Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
04 March 1994 to 18 April 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to recent EU & OECD test guidelines in compliance with GLP.

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)
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:
Conducted in accordance with Salmonella/Microsome Test Plate Incorporation and the Privall Mitchell methodology.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Reactive Blue FC 75311 (tested as sodium salt)

Method

Target gene:
Bacterial reverse mutation assay
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: histidine auxotrophic
Metabolic activation:
with and without
Metabolic activation system:
Araclor 1254 induced rat liver, S9
Test concentrations with justification for top dose:
16, 50, 158, 500, 1581, 5000 ug/l
Vehicle / solvent:
The solvent employed for the Congo red positive control was deionized water, and for other positive controls DMSO. Reactive Blue FC 75311 was dissolved in deionized water.
Solvent use is based on historical acceptability from within the laboratory.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / deionised water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, 2-aminoanthracene. Congo red and benzidine were used instead of 2-aminoanthracene for TA 98 in the Prival modification.
Details on test system and experimental conditions:
Positive Controls:
Sodium azide (Na-azide, SERVA), order no. 30175 (Control:D), a direct-acting mutagen used as specific positive control for TA 153 5.

Nitrofurantoin (NF, SERVA), order no. 30600 (Control:A), a direct-acting mutagen used as specific positive control for TA 100.

4-nitro-l, 2-phenylene diamine (4-NPDA, Fluka) , Analysis no. 247722/1 194, a direct-acting mutagen used as specific positive control for TA 98.

2-aminoanthracene (2-AA, EGA-Chemie), batch no. 7413406, a promutagen which reverts all the strains and serves as a control for the activating effect of the S9 mix.

Benzidine (Serva) , order no. 14542... (Control: H8) , a promutagen used as specific positive control for TA 98 in the modification of Prival and Mitchell (1982).

Congo red (Serva), order no 527215 (Control: A9) , a promutagen used as specific positive control for TA 93 in the modification of Prival and Mitchell (1982).

The positive controls sodium azide, nitrofurantoin and 4-nitro-1,2-phenylene diamine were only used without S9 mix; the positive controls Congo red, benzidine and 2-aminoanthracene were only used with S9 mix.

Description of Test Strains
Histidine-deficient mutants of Salmonella typhimurium served' as indicators to demonstrate point mutagenic effects. These strains were selected specifically for the Salmonella/ microsome test. Since point mutations can be divided into two basic classes, base-pair substitutions and frameshift muta¬tions, several strains were used which cover both types.

These included the strains selected by Ames et al. (1973b), Salmonella typhimurium TA 1535 and TA 1537, as well as Salmonella typhimurium TA 100 and TA 98, developed by McCann et al. (1975b). TA 1535 and TA 100 bear the base-pair substitu¬tion, his G 46, and TA 100 additionally contains the plasmid pKM 101. This R factor, also contained in TA 98, codes for an ampicillin resistance and should raise the sensitivity of both strains. TA 1537 and TA 98 bear frameshift markers. TA 1537 exhibits the +1 mutant, his c 3076, while TA 98 bears the + 2 type, his D 3052.
Furthermore, all the strains have two other properties in common, which should increase their sensitivity. Firstly, they are deep rough, i.e. partly deficient in lipopolysaccharide side chains in their cell walls, enabling larger molecules to penetrate the bacterial cell wall and produce muta-tions, secondly, their reduced UV repairability partly prevents the repair of damage, such as that triggered by UV light (e.g. thymine dimers), and thus gives rise to mutations.
Whereas TA 1535 was used in addition to TA 100, TA 1538 is not normally used in addition' to TA 98. This has two reasons:
a)There is no relevant increase in the spontaneous mutant counts of TA 98, compared to the spontaneous range of TA 1538. Special differences in sensitivity existing between TA 1535 and TA 100, and attributed to the relatively high spontaneous rate of TA 100 (10 times that of TA 1535), do not exist between TA 1538 and TA 98.

b)An international general inquiry had shown, that using TA 1538 in addition to any of the test strains in this study would not provide further information of biological relevance (Herbold, 1983).

Origin of Strains
The original strains were obtained from Prof. Bruce Ames and arrived at Fachbereich Toxicology, BAYER AG, on February 9, 1993.

Production of Stock Cultures
Immediately upon receipt, the samples were inoculated on nutrient agar plates, to which ampicillin had been added for the TA 100 and TA 98 strains.These plates were incubated at 37 deg C for approximately 24 hours. Samples were taken from individual colonies with a sterile inoculation loop, and transferred to nutrient broth. In the case of TA 100 and TA 98, ampicillin had also been added tp this broth. The samples where again incubated overnight at 37 deg C. After an incubation period of approximately 24 hours at 37°C, new samples of individual colonies from these plates were transferred to flasks containing approximately 20 ml normal nutrient broth. This inoculum was incubated overnight at 37 deg C, after which a small sample was taken to check the geno¬type. At the same time, the remaining cultures were treated with DMSO to protect against the effects of freezing, and im¬mediately frozen at -80 deg C in 1 ml portions (Ames et al., 1973b; McCann et al. , 1975b). No ampicillin resistance test was done on-the samples used for testing genotype since the cultures had already been sufficiently selected by the ampicillin.

