2-ethoxy-5-(4-methyl-1-piperazinylsulfonyl)benzoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 23 October and 19 December 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Done under GLP and OECD Methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EEC, Method B10.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

In this study, blood taken from healthy male donors was pooled and diluted with tissue culture medium. The cultures were incubated in the presence of PHA before being treated with the test substance. Following treatment the cells were arrested at metaphase using the mitotic inhibitor, Colcemid•. Chromosomes in these metaphase cells were then examined for the presence of chromosome aberrations. The best estimate of the aberration frequency is at the first cell division after initiation of treatment since certain types of damage may be lost during subsequent cell divisions. In this laboratory the cell cycle time for human lymphocytes in whole blood culture is approximately 15 hours. On this basis the cells are examined after 21 hours. However, some test substances may delay the cell cycle and, for this reason, cells are also examined 45 hours after initiation of treatment, in the event of a negative or equivocal response at 21 hours. Additionally, the 21 hour part of the test is repeated.

Aberrations were scored according to the classification of the ISCN (1985). Traditionally gaps have been excluded from the quantitation of chromosome aberrations

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Intended use: Pharmaceutical intermediate
Appearance: White powder
Storage conditions: Room temperature
Expiry: Not supplied



Purity: 101.3%



Date received: 2 October 1996



Supplier: Pfizer Ltd

Method

Target gene:

No data

Species / strain

Species / strain / cell type:

mammalian cell line, other: Human lymphocytes

Details on mammalian cell type (if applicable):

Human blood was collected aseptically from healthy male donors, pooled and diluted with RPMI 1640 tissue culture medium (Imperial Laboratories) containing 16.7% foetal calf serum (PAA). Aliquots (0.4m1 blood : 4.5m1 media : O. lml phytohaemagglutin (Wellcome)) of the cell suspension were placed in sterile universal containers and incubated at 37°C in a slanted position, for approximately 48 hours. The cultures were gently shaken once daily to resuspend the cells.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphthoflavone induced rat-liver S9.

Test concentrations with justification for top dose:

Without S-9 mix, 21 hour harvest: 1250, 2500, 3750 and 5000 µg/ml.

With S-9 mix, 21 hour harvest: 1250, 2500, 3750 and 5000 µg/ml.



cultured medium

for each culture heparinsed whole blood was added to culture medium containing a mutagen (photohaemogglutinin) and incubated at 37C in a humidified atmosphere at 5% CO2/95% air for 48 hours
dose levels for positive controls
without S9 mix- mitomycin C 0.125 ug/ml ( 3 hour of treatment or 2 ug/ml ( continuous treatment)
with S9, cyclophosphamide: 12.5 ug/ml

Vehicle / solvent:

Prior to commencing testing, the solubility of the test substance was assessed in solvents compatible with the test system. As UK-143,108 was found to be insoluble in all aqueous and organic solvents tested, it was formulated directly in tissue culture medium at a highest final concentration of 5000 µglml, from which halving dilutions were made. No precipitate was observed when UK-143,108 was dissolved directly in the culture medium at 5000 µg/ml.

Controls

Controls 1

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

TREATMENT OF CELLS WITH TEST SUBSTANCE - FIRST TEST
After 48 hours, the cultures were centrifuged and the supernatant replaced by 5 ml culture medium which contained the appropriate treatment. To one set of cultures 1.25 ml S-9 mix was added to each culture. For cultures to be treated in the absence of S-9 mix, 1.25 ml of untreated culture medium was added. This diluted the various concentrations of UK-143,108 to give final concentrations of 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/ml. Three hours after dosing, the cultures containing the S-9 mix were centrifuged and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours. The cultures treated in the absence of S-9 mix were incubated for 21 hours.
SECOND TEST
From the results obtained in the first test it was not deemed necessary to include a 45 hour harvest in the second test.

Cultures were initiated and maintained as previously described. Concentrations of UK-143,108 were as follows:

Without S-9 mix, 21 hour harvest: 1250, 2500, 3750 and 5000 µg/ml.

With S-9 mix, 21 hour harvest: 1250, 2500, 3750 and 5000 µg/ml.

Duplicate cultures were used for each treatment and four cultures in each set were untreated. Positive control cultures were treated as in the first test.

Three hours after dosing, the cultures containing S-9 mix were centrifuged and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours. Cultures treated in the absence of S-9 mix were incubated for 21 hours.

All cultures were treated with Colcemid at a final concentration of 0.1 µg/ml two hours before the end of the incubation period. They were then harvested, fixed and the slides prepared as previously described. The slides were then examined microscopically.

Evaluation criteria:

Chromosomes in these metaphase cells were then examined for the presence of chromosome aberrations. The best estimate of the aberration frequency is at the first cell division after initiation of treatment since certain types of damage may be lost during subsequent cell divisions
Cytotoxicity evaluated based on mitotic index which is number cells with mitosis indicating mitotic inhibition. 1000 cells with mitosis were evaluated with no blind scoring.

analysis of 200 metaphases/dose-level with 44 to 46 chromosome were made with 100 metaphases/culture whenever possible. Only 50 metaphases/culture were analyst when at least 10% with structural chromosome aberrations were observed. Blind scoring was done.

Statistics:

No data

Results and discussion

Test results

Species / strain:

lymphocytes: Mitotic indices of cultured human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the absence of S-9 mix, UK-143, 108 caused no statistically significant increase in the proportion of aberrant cells compared to the untreated cultures at the lowest two concentration analysed, 2500 and 3750 µg/ml. There was a statistically significant increase (P<0.001) in the proportion of aberrant cells at the highest concentration analysed. This increase, to 8.5%, lies outside the historical control range and is therefore considered to be indicative of a clastogenic response.

In the presence of S-9 mix, UK-143, 108 did not cause any statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations when compared to the untreated control at any dose level analysed.

Both positive control compounds, mitomycin C and cyclophosphamide, caused large, statistically significant increases (P <0.001) in the proportion of aberrant cells

Remarks on result:

other: other: METAPHASE CHROMOSOME ANALYSIS OF HUMAN LYMPHOCYTES CULTURED IN VITRO

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation
negative without metabolic activation

UK-143, 108 has shown evidence of clastogenic activity in the absence of S-9 mix, in this in vitro
cytogenetic test system, but not in its presence.