2-phthalimidoethyl N-[4-(2-cyano-4-nitrophenylazo)phenyl]-N-methyl-β-alaninate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From March 31 to July 14, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1992

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Guidelines stipulated by the Japanese Ministry of Health and Welfare and the Japanese Ministry of lnternational Trade and lndustry.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Red RA 10463

IUPAC Name:

Red RA 10463

Method

Species / strain

Species / strain / cell type:

lymphocytes: cultured peripheral human

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats from Charles River, Sulzfeld, Germany, and housed at the testing facility under standard laboratory conditions. Rats were injected intraperitoneally with 20 % w/v Aroclor 1254 (500 mg/kg bw) in corn oil then sacrificed by decapitation 5 days later. Rat livers were removed and washed in cold (0 °C), sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Livers were then blended and homogenised in 3 volumes of phosphate buffer with a Potter homogeniser. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196 °C).

PREPARATION:
Prepared immediately before use and kept on ice. S9-mix contained per ml: 1.63 mg MgCl2.6H2O 2.46 mg KCI; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES. The above solution was filter (0.22 µm)-sterilised. To 0.5 ml S9-mix components, 0.5 ml S9-fraction was added (50 % (v/v) S9 fraction).
Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml exposition medium (4.8 ml F10 complete culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin). The concentration of the S9-fraction in the exposition medium was 1.8 % (v/v).

Test concentrations with justification for top dose:

- Test concentrations: 0, 1, 3 and 10 µg/ml
- Justification: based on a dose range finding test (0.11, 0.33, 1.1, 3.3 and 1.1 µg/ml, both with and without S9-mix), precipitation is observed at 10 µg/ml

Vehicle / solvent:

DMSO (0.9 % v/v final concentration)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

- BLOOD SAMPLES: taken from healthy adult male volunteers by venapuncture and stored at between 4 and 25 °C. Within 4 h, lymphocyte cultures were started.
- F10 COMPLETE CULTURE MEDIUM: consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively), sodium bicarbonate (1.2 g/l) and 30 U/ml heparin.
- CELL CULTURE CONDITIONS: whole blood was cultured in F10 complete culture medium with Phytohaemagglutinin (Murex). In the absence of S9-mix 5 mL F10 complete culture medium and in the presence of S9-mix 4.8 ml F10 complete culture medium and 0.4 ml whole blood, 0.1 ml per culture (9 mg/ml) phytohaemagglutinin was added.
- ENVIRONMENTAL CONDITIONS: all incubations were carried out in a humid atmosphere (80-100 %) containing 5 % CO2 in air in the dark at 37 °C. The temperature, humidity and %-CO2 were monitored.
- EXPERIMENTS: two experiments were performed. In both, lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected test item doses (0.4 ml blood of a healthy male donor added to 5 ml or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) phytohaemagglutinin).
In Experiment I, after 3 hours, cells were washed with 5 ml of HBSS and incubated in 5 ml culture medium for another 20-22 h (24 h fixation time). Based on the mitotic index of the dose range finding test and the first cytogenetic assay, appropriate dose levels were selected for the second cytogenetic assay.
The independent repeat was performed with the following modifications of experimental conditions. In Experiment II, after 3 h treatment, cells in the presence of S9-mix were rinsed once wlth 5 ml of HBSS and incubated in 5 ml culture medium for another 44-46 h (48 h fixation time). The cells which were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after treatment but were worked up immediately after 24 h and 48 h (24 h and 48 h fixation time).
- CHROMOSOME PREPARATION: during the last 3 h of the culture period, cell division was arrested by addition of the spindle inhibitor colchicine (0.5 µg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cellpellet were
swollen by a 5 min treatment with hypotonic 0.56 % (w/v) potassium chloride solution at 37 °C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).

Rationale for test conditions:

Recommended test system

Evaluation criteria:

ACCEPTABILITY OF ASSAY
a) The number of chromosome aberrations found in the solvent control cultures should be reasonably within the laboratory historical control data range of 0 to 9 (mean = 0.9; standard deviation = 1.0) aberrant cells per 100 metaphases in the absence of S9-mix, gaps excluded and 0 to 5 (mean = 0.7; standard deviation = 0.9) aberrant cells per 100 metaphases in the presence of S9-mix; gaps excluded).
b) The positive control substances should produce slatistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.

DATA EVALUATION AND STATISTICAL PROCEDURES
Positive if:
a) induces a dose-related statistically significant (Chi-square test, P < 0.05) increase in number of cells with chromosome aberrations.
b) a statistically significant increase in frequencies of the number of cells with chromosome aberrations is observed in the absence of a clear dose-response relationship.
Negative (not clastogenic) if:
- None of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: cultured peripheral human

Remarks:

exp. I

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: cultured peripheral human

Remarks:

exp. II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Dose range finding study: test item precipitated at top nominal dose of 10 µg/mL, both with and without metabolic activation.
- Experiment I, with and without S9 mix, 3 h treatment time, 24 h fixation time: no statistically or biologically significant increase in the number of cells with chromosome aberrations.
- Experiment II, without S9 mix, 24 h treatment time, 24 h fixation time: no statistically or biologically significant increase in the number of cells with chromosome aberrations.
- Experiment II, without S9 mix, 48 h treatment time, 48 h fixation time: induced a statistically significant increase in the number of cells with chromosome aberrations, when gaps were included only, at the highest tested precipitating concentration of 10 µg/mL only. Since the types of aberrations observed were mainly breaks and gaps, the increase is not dose-related and moreover the number of cells with chromosome aberrations was within the historical solvent control data range, the increase is not considered biologically relevant.
- Experiment II, with S9 mix, 3 h treatment time, 48 h fixation time: no statistically or biologically significant increase in the number of cells with chromosome aberrations.

Applicant's summary and conclusion

Conclusions:

Test substance is not considered clastogenic as it did not induce chromosome aberrations in cultured peripheral human lymphocytes up to 10 µg/ml with and without metabolic activation under the described experimental conditions.