Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro studies with reliability 1 are available: an Ames test, an in vitro gene mutation study in mammalian cells and a chromosome aberration study in mammalian cells. These three tests gave negative results.

Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
not applicable
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%
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.
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

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.

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 27-04-1999 to 23-06-1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GPL study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT (hypoxanthine-guaninphosphoribosyl-transferase)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type: Chinese hamster V79 cells, derived from a culture of embryonic lung tissue of male Chinese hamster (Cricetulus griseus)
- Source: from Dr. J. Thacker, MRC Radiobiology Unit, Harwell, UK.
- Type and identity of media: EMEM Complete (900 mL Minimal medium + 100  mL Foetal calf serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
- The karyotype, generation time, plating efficiency and mutation rates  (spontaneous and induced) have been checked in this laboratory.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 TISSUE HOMOGENATE: The S9 tissue fraction was prepared using liver from young male Sprague-Dawley rats pretreated with phenobarbitone and betanaphthoflavone.
Test concentrations with justification for top dose:
Pre-Test, with and without metabolic activation: 19.5, 39.1, 78.1, 156,  313, 625, 1250, 2500 and 5000 µg/mL
Main test; 2 assays were performed:
- in the absence of S9 mix: 2500, 1250, 625, 313, 156 and 78.1 µg/mL
- in the presence of S9 mix: 3750, 2500, 1250, 625, 313 and 156 µg/mL for the first experiment 
and 2500, 1900, 1250, 625, 313 and 156 µg/mL for the second experiment.
Vehicle / solvent:
solvant: DMSO
- Justification for choice of solvent/vehicle: no
- Vehicle controls tested: medium with solvent 
- volume of vehicle/solvent in the medium: no more than 1%
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(treated with the maximum amount of solvent used in any test substance treatment)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - 7,12- dimethylbenzanthracene, in DMSO (experiments in the presence of S9 mix), at 0.1 mL per 10 mL  - and ethylmethanesulphonate, in ethanol (experiments in the absence of S9 mix), at 0.1 mL per 10 mL 
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours
- Expression time: 6 and 9 days 
- Selection time: not applicable 
- Fixation time: not applicable
MAIN EXPERIMENT details: 
- Number of experiments: 3.
- Number of replicates: 2 at each test point, with the exception of the positive controls (single culture).
- Volume of test solution added: 0.1 mL.
- CO concentration: 5%.
- Incubation temperature: 37°C.
SELECTION AGENT: 6-thioguanine (at 7.5 µg/mL)  
SPINDLE INHIBITOR: not applicable 
STAIN: Giemsa solution 
NUMBER OF REPLICATIONS: not applicable
NUMBER OF CELLS EVALUATED: 200 cells/plate
DETERMINATION OF CYTOTOXICITY: survivals count
OTHER EXAMINATIONS: not applicable
OTHER: SCORING METHOD: no data  
Evaluation criteria:
Criteria for outcome of assay: the test substance was considered mutagenic if  (1) there is a 5-fold increase in mutation frequence compared with 
the solvent controls, over 2 consecutive doses, and (2) there must be evidence for a dose-relation.
Statistics:
The results were subjected to an Analysis of Variance in which the effect of replicate culture, expression time and dose-level in explaining the  
observed variation was examined.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: > or = 2500 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The addition of the test substance solution did not have any obvious effect on the pH of the treatment medium.
- Effects of osmolality: The addition of the test substance solution did not have any obvious effect 
on the osmolality of the treatment medium.
- Evaporation from medium: no data
- Water solubility: not applicable
- solvent solubility: the Test Substance was found to be soluble in DMSO at a maximum concentration of 500 mg/mL.
- Precipitation: no data
- Other confounding effects: no
RANGE-FINDING/SCREENING STUDIES:  
Treatment with the TS at a maximum dose level of 5000 µg/mL resulted in a severe toxicity. 
Maximum concentrations of 2500 and 3750 µg/mL were selected as the maximum dose-levels to be used in the first mutation assay in the absence and presence of S9 metabolism, respectively.
COMPARISON WITH HISTORICAL CONTROL DATA: no
ADDITIONAL INFORMATION ON CYTOTOXICITY:  In both mutation assays dose related toxicity was observed in the absence and presence of S9 
metabolism.
Remarks on result:
other: all strains/cell types tested

RESULTS OF EXPERIMENT No. 1:

* Without metabolic activation:

Dose level (µg/mL) 

%RS

MF (day 6) 

MF (day 9)

