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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
Study period:
18 February 1994 to 7 March 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No Prival modification, 4 strains tested

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
First Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 4 71, "Salmonella typhimurium, Reverse Mutation Assay", adopted May 26, 1983
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EEC Directive 92/69, L 383 A, Annex V, B 14, dated December 29, 1992.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
According to the results of this pre-experiment the concentrations applied in the main experiments were chosen. The concentration range covered two logarithmic decades.
The maximum concentration was 5000.0 μg/plate in experiment I and 1000.0 μg/plate in experiment I. The concentration range included two logarithmic decades. In this study six adequately spaced concentrations were tested. Two independent experiments were performed.
As the results of the pre-experiment were in accordance with the criteria described, these data are reported as a part of the main experiment I.
According to the dose selection criteria the test article was tested at the following concentrations:
Exp. I: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 μg/plate
Exp. II: 33.3; 66.6; 100.0; 333.3; 666.6; and 1000.0 μg/plate
Vehicle / solvent:
On the day of experiment, the test article was dissolved in DMF.
The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
THE TEST SYSTEM
Characterisation of the Salmonella typhimurium Strains
The strains are derived from s. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.

In summary, the mutations of the TA strains used in this study can be described as follows:
Salmonella typhimurium
TA 1537: his c 3076; rfa-; uvrB-; frame shift mutations
TA 98: his D 3052; rfa-; uvrB-;R-factor: frame shift mutations
TA 1535: his G 46; rfa-; uvrB-; base-pair substitutions
TA 100: his G 46; rfa-; uvrB-;R-factor: base-pair substitution

Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in C C R according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

The bacterial strains were obtained from Dr. Heinz Trager, Knoll AG, D-67008 Ludwigshafen, F.R.G.

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Precultures
From the thawed ampoules of the strains 0.5 ml suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 ml ampicillin was added to the strains
TA 98 and TA 100*. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial culture was incubated in a shaking water bath for 10 hours at 37 °C.
* in deviation to protocol, updating

Selective Agar
2. 0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium.
Sterilisations were performed at 121° C in an autoclave.

Overlay Agar
The overlay agar contains per litre:
6.0 g Merck Agar Agar*
6.0 g NaCl*
10.5 mg L-histidine x HCl X H20*
12.2 mg biotin*
* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121° C in an autoclave.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
The bacteria used in these assays do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

S9 (Preparation by C C R)
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats, strain WU (Charles River Wiga GmbH, D-97633 Sulzfeld, F.R.G.; weight approx. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Ettlingen, F.R.G.) in olive oil 5 days previously.
After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCI and centrifuged cold at 9, 000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80° C. Small numbers of the ampoules are kept at -20° C for only one week before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 München:
Bio-Rad protein assay, Catalogue 500 000 6.
The protein concentration in the S9 preparation was 38.2 mg/ml (lot no. 220993).

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v. The composition of the co-factor solution was concentrated to yield the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
5 mM NADP
in 100 mM ~odium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same for the experiment I (plate incorporation test).
Toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

EXPERIMENTAL PERFORMANCE
For each strain and dose level, including the controls three plates were used-as a minimum.
The following materials were mixed in a test tube and poured onto the selective agar plates:

100 μl
Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),

500 μl
S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),

100 μl
Bacteria suspension (cf. test system, pre-culture of the strains),

2000 μl
Overlay agar

After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

DATA RECORDING
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben; F.R.G.). The counter was connected to an IBM AT compatible PC with printer which printed out the individual values and the means from the plates for each concentration together with standard deviations and (enhancement factors as compared to the spontaneous reversion rates (see tables of results). If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.
Rationale for test conditions:
The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations.
In spite of great differences between bacterial and eukaryotic cells with respect to structure and function there is a relationship between mutagenicity in bacteria and carcinogenicity in mammals described in literature.
Reverse mutation assays determine the frequency with which an agent repairs or suppresses the effect of the forward mutation. The genetic target presented to an agent is therefore small, specific and selective. Several bacterial strains, or a single strain with multiple markers are necessary to overcome the effects of mutagen specificity. The reversion of bacteria from growth-dependence on a particular amino acid to growth in the absence of that amino acid (reversion from auxothrophy to prototrophy) is the most widely used marker. The Salmonella typhimurium histidine (his) reversion system measures his- --> his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.
According to the direct plate incorporation or the pre-incubation method the bacteria are exposed to the test article with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies are counted.
To establish a dose response effect at least 5 dose levels with adequately spaced intervals are to be tested. The maximum dose level was 5000.0 μg/plate (active ingredient), unless limited by toxicity or solubility of the test article.
To validate the test, reference mutagens are tested in parallel to the test article.
Evaluation criteria:
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative controls and test plates
- normal range of spontaneous reversion rates.
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
A test article is considered mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100.

