[4-[[4-anilino-1-naphthyl][4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Detailed read across justification is attached in section 13. Source study has reliability 4, due to the lack of data on composition of test substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9-mix, 10 µg in DMSO

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9-mix

Controls 3

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: N-methyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9-mix

Controls 4

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

without S9-mix

Details on test system and experimental conditions:

Tissue preparation
S9 fraction
5 male Sprague-Dawley rats (200 - 300 g) receive a single intraperitoneal injection of 500 mg Aroclor 1254 (as a 20 % solution in peanut oil - w/v) per kg bw 5 days before sacrifice.
During this time the animais are housed in Makrolon cages in air-conditioned rooms. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours).
Standardized pelleted feed and tap water from bottles are available ad libitum.
5 days after administration the rats are sacrificed, and the livers are prepared (all preparation steps for obtaining the liver microsome enzymes are carried out using sterile solvents and glassware at a temperature of + 40 °C).
The livers are weighed and washed in an equivalent volume of a 150 mM KCl solution (1 ml ~ 1 g wet liver), then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 × g for 10 minutes at + 40 °C, 5 ml portions of the supernatant (so-called S9 fraction) are quickly deep-frozen in dry ice and stored at - 70 °C to - 80 °C for 2 months at the most.

S9-mix
The S9-mix is prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction is thawed at room temperature and 3 volumes of S9 fraction are mixed with 7 volumes of S9 supplement (cofactors). This preparation, the so-called S9-mix, is kept an ice until used. The concentrations of the cofactors in the S9-mix are:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 100 mM.
The phosphate buffer is prepared by mixing an NS2HPO4 solution with an NaH2PO4 solution in a ratio of about 4 : 1.

Bacteria
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy to histidine prototrophy is determined. The indicator organisms TA 1535, TA 1537, TA 98 and TA 100 selected by Ames especially far this purpose are derivatives of Salmonella typhimurium. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a cansiderably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.
The strains TA 1535 and TA 100 are derived from histidine prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions.
TA 1537 and TA 98 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the +1 mutant bis C 3076 in the case of TA 1537 and the +2 type his 0 3052 in the case of TA 98.
The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system. which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.
For testing, deep-frozen (- 70 °C to - 80 °C) bacterial cultures (1 ml in 15 ml glass tubes) are thawed at room temperature, 0.1 ml of this bacterial suspension is inoculated in nutrient broth solution (8 g Difco nutrient broth + 5 g NaCl/liter) and incubated in the shaking water bath at 37 °C for 16 hours. As a rule, a germ density of above 10^8 bacteria/ml is reached. These cultures grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth.

Standard plate test
Test tubes containing 2 ml portions af soft agar which consists af 100 ml agar (0.6% agar 0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination af mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml S9 mix (in tests with metabolic activation) or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, samples are poured onto Vogel-Banner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Preincubation test
0.1 ml. test solution, 0.1 ml bacterial suspension and 0.5 ml S9 mix are incubated at 37 °C for the duratian of 20 minutes. Subsequently, 2 ml. of soft agar is added and, after mixing, the samples are poured onta the Vogel-Bonner agar plates within approx. 30 seconds.
Composition of the minimal glucose agar:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37 °C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Titer determination
In general, the titer is determined only in the experiments with S9-mix both without test substance (solvent only) and after adding the two highest amounts of substance.
For this purpose, 0.1 ml of the overnight cultures is diluted to 10^-6 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution (5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining
components are added in the following order:
0.1 ml solvent (without and with test substance)
0.1 ml bacterial suspension (dilution: 10^-6)
0.5 ml S9-mix
After mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds. After incubation at 37 °C for 48 hours in the dark, the bacterial colonies are counted.

Checking out the tester strains
The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (∆ uvrB); ampicillin resistance (R factor plasmid). Histidine auxotrophy is automatically checked in each experiment via the spontaneous rate.

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was observed depending an the strain, experiment and test conditions in the standard plate test fram about 100 µg - 500 µg/plate onward. In the preincubation study, bacteritoxicity was found without S9-mix from about 4 µg - 20 µg/plate onward or with metabolic activation at doses ≥ 20 µg/plate (TA 1535), ≥ 100 µg/plate (TA 100, TA 98) or ≥ 500 µg/plate (TA 1537).

Solubility
Incomplete solubility of test substance in DMSO from about 500 µg/plate onward.

Remarks on result:

other: standard plate test and preincubation test; with and without S9-mix

Applicant's summary and conclusion

Conclusions:

Non mutagenic in Ames test.

Executive summary:

Method

Ames test using TA 1535, TA 100, TA 1537, TA 98 strains in the dose range 0.16 µg - 5000 µg/plate for the standard plate test and 0.16 - 750 µg/plate for the plate incorporation test. Both types of test were conducted with and without metabolic activation by rat liver S9-mix. Positive and solvent controls were used.

Results

Solubility: incomplete solubility of test substance in DMSO from about 500 µg/plate onward.

Toxicity: a bacteriotoxic effect was observed in the standard plate test from about 100 - 500 µg/plate onward and in the preincubation test from about 1 - 20 µg/plate onward without S9-mix or at doses ≥ 20 µg (TA 1535), ≥ 100 µg (TA 100, TA 98) or ≥ 500 µg (TA 1537) with metabolic activation.

Mutagenicity: an increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S9-mix or after the addition of a metabolising system.

Overall, test substance resulted as non mutagenic in the Ames test under these experimental conditions.