[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Acid Red 195 showed weak mutagenic effects in the Salmonella typhimurium reverse mutation assay, however, failed to induce gene mutations in mammalian cells in vitro. It also had positive results in an in vitro mammalian chromosome aberrations assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

07 February, 1994 to 02 June, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 tester strains tested; no tester strain to detect cross-linking mutagens was included

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: 175
- Expiration date of the batch: June 30, 1998

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable

Target gene:

Histidine gene

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Salmonella typhimurium (TA 98, TA 1535, TA 1537) were obtained from Prof. B. Ames, Berkeley, USA. Strain TA 100 was obtained from Dr. M. Schüpbach, Hoffmann-La Roche Limited, Basel, Switzerland.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Original and confirmatory experiment: 61.73, 185.19, 555.56, 1666.67 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Bidistilled water

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 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: three per test substance concentration and controls

OTHER EXAMINATIONS:
Colony counting and scoring of the plates
Colonies were counted electronically using an Artek Colony Counter (Fisher Scientific), or manually where minor agar damage or test chemical precipitates or strong coloration of the agar plates 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. At present the use of statistical methods concerning this particular test system is not generally recommended

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

other: Weak mutagenic

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 5000.0 µg/plate with and without metabolic activation.

Mutagenicity test, original experiment
In the experiment performed with metabolic activation, treatment of strain TA 1537 with FAT 20042/C (Neolan Rosa BE roh trocken SFO) led to a marginal increase in the number of revertant counts at the concentration of 5000 µg/plate . In the experiments conducted without activation on strains TA 98 and TA 1537, a similar effect was observed at the same concentration. No effects occurred on the other strains.

Mutagenicity test, confirmatory experiment
In the experiment performed without metabolic activation on strain TA 98, treatment with FAT 20042/C (Neolan Rosa BE roh trocken SFO) led to a slight increase in the number of back-mutants at the concentrations of 555.6 µg/plate and above . No increased revertant counts were observed with the other strains . The marginal effect observed in the original experiment with and without activation on strain TA 1537 could not be reproduced.

In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced with increasing concentration. Therefore, the test substance exerted no toxic effect on the growth of the bacteria.

SUMMARY OF THE MUTAGENICITY EXPERIMENTS

• Original experiments with metabolic activation

  Strain	Treatment	Mean counts
  TA 100	Negative control	126.67
  	61.73 µg/plate	132.00
  	185.19 µg/plate	139.33
  	555.56 µg/plate	140.00
  	1666.67 µg/plate	115.33
  	5000.00 µg/plate	99.00
  	Positive control	1775.33

  Strain	Treatment	Mean counts
  TA 1535	Negative control	16.67
  	61.73 µg/plate	15.33
  	185.19 µg/plate	17.67
  	555.56 µg/plate	12.00
  	1666.67 µg/plate	14.33
  	5000.00 µg/plate	14.00
  	Positive control	340.67

  Strain	Treatment	Mean counts
  TA 98	Negative control	29.67
  	61.37 µg/plate	37.33
  	185.19 µg/plate	29.00
  	555.56 µg/plate	26.00
  	1666.67 µg/plate	40.33
  	5000.00 µg/plate	36.33
  	Positive control	1388.67

  Strain	Treatment	Mean counts
  TA 1537	Negative control	7.00
  	61.73 µg/plate	6.00
  	185.19 µg/plate	4.67
  	555.56 µg/plate	9.33
  	1666.67 µg/plate	9.67
  	5000.00 µg/plate	14.67
  	Positive control	239.00

• Original experiments without metabolic activation

  Strain	Treatment	Mean counts
  TA 100	Negative control	105.33
  	61.73 µg/plate	105.33
  	185.19 µg/plate	112.33
  	555.56 µg/plate	111.33
  	1666.67 µg/plate	116.00
  	5000.00 µg/plate	101.67
  	Positive control	816.00

   

  Strain	Treatment	Mean counts
  TA 1535	Negative control	19.00
  	61.73 µg/plate	20.67
  	185.19 µg/plate	25.33
  	555.56 µg/plate	20.00
  	1666.67 µg/plate	18.67
  	5000.00 µg/plate	9.00
  	Positive control	754.67

   

   

  Strain	Treatment	Mean counts
  TA 98	Negative control	26.00
  	61.73 µg/plate	20.67
  	185.19 µg/plate	20.67
  	555.56 µg/plate	28.00
  	1666.67 µg/plate	40.00
  	5000.00 µg/plate	53.67
  	Positive control	1462.67