0

100        

3.73 

4.51

78.1                        

50        

1.87   

14.51

156

33        

9.84     

6.94

313

37        

5.53       

7.32

625

32        

4.55        

7.81

1250

21        

7.69      

9.18

2500

15        

16.51     

11.28

Ethylmethanesuphonate

45        

1270.59   

1181.65


* With metabolic activation:

Dose level (µg/mL) 

%RS

MF (day 6) 

MF (day 9)

0                              

100 

8.04        

18.02

156 

55 

4.98 

8.09

313                              

39

9.05 

16.04

625                                

38

9.55 

14.59

1250                               

34 

10.67 

18.26

2500                               

5

6.81 

5-37

3750         

0

-

-

DMBA   

55 

581.01  

578.05


RESULTS OF EXPERIMENT No. 2:


* Without metabolic activation:

 

 Dose level (µg/mL) 

%RS

MF (day 6) 

MF (day 9)

0

100       

7.27         

14.02

78.1         

83        

5.42        

12.21

156

59      

11.41        

13.13

313

71       

2.62        

4.70

625

48       

4.78        

1.55

1250

19     

8.43         

6.53

2500

23     

3.05         

3.93

Ethylmethanesuphonate

41            

1070.33   

1422.71


* With metabolic activation:

 

Dose level (µg/mL) 

%RS

MF (day 6) 

MF (day 9)

0                              

100       

7.61         

4.63

156 

85    

10.10        

21.91

313                              

66     

7.30         

9.53

625                                

52     

3.30         

13.79

1250                               

42     

5.73        

4.61

1900                             

32   

7.61         

1.63

2500        

2       

2.84        

8.45

DMBA   

49      

735.32     

983.89


%RS = Percentage of relative survival
MF = Mutation frequency per million surviving cells

No significant increases in mutant frequency were observed either in the absence or presence of metabolic activation at any test point.



 

Conclusions:

negative with metabolic activation
negative without metabolic activation

Paramethoxyphenol ecaille does not induce mutation in Chinese hamster V79 cells after in-vitro treatment, either in the absence or presence of S9 metabolic activation, under the reported experimental conditions.


Executive summary:

In a mammalian cell gene mutation assay for 6-thioguanine resistance, chinese hamster lung V79 cells cultured in vitro were exposed to paramethoxyphenol ecaille (DMSO as solvent), at concentrations of 2500, 1250, 625, 313, 156 and 78.1 µg/mL, in the absence of S9 mix 2500, 1250, 625, 313 and 156 µg/mL in the presence of S9 mix, for the first experiment and 2500, 1900, 1250, 625, 313 and 156 µg/mL for the second experiment. Paramethoxyphenol ecaille was tested up to 2500 and 3750 µg/mL in the first mutation assay in the absence and presence of S9 metabolism, respectively (treatment with the test substance at a maximum dose level of 5000 µg/mL resulted in a severe toxicity). The positive controls did induce the appropriate response. With paramethoxyphenol ecaille, there was no evidence of induced mutant colonies over background. This study is classified as acceptable; it satisfies the requirements for test guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 26-03-1999 to 12-04-1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GPL study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
not applicable
Species / strain / cell type:
other: Salmonella typhimurium TA1535, TA1537, TA98, TA100, TA102
Details on mammalian cell type (if applicable):
stock of Salmonella tester strains were obtained from Dr. BN.Ames, University of California. Permanent stocks are kept at -80°C, and overnight 
subcultures of these stocks are prepared for each day's work.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 - from induce - phenobarbital and betanaphthoflavone - Sprague Dawley rats - liver
Test concentrations with justification for top dose:
Pre-Test: 50, 158, 500, 1580, 5000 µg/plate
main test: 0, 313, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
DMSO
- Justification for choice of solvent/vehicle: no
- Vehicle controls tested: medium with DMSO alone
- volume of vehicle/solvent in the medium: 100 µL/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation 
DURATION
- Preincubation period: 30 min at 37°C
- Exposure duration: 72h at 37°C
NUMBER OF CELLS EVALUATED: not applicable
OTHER: SCORING METHOD: Artek colony counter
Evaluation criteria:
for a mutagenic test substance, two-fold (or more) increase in the mean revertant numbers must be observed at two  
consecutive dose levels or at the highest practicable dose level only. 
In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose-levels. 
The effect must be reproduced in an independant experiment.
Statistics:
regression line method (includes the solvent control data but not the untreated control data)
Parameters given:
- individual plate counts
- mean number of revertant colonies
- standard error of the mean
- titre of bacterial cultures
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102 
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
The test substance was found to be soluble in DMSO at a concentration of 100 mg/mL. Since 100µL of the TS solution are used in the preparation 
of each plate, this permitted the maximum concentration of 5000 µg/plate to be used in the toxicity test.
RANGE-FINDING/SCREENING STUDIES:  
The test substance was assayed at a maximum dose-level of 5000 µg/plate and at 4 lower dose-levels spaced at approximately half-log intervals:  
1580, 500, 158 and 50 µg/plate. No sign of toxicity were observed at the dose-levels tested in any tester strain in the absence or presence of S9 
metabolism
COMPARISON WITH HISTORICAL CONTROL DATA: no
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the plate incorporation assay, slight toxicity was observed with TA1537 strain both in the 
presence and absence of S9 metabolism at the highest dose level. In the preincubation assay, with all tester strains, severe toxicity was observed 
in the absence of S9 mix at the highest dose level, while marked toxicity was observed in the presence of S9 mix. Moderate toxicity was observed  
at the dose level of 2500 µg/plate with all tester strain (S9 +/-).
Remarks on result:
other: all strains/cell types tested