In addition to the test for crystal-violet sensitivity (deep rough character), a test was done for UV sensitivity (uvrB). The crystal-violet and UV sensitivity tests are described be¬low. The frozen cultures which did not produce satisfactory results here were discarded. Remaining cultures were stored for future tests. In addition, frozen cultures of batches with unsatisfactory negative and/or positive control results in the definitive tests were also discarded.
Whenever new stock cultures needed to be produced; individual cultures grown on nutrient agar were used, to which ampicil¬lin had been added for the TA 100 and TA 98 strains. Samples of these individual colonies were then transferred to approx¬imately 20 ml nutrient broth, incubated, divided up, and checked for crystal-violet and UV sensitivity.
One 1 ml-portion was thawed for each test and strain, and quantities of 0.2 ml of the thawed,-culture were added to 10 ml nutrient broth. This culture was' incubated overnight at 37'C and used only on the same day. A new, small stock cul¬ture, which had been checked for its properties directly before freezing, was thus available for each individual test. This obviated any need to recheck the genotype for each Salmonella/microsome test. This procedure is in accordance with the methods described by Ames et al. (1975) and Maron and Ames (1983) .

Checking of Genotype

Histidine Requirement

In each individual test, histidine dependence of the cultures was automatically checked by the accompanying negative controls . The number of mutants per individual plate was documented

Ampicillin Resistance (pKM 101)
A special test for ampicillin resistance was not necessary since strains TA 100 and TA 98 were incubated on ampicillin containing nutrient agar and formed individual colonies. Consequently surviving bacteria were ampicillin resistant.

Crystal-Violet Sensitivity (deep rough)
A quantity of 0.1 ml was taken from the samples of individual stocks and spread onto nutrient agar, using four plates per strain. After a few minutes, filter papers, to which 10 ul of an aqueous, crystal-violet solution had been added at a Con¬centration of 1 rug/ml, were placed in the middle of the plates. The plates were then incubated overnight at 37*C. The diameters of the inhibition zones that had formed were then measured. The inhibition zones of all stock batches used in¬dicated an adequate sensitivity to crystal-violet.

UV Sensitivity (uvrB)
As described under 4.2.4.3, samples were spread onto nutrient agar plates. One half of each plate was covered with alumini¬um foil and irradiated without a lid for six seconds (TA 1535 and TA 1537) or eight seconds (TA ,100 and TA 98) with UV light of a wavelength of 254 nm at a distance of 33 cm. The irradiated plates were incubated and checked. To demonstrate adequate sensitivity in this test, cultures hsd to show an inhibition of growth over half their area, i.e. no bacteria should have grown on the irradi¬ated half. This was the case with all the stock batches used.

S9 mix was used to simulate the mammalian metabolism of &e test substance. It was made from the livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a sin¬gle intraperitoneal injection of vehicle, dissolved in corn oil, at a dose of 500 mg/kg body weight, five days prior to sacrifice. The animals were prepared unfasted , following the directions of Ames et al. (1975) and Maron and Ames (1983).

The rats were terminated. Livers were removed under sterile conditions immediately after sacrifice and kept at 4 deg C until all animals had been prepared. All the remaining steps were carried out under sterile conditions at 4 deg c.
The livers were washed with cold (4 deg C)0.15 M KCl solution (approximately 1 ml KCl per 1 g liver), and then homogenized in fresh, cold (4°C) 0.15 M KCl {approximately 3 ml KCl per 1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 4 deg C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was stored at -80 deg C in small portions.

These portions were slowly thawed before use. The S9 mix was freshly prepared and used only on the same day. It was placed in a vessel with a double glass wall until used. The hollow wall was filled with ice to keep the S9 mix cold.

S9 mix consists of a cofactor solution, S9 fraction and, if needed, 0.15 M KCl. The amount of S9 fraction in S9 mix is indicated in Tables 1 to 9 in percent. The S9 fraction was derived from the preparation dated September 27, L993 (protein content 27.5 mg per ml). Prior to first use, each batch was checked for its metabolizing capacity by using ref¬erence mutagen(s); appropriate activity was demonstrated. At the beginning of each experiment 4 aliquots of t the S9 mix were plated (0.5 ml per plate) in order to assessed its steril¬ity. This was repeated after completion of test-tube plating. The sterility control plates were then incubated for 48 hours at 37 deg C. No indication of contamination of S9 mix was found.

S9 Mix According to Prival
S9 mix was used to simulate the mammalian metabolism of the test substance. It was made from the livers of at least six adult male Syrian hamsters, approximately 8 weeks old. No enzyme induction was performed. The animals were prepared unfasted, following the directions of Ames et al, (1975) and Maron and Ames (1983).
The hamsters were terminated. Livers were removed under ste¬ile conditions immediately after sacrifice and kept at 4 deg C until all animals had been prepared. All the remaining steps were carried out under sterile conditions at 4deg C.
The livers were washed with cold (4 deg C), 0.15 M KCl solution (approximately 1 ml KCl per 1 g liver), and then homogenized in fresh, cold. (4deg C), 0.15 M KCl (approximately 3 ml KCl per 1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 4 deg C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was stored at -80°c in small portions.
These - portions were slowly thawed before use. The S9 mix was freshly prepared (Prival and Mitchell, 1982) and used only on the same day. It was placed in a vessel with a double glass wall until used. The hollow wall was filled with ice to keep the S9 mix cold.