   

  Strain	Treatment	Mean counts
  TA 1537	Negative control	8.33
  	61.73 µg/plate	11.67
  	185.19 µg/plate	12.00
  	555.56 µg/plate	10.00
  	1666.67 µg/plate	13.00
  	5000.00 µg/plate	19.33
  	Positive control	1594.33

   

• Confirmatory experiments with metabolic activation

Strain	Treatment	Mean counts
TA 100	Negative control	159.00
	61.73 µg/plate	177.33
	185.19 µg/plate	159.00
	555.56 µg/plate	168.67
	1666.67 µg/plate	180.00
	5000.00 µg/plate	170.00
	Positive control	1373.00

 

Strain	Treatment	Mean counts
TA 1535	Negative control	18.33
	61.73 µg/plate	18.33
	185.19 µg/plate	8.00
	555.56 µg/plate	12.67
	1666.67 µg/plate	15.67
	5000.00 µg/plate	11.33
	Positive control	437.33

 

 

Strain	Treatment	Mean counts
TA 98	Negative control	42.67
	61.73 µg/plate	33.33
	185.19 µg/plate	39.00
	555.56 µg/plate	42.00
	1666.67 µg/plate	43.67
	5000.00 µg/plate	53.00
	Positive control	1843.67

 

Strain	Treatment	Mean counts
TA 1537	Negative control	7.33
	61.73 µg/plate	8.33
	185.19 µg/plate	6.00
	555.56 µg/plate	6.00
	1666.67 µg/plate	12.00
	5000.00 µg/plate	13.67
	Positive control	409.33

 

 

Confirmatory experiments without metabolic activation

 

Strain	Treatment	Mean counts
TA 100	Negative control	171.67
	61.73 µg/plate	173.00
	185.19 µg/plate	171.67
	555.56 µg/plate	176.67
	1666.67 µg/plate	173.33
	5000.00 µg/plate	164.33
	Positive control	866.33

 

Strain	Treatment	Mean counts
TA 1535	Negative control	11.33
	61.73 µg/plate	8.33
	185.19 µg/plate	12.67
	555.56 µg/plate	10.67
	1666.67 µg/plate	12.33
	5000.00 µg/plate	8.33
	Positive control	777.33

 

 

Strain	Treatment	Mean counts
TA 98	Negative control	16.67
	61.73 µg/plate	20.00
	185.19 µg/plate	26.33
	555.56 µg/plate	33.67
	1666.67 µg/plate	61.67
	5000.00 µg/plate	72.00
	Positive control	1589.33

 

Strain	Treatment	Mean counts
TA 1537	Negative control	10.00
	61.73 µg/plate	6.33
	185.19 µg/plate	12.67
	555.56 µg/plate	12.00
	1666.67 µg/plate	12.00
	5000.00 µg/plate	16.00
	Positive control	1728.00

 

Conclusions:

FAT 20042/C (Neolan Rosa BE roh trocken SFO) exerted a weak mutagenic effect on strain TA 98 of Salmonella typhimurium without metabolic activation only.

Executive summary:

An in vitro bacterial reverse mutation study was performed to investigate the potential of FAT 20042/C (ca. 80 % purity) to induce gene mutations according to OECD Guideline 471, EU Method B.14 and EPA OTS 798.5265 in compliance with GLP. The concentration range to be tested was determined in a preliminary toxicity study. The substance was tested for mutagenic effects without and with metabolic activation at doses ranging from 20.6 to 5000 µg active ingredient/plate on Salmonella typhimurium strains TA 100, TA 1535, TA 98 and TA 1537. An independent repetition of the experiments was performed with the same concentrations.

Mutagenicity test, original experiment: In the experiment performed with metabolic activation, treatment of strain TA 1537 with FAT 20042/C (Neolan Rosa BE roh trocken SFO) led to a marginal increase in the number of revertant counts at the concentration of 5000 µg/plate. In the experiments conducted without activation with strains TA 98 and TA 1537, a similar effect was observed at the same concentration. No effects occurred on the other strains.

Mutagenicity test, confirmatory experiment: In the experiment performed without metabolic activation, treatment with FAT 20042/C (Neolan Rosa BE roh trocken SFO) with strain TA 98 led to a slight increase in the number of back-mutants at the concentrations of 555.6 µg/plate and above. No increased revertant counts were observed with the other strains. The marginal effect observed in the original experiment with and without metabolic activation on strain TA 1537 could not be reproduced. Based on the results of these experiments and using standard evaluation criteria, it is concluded that FAT 20042/C (Neolan Rosa BE roh trocken SFO) exerted a weak mutagenic effect on strain TA 98 of Salmonella typhimurium without metabolic activation only.