RESULTS IN THE PLATE INCORPORATION METHOD:

* In TA1535

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

18

15

0 (DMSO)                

18

18

313

16

16

625

14

15

1250

16

13

2500

15

14

5000

17

12

Sodium azide                

476

-

2-Aminoanthracene        

-

113


* In TA1537

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

16

24

0 (DMSO)                

16

24

313

13

22

625

18

21

1250

15

22

2500

14

20

5000

9

14

9-Aminoacridine     

101

-

2-Aminoanthracene        

-

95


* In TA102

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

434

476

0 (DMSO)                

442

502

313

450

478

625

443

494

1250

443

535

2500

467

535

5000

453

503

Cumene hydroperoxide

1029

-

2-Aminoanthracene        

-

1712


* In TA98

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

31

41

0 (DMSO)                

31

40

313

29

39

625

30

38

1250

31

38

2500

29

38

5000

31

37

2-Nitrofluorene       

220

-

2-Aminoanthracene        

-

981


* In TA100

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

134

138

0 (DMSO)                

121

131

313

117

132

625

129

136

1250

116

133

2500

121

127

5000

117

125

Sodium azide                

707

-

2-Aminoanthracene        

-

1239


RESULTS IN THE PREINCUBATION METHOD:

* In TA1535

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

17

16

0 (DMSO)                

18

16

313

19

17

625

20

16

1250

17

15

2500

15

13

5000

-

13

Sodium azide                

479

-

2-Aminoanthracene        

-

96


* In TA1537

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

17

23

0 (DMSO)                

25

24

313

17

23

625

16

26

1250

15

25

2500

9

15

5000

-

6

9-Aminoacridine     

109

-

2-Aminoanthracene        

-

88


* In TA 102

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

340

421

0 (DMSO)                

302

405

313

301

397

625

309

413

1250

288

403

2500

238

355

5000

-

270

Cumene hydroperoxide

1013

-

2-Aminoanthracene        

-

1586


* In TA98

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

33

43

0 (DMSO)                

30

40

313

31

39

625

30

39

1250

29

43

2500

19

39

5000

-

28

2-Nitrofluorene       

204

-

2-Aminoanthracene        

-

970


* In TA100

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

126

141

0 (DMSO)                

115

135

313

114

133

625

118

130

1250

116

133

2500

86

131

5000

-

68

Sodium azide                

732

-

2-Aminoanthracene        

-

1191



Conclusions:
negative with metabolic activation
negative without metabolic activation

Paramethoxyphenol ecaille does not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.
Executive summary:

Two experiments were performed, one using a plate incorporation method, the other using a pre incubation method. In both experiments the test substance was assayed at the dose levels of 5000, 2500, 1250, 625 and 313 µg/plate. In the plate incorporation assay slight toxicity, indicated by a reduction in revertant numbers, was observed with tester strain TA1537 both in the absence or presence of S9 metabolism at the highest dose level. In the pre incubation assay, with all tester strains, severe toxicity, as indicated by microcolony formation, was observed at the highest dose level in the absence of S9 metabolism. Marked toxicity, as indicated by thinning of the background lawn and reduction in revertant numbers, was observed at the highest dose level in the presence of S9 metabolism. The test substance did not induce two-fold increases in the number of revertant colonies at any dose level, in any tester strain, in the plate incorporation or pre incubation assay, in the absence or presence of S9 metabolism. The sterility of the S9 mix and the test substance solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control substances, indicating that the assay system was functioning correctly.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Only one in vivo test is available with reliability 3.

