Strain	Treatment	Mean counts
  TA 100	Negative control	111.67
  	123.46 µg/plate	119.00
  	370.37 µg/plate	112.33
  	1111.11 µg/plate	137.67
  	3333.33 µg/plate	126.00
  	10000.00 µg/plate	113.67
  	Positive control	1090.67

   

  Strain	Treatment	Mean counts
  TA 1535	Negative control	14.67
  	123.46 µg/plate	14.33
  	370.37 µg/plate	11.33
  	1111.11 µg/plate	12.33
  	3333.33 µg/plate	11.67
  	10000.00 µg/plate	15.33
  	Positive control	1127.00

   

   

  Strain	Treatment	Mean counts
  TA 98	Negative control	17.67
  	123.46 µg/plate	19.67
  	370.37 µg/plate	20.67
  	1111.11 µg/plate	21.33
  	3333.33 µg/plate	24.00
  	10000.00 µg/plate	35.33
  	Positive control	1460.00

   

  Strain	Treatment	Mean counts
  TA 1537	Negative control	8.00
  	123.46 µg/plate	8.00
  	370.37 µg/plate	9.00
  	1111.11 µg/plate	10.00
  	3333.33 µg/plate	9.67
  	10000.00 µg/plate	9.33
  	Positive control	2477.00

   

  Strain	Treatment	Mean counts
  TA 102	Negative control	204.33
  	123.46 µg/plate	282.00
  	370.37 µg/plate	191.67
  	1111.11 µg/plate	323.67
  	3333.33 µg/plate	305.67
  	10000.00 µg/plate	127.67
  	Positive control	1416.67

   

• Original experiments without metabolic activation

Strain	Treatment	Mean counts
TA 100	Negative control	145.00
	123.46 µg/plate	128.67
	370.37 µg/plate	141.67
	1111.11 µg/plate	143.00
	3333.33 µg/plate	152.33
	10000.00 µg/plate	147.67
	Positive control	2587.00

 

Strain	Treatment	Mean counts
TA 1535	Negative control	12.33
	123.46 µg/plate	13.00
	370.37 µg/plate	19.00
	1111.11 µg/plate	15.00
	3333.33 µg/plate	15.67
	10000.00 µg/plate	9.00
	Positive control	327.33

 

 

Strain	Treatment	Mean counts
TA 98	Negative control	45.00
	123.46 µg/plate	41.33
	370.37 µg/plate	35.67
	1111.11 µg/plate	38.00
	3333.33 µg/plate	50.67
	10000.00 µg/plate	45.67
	Positive control	1992.33

 

Strain	Treatment	Mean counts
TA 1537	Negative control	14.67
	123.46 µg/plate	9.00
	370.37 µg/plate	15.67
	1111.11 µg/plate	12.00
	3333.33 µg/plate	16.00
	10000.00 µg/plate	7.67
	Positive control	309.67

 

Strain	Treatment	Mean counts
TA 102	Negative control	222.33
	123.46 µg/plate	248.33
	370.37 µg/plate	239.33
	1111.11 µg/plate	197.00
	3333.33 µg/plate	24.00
	10000.00 µg/plate	127.67
	Positive control	1600.33

 

Confirmatory experiments without metabolic activation

 

Strain	Treatment	Mean counts
TA 100	Negative control	139.67
	123.46 µg/plate	130.33
	370.37 µg/plate	135.67
	1111.11 µg/plate	124.67
	3333.33 µg/plate	134.67
	10000.00 µg/plate	146.00
	Positive control	1197.00

 

Strain	Treatment	Mean counts
TA 1535	Negative control	12.00
	123.46 µg/plate	19.33
	370.37 µg/plate	20.00
	1111.11 µg/plate	21.67
	3333.33 µg/plate	24.00
	10000.00 µg/plate	8.33
	Positive control	947.00

 

 

Strain	Treatment	Mean counts
TA 98	Negative control	28.00
	123.46 µg/plate	24.67
	370.37 µg/plate	29.33
	1111.11 µg/plate	26.33
	3333.33 µg/plate	40.33
	10000.00 µg/plate	59.00
	Positive control	1750.00

 

Strain	Treatment	Mean counts
TA 1537	Negative control	8.67
	123.46 µg/plate	12.33
	370.37 µg/plate	13.67
	1111.11 µg/plate	9.33
	3333.33 µg/plate	10.67
	10000.00 µg/plate	12.67
	Positive control	2836.33

 

Strain	Treatment	Mean counts
TA 102	Negative control	291.33
	123.46 µg/plate	299.00
	370.37 µg/plate	381.33
	1111.11 µg/plate	354.67
	3333.33 µg/plate	331.00
	10000.00 µg/plate	228.00
	Positive control	1543.67

 

Applicant's summary and conclusion

Conclusions:

FAT 20042/D (Neolan Rosa BE ZP feucht) exerted a marginal mutagenic effect on strain TA 98 of Salmonella typhimurium without metabolic activation only.

Executive summary:

FAT 20042/D, identified as black-reddish humid pieces, purity approx. 50 %, batch no. 276, was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at five concentrations in the range of 123.5 to 10000 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 123.5 to 10000 µg/plate. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. FAT 20042/D led to a slight increase in the incidence of histidine-prototrophic mutants in comparison to the negative control in strain TA 98 at the highest concentration in both original as well as confirmatory experiments when tested in absence of metabolic activation. No relevant effects occurred with the other strains. Based on the results of these experiments and using standard evaluation criteria, it is concluded that FAT 20042/D (Neolan Rosa BE ZP feucht) exerted a marginal mutagenic effect on strain TA 98 of Salmonella typhimurium without metabolic activation only.