6-chloro-5-(2-chloroethyl)-1,3-dihydroindol-2-one

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:

24 October 1997

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:

Human lymphocytes in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the presence and absence of S9 mix derived from rat livers .
Solvent and positive control cultures were also prepared.
After the appropriate incuhalion time, the cells were harvested and slides prepared so that metaphase figures could be
examined for chromosonral damage.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

6-chloro-5-(2-chloroethyl)-1,3-dihydroindol-2-one

EC Number:

421-320-0

EC Name:

6-chloro-5-(2-chloroethyl)-1,3-dihydroindol-2-one

Cas Number:

118289-55-7

Molecular formula:

Hill formula: C10 H9 Cl2 N O CAS formula: C10 H9 Cl2 N O

IUPAC Name:

6-chloro-5-(2-chloroethyl)-2,3-dihydro-1H-indol-2-one

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Off-white powder
Date received: 2 June 1997

Method

Target gene:

NA

Species / strain

Species / strain / cell type:

mammalian cell line, other: Human lymphocytes

Details on mammalian cell type (if applicable):

Hunan blood was collected aseptically from healthy male donors, pooled and diluted with RPMI 1640 tissue culture medium (Imperial) containing 10% foetal calf serum (PAA). Aliquots (0.4 ml blood : 4.5ml medium : 0.1 ml phytohaemagglutinin (Gibco)) of the cell suspension were placed in sterile universal containers and incubated at 37C in a slanted position for approx. 48 hours. The cultures were gently shaken once daily to resuspended the cells.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat-liver S9 mix

Test concentrations with justification for top dose:

First test
Without S9 mix- 3 hours treatment, 18 hours recovery: I00, 200 and 400 µg/ml
With S9 mix- 3 hours treatment. 18 hours recovery: I 00, 200 and 400 µg/ml.

Second test
.Without S9 mix - 2 1 hour> continuous treatment : I00, 200 and 400 µg/ml.
With S9 mix- 3 hours treatment ,18 hours recovery: 100,200 and 400 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:

- Vehicle(s)/solvent(s) used: DMSO

Prior to commencing testing the solubility of the test substance in solvents compatible with the test system was assessed. CP-85,575 was found to be soluble in dimethyl sulphoxic at 14 1 .7 mg/ml. On dosing at 1% v/v into aqueous tissue culture medium giving a final concentration of 1417 ug/ml , a heavy precipitate was observed . A precipitate was still observed at 88.6 ug/ml, but not at 44.3 1u/ml. In this study, the highest final concentration used for subsequent testing was 400 ug/ml , so that several dose levels would be in the insoluble range.

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:

Initial assay:
Exposure period (with metabolic activation): 21 hr
Exposure period (without metabolic activation): 21 hr

Confirmatory assay:
Exposure period (with metabolic activation): 21 hrs
Exposure period (without metabolic activation): 21 hrs

Before harvest each cultured treated with colcemid solution ( 10 mg/ml) to block cells at the metaphase stage of mitosis.

Evaluation criteria:

Reproducible and statistically significant increase in frequency of cells with structural chromosome aberrations for at least one dose level and for 1 of 2 harvest times was considered as positive results.

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:

Aberrations were scored according to the classification of the I SCN ( 1985).

mean aberrations per cells, number of cells with aberrations and any evidence for increasing amount of damage with increasing dose.

Results and discussion

Test results

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
Analytical analysis completed:
intial assay : 90.4% and 100.5% ( acceptable)
confirmatory assay: 70.4% to 98.7% ( lower assay results in lower concentrations but did not impact the results)

vehicle and postive controls were acceptable and valid

Remarks on result:

other: other:

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

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

CP-85.575 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system. However. it was observed that ClP-89.575 caused increases in mitotic index in all parts of the test.