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Toxicological information

Genetic toxicity: in vitro

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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:
from 29-04-1999 to 23-07-1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GPL study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes: Human lymphocytes
Details on mammalian cell type (if applicable):
- Source: from fresh venous blood drawn from a healthy donor.
- treatment: Heparin in the ratio of 1 part to 9 parts blood, and phytohaemagglutinin.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The S9 homogenate was prepared from the livers of five young male Sprague-Dawley rats which had received prior treatment with phenobarbital  and betanaphthoflavone.
Test concentrations with justification for top dose:
5000, 2500, 1250, 625, 313, 156, 78.1, 39.1 µg/mL with/without S9 (experiment 1)
156, 78.1, 39.1, 19.5, 9.77, 4.88, 2.44, 1.22, 0.611 µg/mL without S9 (experiment 2)
Vehicle / solvent:
VEHICLE: DMSO
- Justification for choice of solvent/vehicle: no
- Vehicle controls tested: culture medium with DMSO
- volume of vehicle/solvent in the medium: 1%
Controls
Untreated negative controls:
yes
Remarks:
untreated cultures
Negative solvent / vehicle controls:
yes
Remarks:
cultures treated with DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - mitomycin C (0.5 µg/mL, for treatment time of 3 hours, and 0.3 µg/mL  for treatment time of 24 hours), in sterile distilled water, in the absence of S9 metabolism - cyclophosphamide (18 µg/mL), in distilled water, in the presence of S9 metabolism
Remarks:
no
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION - Preincubation period: not applicable
- Exposure duration: 3 hours (without S9) or 2 hours (with S9) in experiment 1, 24h (without S9) in experiment 2.
- Expression time (cells in growth medium): 21 hours (without S9 mix) and 22 hours (with S9 mix)
- Selection time: not applicable 
- Fixation time: the last 3 hours of the recovery period 
SELECTION AGENT: not applicable 
SPINDLE INHIBITOR: colcemid (0.2 µg/mL final concentration) 
STAIN: 3% Giemsa 
NUMBER OF REPLICATIONS: 1.5 cell cycle
NUMBER OF CELLS EVALUATED: 100 cells/ culture (2 replicates)
DETERMINATION OF CYTOTOXICITY: mitotic index
OTHER EXAMINATIONS: 
- Determination of polyploidy: only metaphases containing 46 chromosomes are scored 
OTHER: 
-selection of dose levels: the highest dose level is determined according to the solubility of the TS in the culture medium and solvent vehicle,  
but will not exceed a maximum concentration of 5 mg/mL
-volume of test solution added: 0.05 mL
-incubation temperature: 37°C
-number of replicates: 2 at each test point
-SCORING METHOD: no data
Evaluation criteria:
For a substance to be considered clastogenic, 4 criteria must be met:
- Increases over the concurrent controls;
- Increase over historical controls;
- Reproducibility;
- Biological significance.
Statistics:
The number of cells bearing aberrations in the control and treated cultures are compared 
using Fisher's exact test.

Results and discussion

Test results
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the TS had no obvious effect on pH
- Effects of osmolality: the TS had no obvious effect on osmolality
- DMSO solubility: TS was found soluble in DMSO at a maximum concentration of 500 mg/mL
RANGE-FINDING/SCREENING STUDIES:  yes
COMPARISON WITH HISTORICAL CONTROL DATA: no 
ADDITIONAL INFORMATION ON CYTOTOXICITY:  In the first experiment, both in the presence or absence of S9 mix, no mitoses were observed 
at the highest dose level (5000 µg/mL). Moderate depression of the Mitotic Index (MI) was observed at the next lower dose level (2500 µg/mL).
In the second experiment, dose related reductions in (MI) were observed.  At the 2 higher dose levels no metaphases were recovered. 
A marked  depression of MI (13% of the control) was observed at 39.1 µg/mL, while a  moderate reduction (57% of the control) was observed at the 
next lower concentration (19.5 µg/mL).
Remarks on result:
other: other: Human lymphocytes

Any other information on results incl. tables

RESULTS OF EXPERIMENT 1:

Treatment                

Dose (µg/mL) 

With S9                

Without S9

%CA  

MI

%CA     

MI

Untreated                

-        

0.0        

105

0.0        

100

Solvent         

1%        

0.0        

100

0.0        

100

PMP                        

2500        

0.0        

56

0.0        

55

PMP                        

1250        

0.0        

73

0.0        

80

PMP                        

625        

0.0        

90

0.0        

73

Cyclophosphamide  

18

8.0

41

-

-

Mitomycin-C           

0.50

-     

-

8.0        

45

 


RESULTS OF EXPERIMENT 2:

Treatment                

Dose (µg/mL) 

Without S9          

%CA  

MI

Untreated                

-        

0.0        

126

Solvent         

1%        

0.0        

100

PMP                        

19.5        

1.0        

57

PMP                        

9.77        

0.0        

73

PMP                        

4.88        

0.0        

75

Mitomycin-C           

0.30        

18.5      

22


%CA = Percentage of cells bearing aberrations (excluding gaps)
MI = Mitotic index relative to solvent controls (%)

Following treatment with PMP, no statistically significant increases in the number of cells bearing aberrations were observed at any dose-level  

selected for scoring.
Statistically significant increases in the number of cell bearing aberrations were observed following treatments with the positive controls, 

indicating the correct functioning of the test system.

Applicant's summary and conclusion

Conclusions:

negative with metabolic activation
negative without metabolic activation

PMP does not induce chromosomal aberrations in cultured human lymphocytes after in-vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Executive summary:

In a mammalian cell cytogenetics assay [Chromosome aberration], primary human lymphocyte cultures were exposed to Paramethoxyphenol ecaille, prepared in DMSO, at concentrations of: 5000, 2500, 1250, 625, 313, 156, 78.1, 39.1 µg/mL (experiment 1), with and without metabolic activation 156, 78.1, 39.1, 19.5, 9.77, 4.88, 2.44, 1.22, 0.611 µg/mL (experiment 2), without metabolic activation. Paramethoxyphenol ecaille was tested up to cytotoxic concentration: 5000 µg/ml. Positive controls induced the appropriate response: statistically significant increases in the number of cells bearing aberrations (including and excluding gaps) were observed following treatments with cyclophosphamide and mitomycin-C. With Paramethoxyphenol ecaille, there was no evidence of chromosome aberration induced over background. This study is classified as acceptable: it satisfies the requirement for Test Guideline In vitro mammalian cytogenetics assay OPPTS 798.5375, EEC Council Directive 92/69, Part B and OECD Test Guideline 473.