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

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

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

Target gene:

Histidine gene

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)

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.

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

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

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

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

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

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.

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

 

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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell gene mutation assay

Specific details on test material used for the study:

NoneSOURCE OF TEST MATERIAL
- Source and batch No.of test material: 175
- Expiration date of the batch: June 30, 1998

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable

Target gene:

Chinese Hamster Cells V79

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F10 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

Pre-experiment:
Cytotoxicity test: with and without metabolic activation: 0.24 to 500 µg/mL

Mutagenicity test:

Original experiment:
Range with metabolic activation: 14.81 to 400.0 µg/ml
Range without metabolic activation: 7.41 to 200.0 µg/ml

Confirmatory experiment:
Range with metabolic activation: 11.11 to 300.0 µg/ml
Range without metabolic activation: 9.26 to 250.0 µg/ml

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with metabolic activation

Details on test system and experimental conditions:

DURATION
- Exposure duration: Overnight
- Expression time (cells in growth medium): 2.5-5.0E+6 cells

- SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)
- STAIN (for cytogenetic assays): Giemsa

DETERMINATION OF CYTOTOXICITY: Cell count

Rationale for test conditions:

None

Evaluation criteria:

All mutant frequencies were normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency of the viability cultures was lower than 15%, the corresponding mutant frequency was usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mean mutant factor, which was defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures,was calculated.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDY
In the preliminary toxicity test conducted with and without metabolic activation, 12 concentrations of FAT 20042/C were tested. The concentrations selected ranged from 0.24 to 500.0 µg/ml and separated
by 2-fold intervals. In the part with metabolic activation, the highest concentration was toxic. The next lower concentration of 250.0 µg/ml produced an acute growth inhibition of 50.55%. In the part without metabolic activation FAT 20042/C exerted a complete growth inhibitory effect at the concentration of 500.0 µg/ml. The next lower concentration of 250.0 µg/ml revealed an acute growth inhibitory effect of 95.12%.

MAIN EXPERIMENTS - Toxicity
Accordingly, four concentrations were selected for the original experiment ranging from 14.81 to 400.0 µg/ml and from 7.41 to 200.0 µg/ml in the presence and absence of metabolic activation, respectively.
In the part with metabolic activation the highest concentration was toxic. At the next lower concentration, growth inhibition determined after treatment and expression revealed values of 19.87% and less than 9.06%.
In the absence of metabolic activation the mean growth inhibitory effect determined after treatment was 45.09% at the highest concentration. After the expression period the determined cytotoxicity revealed a value of less than 10.26%.
Due to the pronounced growth inhibitory response in the part with metabolic activation the concentration range was decreased in the confirmatory experiment ranging from 11.11 to 300.0 µg/ml. In
the part without metabolic activation a concentration range of 9.26 to 250.0 µg/ml was selected in order to reach a complete toxicity at the highest concentration. In the presence of metabolic activation the highest concentration was still fully toxic. At the next lower concentration, the mean growth inhibition determined after treatment and expression revealed values of 74.96 and 24.11%. In the part without activation, the highest concentration proved fully growth inhibiting. Growth inhibition at the next lower concentration of 83.33 µg/ml was less than 15.49% and less than 12.89% after treatment and expression respectively.

MAIN EXPERIMENTS - Mutagenicity
In the presence and absence of metabolic activation, no significant increase in mutant frequency was observed at any concentration level of FAT 20042/C tested in the original or the confirmatory experiment in comparison with the negative control.
The statistical significance obtained with the two higher concentrations of the original experiment without metabolic activation is considered to be of no biological relevance. The respective mutant frequency values of 5.83 and 5.94E+6 are clearly within the range of the historical negative controls.
The positive controls induced a clear increase in mutant frequency.

Remarks on result:

other: all strains/cell types tested

None

Conclusions:

FAT 20042/C and its metabolites did not show any mutagenic activity in this forward mutation system.

Executive summary:

FAT 20042/C, was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours.

