The product from the burning of a combination of carbonaceous materials.

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:

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

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

Test material information

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

Controls

Controls 1

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9-mix; 4 and 20-hour treatments

0.25 µg/mL

Controls 2

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)	0	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)	0	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 control^a	Initial conc. inμg/mL	% Mitotic index relative to control^a
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 control^a	Initial conc. inμg/mL	% Mitotic index relative to control^a	Initial conc. inμg/mL	% Mitotic index relative to control^a
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 control^a

NUMERICAL ABERRATIONS /200 CELLS^b

STRUCTURAL ABERRATIONS /200 CELLS^b

Initial conc. inμg/mL

% Mitotic index relative to control^a

NUMERICAL ABERRATIONS /200 CELLS^b

STRUCTURAL ABERRATIONS /200 CELLS^b

Initial conc. inμg/mL

% Mitotic index relative to control^a

NUMERICAL ABERRATIONS /200 CELLS^b

STRUCTURAL ABERRATIONS /200 CELLS^b

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

0.010 ±0.100

2

4

0

-

0

0.010±0.100

2

3

0

-

0

0.010 ±0.100

2

2

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 control^a

NUMERICAL ABERRATIONS /200 CELLS^b

STRUCTURAL ABERRATIONS /200 CELLS^b

Initial conc. inμg/mL

% Mitotic index relative to control^a

NUMERICAL ABERRATIONS /200 CELLS^b

STRUCTURAL ABERRATIONS /200 CELLS^b

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.