The plates with the test article showed normal background growth up to 5000.0 μg/plate in strain TA 98 and TA 100, respectively.
According to the dose selection criteria, the test article was tested at the following concentrations:
Exp. I: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 μg/plate
Exp. II: 33.3; 66.6; 100.0; 333.3; 666.6; and 1000.0 μg/plate
(active ingredient)

Any other information on results incl. tables

PRE-EXPERIMENT FOR TOXICITY

Substance

Concentration per plate

μg

Revertant per plate

TA 98

TA 100

-

+

-

+

Negative control

-

34

41

145

200

Solvent control

-

28

29

147

157

4-NOPD

10.0

501

/

/

/

Sodium azide

10.0

/

/

751

/

2-aminoanthracene

2.5

/

330

/

664

Test article

3.3

10.0

33.3

100.0

333.3

1000.0

2500.0

5000.0

52

169

443

457

1174

1119

1662

960

41

53

70

147

478

860

1176

1463

133

237

255

254

244

325

430

417

167

180

228

373

383

392

426

497

* - without S9 mix; + with S9 mix

/ not performed

 

SUMMARY OF RESULTS

 

Without S9 mix

Revertant/plate

Mean from three plates

Concentration

μg/plate

TA 1535

TA 1537

TA 98

TA 100

I

II

I

II

I

II

I

II

Neg. contr.

17

14

9

9

34

38

145

166

Solv. Contr.

13

17

8

5

28

29

147

132

33.3

16

18

10

8

443

532

255

248

66.6

/

10

/

12

/

1215

/

391

100.0

16

21

27

41

457

862

254

341

333.3

21

24

48

23

1174

1021

244

357

666.6

/

10

/

12

/

1215

/

391

1000.0

10

2

71

0

1119

1390

325

456

2500.0

7

/

74

/

1662

/

430

/

5000.0

0

/

21

/

960

/

417

/

Positive controls

Sodium azide

(10 μg/plate)

782

941

 

 

 

 

751

889

4-nitro-o-pheylene-diamine

(10 μg/plate)

 

 

71

91

501

459

 

 

/ = not performed

 

With S9 mix

Revertant/plate

Mean from three plates

Concentration

μg/plate

TA 1535

TA 1537

TA 98

TA 100

I

II

I

II

I

II

I

II

Neg. contr.

16

22

11

13

41

44

200

171

Solv. Contr.

19

23

6

15

29

38

157

150

33.3

18

23

23

24

70

77

228

226

66.6

/

21

/

41

/

115

/

304

100.0

23

28

53

64

147

132

373

470

333.3

39

32

245

215

478

656

383

663

666.6

/

62

/

223

/

1233

/

558

1000.0

79

95

150

269

860

1119

392

552

2500.0

88

/

54

/

1176

/

426

/

5000.0

42

/

11

/

1463

/

497

/

Positive controls

2-aminoanthracene

(2.5 μg/plate)

214

160

82

58

330

142

664

643

/ = not performed

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98 and TA 100.
Therefore, the test item is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

This study was ·performed to investigate the potential of FAT 36'036/G to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100.

 

The assay was performed in two independent experiments, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

 

Exp. I: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 μg/plate

Exp. II: 33.3; 66.6; 100.0; 333.3; 666.6; and 1000.0 μg/plate

(active ingredient)

 

Distinct toxic effects evidenced by a reduction in spontaneous or induced revertant colonies occurred with and without S9 mix in experiment I and II in nearly all strains used.

 

The plates incubated with the test article showed normal background growth up to 5000.0 μg/plate (experiment I) and 1000.0 μg/plate (experiment II) with and without S9 mix in all strains used.

 

Reproducible, dose-dependent increases in revertant colony numbers were obtained with the tester strains TA 1535 (with S9 mix), TA 1537 (with and without S9 mix), TA 98 (with and without S9 mix) and TA 100 (with and without S9 mix).

 

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

CONCLUSION

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98 and TA 100.

 

Therefore, FAT 36'036/G; Terasil Rot 3BL roh feucht (laborgetrocknet) is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.