Mutagenicity test with metabolic activation: The original experiment was performed at the following concentrations: 14.81, 44.44, 133.33 and 400.0 µg/ml. The highest concentration proved fully growth inhibiting after treatment. At the next lower concentration, the growth inhibiting values found at after treatment and expression were 19.87 % and less than 9.06 %, respectively. In the confirmatory experiment, the concentrations applied were 11.11, 33.33, 100 and 300 µg/ml. The highest concentration was again fully growth inhibiting after treatment. At the next lower concentration a mean acute growth inhibition of 74.96 % was obtained. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

Mutagenicity test without metabolic activation: The original experiment was performed at the following concentrations: 7.41, 22.22, 66.67 and 200 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 45.09 %* and less than 10.26 %* respectively. In the confirmatory experiment the concentrations applied were 9.26, 27.78, 83.33 and 250 µg/ml. The highest concentration proved fully growth inhibiting after treatment. At the next lower concentration, an acute growth inhibitory effect of less than 15.49 % was obtained. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

Hence, based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that FAT 20042/C and its metabolites did not show any mutagenic activity in this forward mutation system.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Guideline followed

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

NoneSOURCE OF TEST MATERIAL
- Source and lot No.of test material: 0000657250 (India)
- Expiration date of the lot: 04 August 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2 to 8 °C, protected from light

Target gene:

chromosomes

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the concentrations tested were 0.2, 0.6, 2, 6, 20, 60, 200, 600 and 2000 µg/mL. In the chromosome aberration assay, the concentations tested were: 250, 500, 1000, 1600, 1800, 1900, and 2000 µg/mL in the non-activated 4-hour exposure group; 800, 1000, 1200, 1400, 1600 and 1800 µg/mL in the S9-activated 4 h exposure group; and 50, 100, 250, 500, 600, 700, and 800 µg/mL in the non-activated 20 h exposure group.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: CHO cells were exposed to the test and control articles for 4 and 20 hours without S9 and for 4 hours with S9, and rinsed. Cells were harvested 20 hours (±30 minutes) after initiation of treatment, which corresponds to 1.5 normal cell cycles.

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 2 in the chromosome aberration assay

NUMBER OF CELLS EVALUATED: a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture), whenever possible

DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in cell counts

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

Rationale for test conditions:

None

Evaluation criteria:

Toxicity induced by treatment was based upon inhibition of cell growth and was reported for the cytotoxicity and chromosome aberration portions of the study. The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but were not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.

A test article was considered positive if it induced a statistically significant and dose dependent increase in the frequency of aberrant metaphases (p ≤0.05), and the results were outside the 95 % control limits of the historical negative control data. If only one criterion was met (statistically significant or dose dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.

Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgement and clearly reported and described any such considerations.

Statistics:

Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

For the induction of structural chromosome aberrations in the 20-hour group without S9

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at doses ≥ 1800 µg/mL in the non activated 4 hour exposure group; at doses ≥ 1400 µg/mL in the S9 activated 4-hour exposure group. In the non-activated 20-hour exposure group, 48% cytotoxicity was observed at 800 µg/mL.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Although requisite toxicity (55 ± 5 % reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non-activated 20-hour exposure group, 48 % cytotoxicity was observed at 800 µg/mL, which was within the rounding margin. Therefore, the Study Director selected 800 µg/mL as the top dose to score in the non activated 20-hour exposure group.

Remarks on result:

other: all strains/cell types tested

None

Conclusions:

FAT 20042/E was positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in the non-activated 20-hour exposure group. FAT 20042/E was negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated 4-hour exposure groups.

Executive summary:

