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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:
The study was carried out between 2 and 4 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

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

Test material

Constituent 1
Reference substance name:
The product from the burning of a combination of carbonaceous materials.
EC Number:
931-597-4
Molecular formula:
UVCB substance, not available. View remarks field.
IUPAC Name:
The product from the burning of a combination of carbonaceous materials.
Details on test material:
- Name of test material (as cited in study report): Aqueous extract of Mixed ash
- Composition range of test material (% (w/w)): Aluminium (Al) 13.07, Calcium (Ca) 45.52 , Iron (Fe) 4.15 , Magnesium (Mg) 3.49, Phosphorus (P) 0.79 , Potassium (K) 2.56, Silicon (Si) 28.07, Sodium (Na) 1.08, Sulphur (S) 1.27.
- The critical minor components examined (mg/kg d.w.): Arsenic (As) 18, Barium (Ba) 670 Cadmium (Cd) 3.6, Copper (Cu) 410, Lead (Pb) 180 and Antimony (Sb) 22.
- Purity test date: the substance is UVCB substance
- Lot/batch No.: Mixed ash 1_01032010
- Expiration date of the lot/batch: end of March 2011
- Storage condition of test material: room temperature
- Other: Mixed Ash is named in dossier as Ash. Mixed in the substance name has meant that there has been several (mixed) fuels when producing ash.
- Extraction of the test item: The test item is extracted in culture medium (toxicity assay) or in distilled water (genotoxicity assay) for one night under stirring at +37°C. After centrifugation, the supernatant was filtered and used for dilutions and/or treatments.

Method

Species / strain
Species / strain / cell type:
lymphocytes: Human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: culture medium RPMI 1640
- Collection of lymphocytes: Human lymphocytes were taken from two healthy non-smoker donors, male and female, receiving no medication and who have not suffered any recent viral infection. The blood was drawn onto lithium-heparin in a sterile Venoject tube.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor1254-induced S9 from rat livers (S9-mix)
Test concentrations with justification for top dose:
PRELIMINARY CYTOTOXICITY ASSAY
Initial concentrations of Mixed ash expressed as μg/mL:
Without S9-mix : 5000 – 2500 – 1250 – 625 – 312.5 – 156.25 – 78.13 (short and continuous treatments)
With S9-mix : 5000 – 2500 – 1250 – 625 – 312.5 – 156.25 – 78.13 (with either 5 or 10% S9-mix)

GENOTOXICITY ASSAY
Initial concentrations of Mixed ash expressed as μg/mL:
Without S9 mix :
5000 – 2500 – 1250 (assay 1: 4-hour treatment)
5000 – 2500 – 1250 (assay 2: 20-hour treatment)
5000 – 2500 – 1250 (assay 2: 44-hour treatment)
With S9 mix :
5000 – 2500 – 1250 (assay 1: with 5% S9-mix)
5000 – 2500 – 1250 (assay 2: with 10% S9-mix)

METAL CONCENTRATIONS OF THE TEST SOLUTION
The test media (aqueous extract of Ash) contained 548 µg/l of aluminium, 0.89 µg/l of antimony, 0.14 µg/l of arsenic, 4202 µg/l of barium, 0.02 µg/l of cadmium, 2.93 µg/l of copper and 68.7 µg/l of lead. The metal concentrations in the solvent control were 5.45, 0.01, <0.01, 0.23, 0.01, 0.12 and 0.01 µg/l for Al, Sb, As, Ba, Cd, Cu and Pb, respectively.
Vehicle / solvent:
Vehicle(s)/solvent(s) used:
- RPMI 1640 (Biochrom, batch 1173T), for toxicity assay
- distilled water (Fresenius, batch 13DBP231), for the main genotoxicity assay
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix; 4 and 20-hour treatments

0.25 µg/mL
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix

10 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Culture medium
To 100 ml of RPMI 1640 culture medium are added 25 ml of inactivated fetal calf serum, 1ml of 30 mg/ml L-glutamine solution, 1 ml of antibiotic solution (penicillin 50000 IU/ml, streptomycin 25 mg/ml), 0.4 ml of 25000 IU/ml heparin and 2.4 ml of phytohaemagglutinin A solution in water (Wellcome).
- In-life: 03/05/2010 - 13/08/2010


DURATION
- Preincubation period: 48 h
- Exposure duration: Without S9-mix: 4 h + 20 h recovery period (short treatment), 20 and 44 h without recovery period (continuous treatments); With S9-mix: 4 h + 20 h recovery period, with 5% or 10 % S9-mix
- Fixation time (start of exposure up to fixation or harvest of cells): in short treatments the cells were harvested 20 h after the beginning of the treatment; in continuous treatments the cells were harvested 20 h and 44 h after the beginning of the treatment.

STAIN (for cytogenetic assays): stained with a 4% dilution of Giemsa reagent in water for 10 minutes.