Additional information

Genotoxicity in vitro:

- Bacterial reverse mutation assay: one study (Scarcella, RTC report, 1999) was chosen as key study,

of reliability 1 and one other study (Haworth et al. 1983) of reliability 2 was chosen as supporting study.

The first is very well described and followed the current guidelines with GLP, the second is equivalent

to the guidelines and well described too.

In the RTC report of 1999, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 were used.

Two experiments were performed, one using a plate incorporation method, the other using a pre incubation method.

In both experiments the test substance was assayed at the dose levels of 5000, 2500, 1250, 625 and 313 µg/plate.

In the plate incorporation assay slight toxicity was observed with tester strain TA1537 both in the absence or presence

of S9 metabolism at the highest dose level. In the pre incubation assay, with all tester strains, severe toxicity,

as indicated by microcolony formation, was observed at the highest dose level in the absence of S9 metabolism.

The test substance did not induce two-fold increases in the number of revertant colonies at any dose level,

in any tester strain, in the plate incorporation or pre incubation assay, in the absence or presence of S9 metabolism.

In the publication of Haworth et al. 1983, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were used,

by 2 different laboratories with a modified preincubation method. The test concentrations were: 0, 3.3, 10, 33,

100 and 167 µg/plate in one laboratory and 0, 100, 333, 1000, 3333 and 5000 µg/plate in another. In the absence

and in the presence of metabolic activation system, PMP does not induce any reverse mutation in all strains tested.

Based on these 2 studies, PMP does not induce reverse mutation in Salmonella typhimurium tested strains.

- mammalian cell gene mutation: one study (Cinelli, RTC report, 1999) was chosen as key study, of reliability 1.

It is a GLP study, which followed the current guidelines. It used Chinese hamster lung fibroblasts (V79) with target

gene HGPRT. In this assay for 6-thioguanine resistance, V79 cells cultured in vitro were exposed to

paramethoxyphenol ecaille (DMSO as solvent), at concentrations of:

- 2500, 1250, 625, 313, 156 and 78.1 µg/mL, in the absence of S9 mix,

- and 3750, 2500, 1250, 625, 313 and 156 µg/mL in the presence of S9 mix, for the first experiment and

- 2500, 1900, 1250, 625, 313 and 156 µg/mL for the second experiment.

Paramethoxyphenol ecaille was tested up to 2500 and 3750 µg/mL in the first mutation assay in the absence

and presence of S9 metabolism, respectively.

Based on this study, PMP does not induce mutation in V79 cells, either in the absence or presence

of S9 metabolic activation.

An other study is available for mammalian cell gene mutation (Rogers-Back A, 1986), but it is of reliability 3

because the test substance is not clearly defined and the effects were observed at highly toxic doses.

In this study on mouse lymphoma L5178Y cells, the test substance produced a positive response in the

absence of exogenous metabolic activation.

- mammalian chromosome aberration: one study (Ciliutti, RTC report, 1999) was chosen as key study,

of reliability 1. It is a GLP study, which followed the current guidelines. It was carried out on primary human

lymphocyte cultures, which 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.

Based on this study, PMP does not induce chromosomal aberration in cultured human lymphocytes,

either in the absence or presence of S9 metabolic activation.

Seven other studies are available but the reliability is 3.

Three Ames test and one sister chromatide exchange assay (in vitro) were performed on PMP and the results

are negative.

One DNA synthesis and repair assay showed that PMP induce a decrease of DNA synthesis and repair but this

result was not PMP specific and the data was too poor to permit conclusions.

The two last studies of reliability 3 are one mammalian cell gene mutation and one mammalian chromosome

aberration tests, but the test substance was not clearly defined in the reports.

Genotoxicity in vivo:

Only one study (Esber, 1986) was available in this endpoint, it is an in vivo mammalian bone marrow chromosome

aberration assay, which led to a negative result. But this study is of reliability 3 because the test material is not

clearly identified and all the test conditions and details were not available to conduct an assessment.

Conclusion for genotoxic potential of PMP: Based on three in vitro studies performed on our substance

(Scarcella , Cinelli and Ciliutti, RTC reports, 1999), PMP ecaille was considered to be not genotoxic.







Justification for classification or non-classification

Several in vitro tests performed on PMP are available. All results coming from reliable studies are negative. Only one in vivo test has been generated on PMP. The result is also negative; however the reliability of this study is 3 according to Klimisch rating.

Based on several reliable in vitro studies performed on our substance, PMP was considered to be not genotoxic.