Test Design

For the mutant count, four plates were used, both with and without S9 mix, for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained four plates per strain. The amount of solvent for the test substance and for the controls was 0.1 ml per plate and tube.
The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 ug or 5 ul per plate were used as the highest dose. At least five additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at least three doses could be used for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment.

Because the first assessable trial showed no mutagenic ef¬fects of the test substance, the repeat was performed according to Prival and Mitchell (1982) due to the chemical structure of the compound. Preincubation was performed in a water bath at 30deg c for 30 minutes. At the end of the pre-incubation period 2 ml of molten soft agar were added to the tubes and the content mixed and plated.

For the mutant count, four plates were used for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained four plates per strain. In experiments without S9 mix buffer was used as replacement.
The doses of this trial were determined on the basis of the results of the plate incorporation assay. Doses are given as ug/tube for better separation of plate incorporation and Pri¬val trials, despite the fact that ug/plate and ug/tube could be used synonymously.


The toxicity of the substance was assessed in three ways.
The first method was a gross appraisal of background growth on the plates for mutant determination. Secondly, a toxic effect of the substance was assumed when, there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bac-terial count was taken without S9 mix.
The bacterial suspensions were obtained from 17-hour cultures in nutrient broth, which had been incubated at 37 deg C and 90 rpm. These suspensions were used for the determination of mutant counts. No standardised procedure was employed to set the bacterial suspensions at a defined density of viable cells per milliliter, since the chosen method of incubation normally produces the desired density. However, the numbers of viable cells were established in a parallel procedure by determining the titers.
The dilution of bacterial suspensions used for the determination of titers was 1:1,000,000. Titers were determined under the same conditions as were the mutations, except that the histidine concentration in the soft agar was increased fivefold to permit the complete growth of bacteria.
The count was made after the plates had been incubated for 48 hours at 37 deg C. If no immediate count was possible, plates were temporarily stored in a refrigerator.

Solvent:
The solvent employed for Congo red was deionized water, and for other positive controls DMSO. Reactive Blue FC 75311 was dissolved in deionized water.
The solvent used was chosen out of the following solvents on the order given: water, methanol, ethanol, acetone, DMSO, DMF, and ethylene glycol dimethylether according to information given by the internal sponsor.

No "untreated" negative control was set up for deionised water since sufficient evidence was available in the literature (e.g. Maron and Ames, 1983) and from the test laboratories own experience, indicating that this solvent had no influence on the spontaneous mutant counts of the bacterial strains used.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase shiould be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
As above.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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 applicable
Positive controls validity:
valid
Additional information on results:
The Salmonella/microsome plate incorporation test, employing doses of up to 5000 ug per plate, showed Reactive Blue FC 75311 not to produce bacteriotoxic effects.

Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility), revealed no biologically relevant variations from the respective negative controls.

In spite of the low doses used, positive controls increased the mutant counts to well over double those of the negative controls, and thus demonstrated the systems high sensitivity.

Despite this sensitivity, no indications of mutagenic effects of Reactive Blue FC 75311 could be found at assessable doses of up to 5000 ug per plate in any of the Salmonella typhimu-rium strains used in the plate incorporation assay.

The Salmonella/microsome test modified according to Prival and Mitchell and employing doses of up to 5000 ug per tube, showed Reactive Blue FC 75311 to only produce bacteriotoxic effects at 5000 (ug per tube and above.

In agreement with the plate incorporation assay, evaluation of individual dose groups of the preincubation assay accord¬ing to Prival and Mitchell, with respect to relevant assessment parameters (dose effect, reproducibility), revealed no biologically relevant variations from the respective negative controls.

In spite of the low doses used, positive controls increased the mutant counts to well over double those of the negative controls, and thus demonstrated the system's high sensitivity.

Despite this sensitivity, no indications of mutagenic effects of Reactive Blue FC 75311 could be found at assessable doses of up to 5000 /ug per tube in any of the Salmonella typhimuri-um strains used in the preincubation assay according to Prival and Mitchell.

Due to these results Reactive Blue FC 75311 has to be regarded as non-mutagenic.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

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

Reactive Blue FC 75311 was initially investigated using the Salmonella/ microsome plate incorporation test for .point mutagenic effects in doses of up to 5000 ug per plate on four Salmonella typhimurium mutants. These comprised the histidine auxotrophic strains TA 1535, TA 100, TA1537 and TA 98.

In the plate incorporation assay doses up to and including 5000 ug per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed.

In the plate incorporation assay evidence of mutagenic activity of Reactive Blue FC 75311 was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant in¬crease in mutant colonies compared to the corresponding negative controls.


Executive summary:

The substance was negative in both assays both with any without metabolic activation.