NUMBER OF REPLICATIONS: 2 assays, 2 replicates per concentration

NUMBER OF CELLS EVALUATED: 100 cells/culture for negative and concentration groups and 50 cells/culture for positive control

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER: Sterility check was valid.
Evaluation criteria:
ACCEPTANCE CRITERIA FOR THE RESULTS
A study is accepted if:
- In the solvent control group, the mean number of cells carrying structural aberrations (excluding cells presenting gaps only) for the two subjects does not exceed 5%,
- In the positive control group (mitomycin C and cyclophosphamide), the number of breaks per cell and number of cells with aberrations (excluding those with gaps only) is significantly increased. The values observed must be close to those of historical data of reference substances.
- At least 2 of the cultures treated with the test item present a mean mitotic index for the 2 subjects greater than about 50% compared to the reference culture.

INTERPRETATION OF THE RESULTS
A test item is found to demonstrate clastogenicity against cultured human lymphocytes if it results in a statistically significant increase in the number of breaks per cell compared with the control or in the number of cells with aberrations (excluding gaps), if this increase amounts to at least a doubling of the control value and if the genotoxicity detected shows a dose-effect relationship.

A test item is found to have no clastogenic effect on cultured human lymphocytes if it does not comply with any of the 3 criteria listed above.
Statistics:
Gaps and breaks are analyzed using Student's t-test. The test can be used for large number of samples although the
standard deviations found do not possess normal distribution.

The total number of damaged cells with and without gaps is analyzed statistically using the χ2 test.

All numerical aberrations (aneuploidy and polyploidy) are analyzed statistically using the χ2 test.

Results and discussion

Test results
Species / strain:
lymphocytes: Human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was comprised in the acceptable range of 6-8.5 at the highest concentrations tested from 5000 to 1250 μg/mL.
- Effects of osmolality: The initial extract and successive dilutions induced no variation in osmolarity higher than 50 mOsmol/kg when compared to the solvent control.

RANGE-FINDING/SCREENING STUDIES: Preliminary cytotoxicity assay

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: strain/cell type: Human peripheral blood lymphocytes

Any other information on results incl. tables

Table 1 Osmolarity and pH in the 4 -hour treatment

 Compound

 Initial concentration inμg/mL

pH

Osmolality (mOsmol/kg)

Osmolality variation  (mOsmol/kg) Compared to solvent control

Culture medium (RPMI)

 7.41

280

Aqueous extract of Mixed ash

5000 

 8.46

277

 -3

2500 

 8.04

279

 -1

1250 

 7.75

279

 -1

Table 2 The pH at the end of the 20 -hour treatment

Compound

Initial concentration inμg/mL

pH

Culture medium (RPMI)

 7.14

Aqueous extract of Mixed ash    

1250 

 7.21

2500 

 7.24

5000 

 7.31

Table 3 Summary of the preliminary toxicity assays with or without metabolic activation

COMPOUND  

Assay S9- / 4h

Assay S9+ / 5% S9-mix

Initial conc. inμg/mL

% Mitotic index relative to controla

Initial conc. inμg/mL

% Mitotic index relative to controla

Solvent control

0

-

0

 -

Aqueous extract of Mixed ash

5000

88.7

5000

68.0

2500

76.8

2500

77.8

1250

69.5

1250

94.1

625

84.2

625

100.0

312.50

92.7

 312.50

103.9

156.25

47.5

156.25

67.3

 78.13

59.9 

78.13 

 62.7

COMPOUND

Assay S9- / 20h

Assay S9- / 44h

Assay S9+ / 10% S9-mix

 Initial conc. inμg/mL

% Mitotic index relative to controla

Initial conc. inμg/mL

% Mitotic index relative to controla

Initial conc. inμg/mL

% Mitotic index relative to controla 

Solvent control

0

-

0

-

0

-

Aqueous extract of Mixed ash

5000

80.8

5000

158.3

5000

70.4

2500

125.6

2500

175.7

2500

57.1

1250

89.7

1250

201.9

1250

81.5

625

82.7

625

119.4

625

83.6

312.50

117.3

312.50

179.6

 312.50

82.0

156.25

58.3

156.25

72.8

 156.25

45.5

78.13

75.0

78.13

51.5

 78.13

49.7

a : expressed as percentage of cells undergoing mitosis compared to solvent control, and based on 1000 cells counted

Table 4 Summary of metaphase analysis assays without metabolic activation

ASSAY 1 SHORT TREATMENT: 4 HOURS

 ASSAY 2 CONTINUOUS TREATMENT: 20 HOURS

ASSAY 2 CONTINUOUS TREATMENT: 44 HOURS

COMPOUND

Initial conc. inμg/mL

 % Mitotic index relative to controla

NUMERICAL ABERRATIONS /200 CELLSb 

 STRUCTURAL ABERRATIONS /200 CELLSb

Initial conc. inμg/mL

 % Mitotic index relative to controla

NUMERICAL ABERRATIONS /200 CELLSb

STRUCTURAL ABERRATIONS /200 CELLSb

 Initial conc. inμg/mL 

  % Mitotic index relative to controla

 NUMERICAL ABERRATIONS /200 CELLSb

 STRUCTURAL ABERRATIONS /200 CELLSb

 Polyploidy

 Break per cell m +/- sd

Abnormal cells excluding gaps only

Abnormal cells including gaps only

Polyploidy

Break per cell m +/- sd 

Abnormal cells excluding gaps only 

Abnormal cells including gaps only 

  Polyploidy

  Break per cell m +/- sd

 Abnormal cells excluding gaps only 

 Abnormal cells including gaps only  

Solvent control

 0

 0.010

±0.100

0.010±0.100

 3

 0

0.010

±0.100 

Mitomycin C

 0.25

103.7 

0

*

0.490

±0.643

<0.001 

45

<0.001 

41

<0.001 

0.25 

49.1 

0

*

0.620

±0.814

<0.001

46

<0.001

47

<0.001

 