The test substance, FAT 20042/E, was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster ovary (CHO) cells in both the absence and presence of an of an exogenous metabolic activation system. Water was used as the vehicle. In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL, which was the limit dose for this assay. Cytotoxicity (≥50 % reduction in cell growth index relative to the vehicle control) was observed at 2000 µg/mL in the non activated and S9-activated 4 hour exposure groups; and at doses ≥600 µg/mL in the non activated 20-hour exposure group. Based upon these results, the doses chosen for the chromosome aberration assay ranged from 250 to 2000 µg/mL for the non activated 4-hour exposure group, from 800 to 1800 µg/mL for the S9-activated 4-hour exposure group, and from 50 to 800 µg/mL for the non-activated 20-hour exposure group. In the chromosome aberration assay, cytotoxicity (55 ± 5 % reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 1800 µg/mL in the non activated 4 hour exposure group; at doses ≥1400 µg/mL in the S9 activated 4-hour exposure group. Although requisite toxicity (55 ± 5 % reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non-activated 20-hour exposure group, 48 % cytotoxicity was observed at 800 µg/mL, which was within the rounding margin. Therefore, 800 µg/mL was selected as the top dose to score in the non activated 20-hour exposure group. The doses selected for evaluation of chromosome aberrations were 500, 1000, and 1800 µg/mL for the non activated 4 hour exposure group; 800, 1000, and 1400 µg/mL for the S9 activated 4-hour exposure group; and 100, 250, and 800 µg/mL for the non activated 20-hour exposure group. In the non-activated and S9-activated 4-hour exposure groups, no significant or dose dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed (p >0.05; Fisher’s Exact and Cochran-Armitage tests). In the non-activated 20-hour exposure group, statistically significant and dose-dependent increases in structural aberrations (5.0 and 5.7 %) were observed at 250 and 800 µg/mL, respectively (p  ≤0.01; Fisher’s Exact test and p  ≤0.05; Cochran-Armitage test). No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed in non-activated 20-hour treatment group. The results for the positive and vehicle controls indicate that all criteria for a valid assay were met. These results indicate FAT 20042/E was positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in the non-activated 20-hour exposure group. FAT 20042/E was negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated 4-hour exposure groups.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Testing proposal

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

An in vitro bacterial reverse mutation assay was performed to investigate the potential of FAT 20042/D to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP. In the original and confirmatory experiments 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. No relevant effects were observed with the other strains.

A second bacterial reverse mutation test also conducted according to OECD 471, demonstrated similar results. In the original as well as confirmatory experiments performed without metabolic activation, treatment of strain TA 98 with FAT 20042/C led to a slight increase in the number of back-mutants at the concentrations of 555.6 µg/plate and above. No relevant increased revertant counts were observed with the other strains. In summary, Acid Red 195 was found to be mutagenic in the bacterial reverse mutation assays.

 

To confirm this, a test on mutagenic effects in an eukaryotic cellular system was conducted. In this in vitro HPRT test conducted according to OECD Guideline 476, comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance in the presence and absence of metabolic activation, revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG). Thus, it was demonstrated that FAT 20042/D did not induce mutagenic effects in this eukaryotic cellular assay, countering the weak mutagenic effects observed in the prokaryotic assays.

 

To investigate if the test substance is likely to induce changes on a chromosomal level, an in vitro mammalian chromosomal aberration assay was conducted according to OECD Guideline 473 and in accordance with GLP. FAT 20042/E did not lead to the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated 4-hour exposure groups. However, it was found to be positive for the induction of structural chromosome aberrations and negative for the induction of numerical chromosome aberrations in the non-activated 20-hour exposure group.

 

Based on the available information, Acid Red 195 may have the potential to induce mutations in bacterial cells, however, it did not induce mutations in the mammalian cells, and hence considered not mutagenic. However, Acid Red 195 was found to be clastogenic, while not being aneugenic, in an in vitro mammalian chromosomal aberration assay. Currently no other data are available to support or negate the clastogenic potential of the substance. Data generated on similar substances with a high structural relationship like Acid Blue 158 (EC 228-875-6 and EC 275-033-9), Acid Blue 314 (EC 289-530-3), Acid Yellow 104 (EC 276-702-8) and Acid Orange 074 (EC 266-657-2) do not provide a clear picture. While Ames test results for Acid Yellow 104 and Acid Blue 314 have been clearly negative,  Acid Red 195 gave a clear response in TA 98 in the presence of metabolic activation. Acid Blue 158 showed a very weak increase in TA 98 at the highest test concentration without metabolic activation while in presence it was clearly negative. Also a second test performed with another test sample of the same material gave clear negative response for all strains and conditions tested. Therefore, it looks like the results regarding mutagenicity obtained with the test material in the Ames test seems to be not reliable. Unfortunately, no data on clastogenicity for the comparable structures are currently available. Therefore this endpoint needs maybe further investigation. Hence, proposal for further testing to clarify if the substance is capable of exerting clastogenic action in mammals is being included in the dossier. To this effect, a testing proposal for conducting micronucleus assay according to OECD Guideline 474 is included in this dossier.

Justification for classification or non-classification

Based on the above mentioned results, a conclusion on the mutagenic and clastogenic activity of Acid Red 195 could not be drawn without the generation of additional data in vivo. Before the results of these tests are available no classification regarding genotoxicity will be applied, since data of comparable chemistry demonstrated in several cases that such molecules are sometimes likely to show false negative results in those test systems.