 

 

 

 

 

Aqueous extract of Mixed ash

5000 

108.8 

 0

*

0.015

±0.122

N.S. 

3

N.S. 

 3

N.S. 

5000 

124.3 

0

*

0.020

±0.140

N.S. 

4

N.S.

 5

N.S.

5000 

109.1 

0

*

0.010

±0.100

N.S.

2

N.S.

3

N.S.

2500 

98.6 

 0

*

0.010

±0.100

N.S. 

2

N.S. 

 2

N.S.

2500 

134.9 

0

*

0.005

±0.071

N.S.

1

N.S.

1

N.S.

2500 

112.5 

 0

*

0.025

±0.157

N.S.

5

N.S.

6

N.S.

1250 

115.3 

 0

*

0.025

±0.186

N.S. 

4

N.S. 

6

N.S. 

1250 

110.1 

0

*

0.020

±0.172

N.S.

3

N.S.

3

N.S.

1250 

92.0 

 0

*

0.010

±0.100

N.S.

2

N.S.

4

N.S.

m : mean

sd : standard deviation

Student's t-test for breaks per cell

Chi2 test for numerical aberrations, and for structural aberrations (cells with aberrations including or excluding cells with gaps only)

N.S.=not statistically significant at the threshold of p<0.05

a : expressed as percentage of cells undergoing mitosis compared to solvent control, and based on 2000 cells counted

b : 100 cells for positive control

*: No statistical analysis could be performed

Table 5 Summary of metaphase analysis assays with metabolic activation

 

ASSAY 1 SHORT TREATMENT: 4 HOURS (5% S9-mix)

  ASSAY 2 SHORT TREATMENT: 4 HOURS (10% S9-mix)

COMPOUND

Initial conc. inμg/mL

 % Mitotic index relative to controla

NUMERICAL ABERRATIONS /200 CELLSb

 STRUCTURAL ABERRATIONS /200 CELLSb

Initial conc. inμg/mL

 % Mitotic index relative to controla

NUMERICAL ABERRATIONS /200 CELLSb

STRUCTURAL ABERRATIONS /200 CELLSb

 Polyploidy

 Break per cell m +/- sd

Abnormal cells excluding gaps only

Abnormal cells including gaps only

Polyploidy

Break per cell m +/- sd 

Abnormal cells excluding gaps only

Abnormal cells including gaps only

Solvent control

0

-

0

0.020

±0.172

3

5

0

-

0

0.005

±0.071

1

2

Cyclophosphamide

10

45.5

0

*

0.630

±0.826

<0.001

44

<0.001

46

<0.001

10

45.7

0

*

0.700

±0.759

<0.001

54

<0.001

54

<0.001

Aqueous extract of Mixed ash

5000

97.0

0

*

0.015

±0.122

N.S.

3

N.S.

4

N.S.

5000

111.4

1

N.S.

0.010

±0.100

N.S.

2

N.S.

3

N.S.

2500

105.9

0

*

0.000

±0.000

N.S.

0

N.S.

0

N.S.

2500

100.0

0

*

0.005

±0.071

N.S.

1

N.S.

2

N.S.

1250

96.0

0

*

 0.010

±0.141

N.S.

1

N.S.

2

N.S.

1250

106.6

1

N.S.

0.005

±0.071

N.S.

1

N.S.

2

N.S.

m : mean

sd : standard deviation

Student's t-test for breaks per cell

Chi2 test for numerical aberrations, and for structural aberrations (cells with aberrations including or excluding cells with gaps only)

N.S.=not statistically significant at the threshold of p<0.05

a : expressed as percentage of cells undergoing mitosis compared to solvent control, and based on 2000 cells counted

b : 100 cells for positive control

*: No statistical analysis could be performed

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Not clastogenic in human lymphocytes with or without S9 metabolic activation.
Executive summary:

Clastogenic potential of Ash was assessed in a GLP compliant guideline in vitro study in human lymphocytes in vitro (OECD Guideline 473 and EU Method B.10).

Ash induced neither statistically nor biologically significant increase in the number of breaks per cell or in the frequency of aberrant cells excluding or including gaps in the treatments with or without metabolic activation. Furthermore, no statistically significant increase in the number of cells with numerical aberrations was noted in any treatment. Under experimental conditions, Ash induced no clastogenic activity in human lymphocytes either in presence or in absence of metabolic activation, in the in vitro human lymphocyte metaphase analysis test.