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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from supporting substance, but study conducted according to OECD guideline 487 and GLP

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD draft proposal for new guideline 487 (2010)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Description Clear yellow liquid (determined at NOTOX)
- Test substance storage: At room temperature in the dark
- Stability under storage conditions: Stable
- Expiry date 18 December 2010 (allocated by NOTOX, 1 year after receipt of the test substance)

Method

Species / strain
Species / strain / cell type:
lymphocytes: peripheral human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix obtained from the liver of rats treated with phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
DOSE RANGE FINDING TEST:
- 3 hours exposure: 10, 33, 100, 333 and 1000 μg/ml culture medium, with and without S9
- 24 hours exposure: 10, 33, 100, 333, 1000 and 1105 μg PAX-18/ml culture medium, without S9

FIRST CYTOGENETIC ASSAY:
- 3 hours exposure, 27 hours harvest time: 100, 300 and 600 μg/ml culture medium, with and without S9
- Repetition due to precipitation observed at 600 μg/ml: 100, 300, 400, 500 and 600 μg/ml culture medium, with and without S9

SECOND CYTOGENETIC ASSAY:
- 24 hours exposure time, 24 hours harvest time: 100, 300 and 600 μg/ml culture medium, without S9
Vehicle / solvent:
Vehicle: exposure medium (RPMI 1640 medium)
Justification for choice of vehicle: Test compound was soluble in medium; culture medium is the recommended test substance vehicle according to OECD Guideline 487.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 1640 medium
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine (CAS# 64-86-8)
Remarks:
Without metabolic activation: Mitomycin C(MMC-C), Colchicine; with metabolic activation: Cyclophosphamide (CP). Solvent for positive controls: Hanks' Balanced Salt Solution (HBSS) without calcium and magnesium.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 46 ± 2 hours
- Exposure duration: 3 hours (first cytogenetic experiment, with and without S9), 24 hours (second cytogenetic experiment, without S9)

ARREST OF CELL DIVISION: Cytochalasin B
STAIN: Giemsa

NUMBER OF REPLICATIONS: at least two slides prepared per culture

NUMBER OF CELLS EVALUATED: at least 1000 binucleated and 1000 mononucleated cells scored per culture

DETERMINATION OF CYTOTOXICITY
- Method: minimum of 500 cells counted per culture (scoring cells with one, two or more nuclei). Cytostasis/cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI): CBPI = ((No. mononucleate cells) + (2x No. binucleate cells) + (3x No. multinucleate cells)) / total number of cells. %Cytostasis = 100-100{(CBPI(t) – 1)/(CBPI(c) –1)}. t = test substance or control treatment culture, c = vehicle control culture.
Evaluation criteria:
The following criteria for scoring of binucleated cells were used:
Main nuclei that
- were separate and of approximately equal size.
- touch and even overlap as long as nuclear boundaries are able to be distinguished.
- were linked by nucleoplasmic bridges.

Not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).

Criteria for scoring micronuclei:
- The diameter of micronuclei should be less than 1/3 of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

Test was considered acceptable if it meets the following criteria:
a) Number of mono and binucleated cells with micronuclei observed in 2000 cells of the solvent control should be <10.
b) Colchicine produced at least a statistically significant increase in the number mononucleated cells with micronuclei and MMC-C and CP produced at least a statistically significant increase in the number of binucleated cells with micronuclei.
c) A homogeneous response between the duplicate cultures is observed.

Test substance was considered positive in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

Test substance was considered negative if:
a) none of the tested concentrations induced a statistically significant increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the historical control data range.
Statistics:
The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics. If p< 0.05 the hypothesis that the incidence of cells with micronuclei is the same for both the treated and the solvent control group is rejected and the number of micronucleated cells in the test group is considered to be significantly different from the control group at the 95% confidence level.

Results and discussion

Test results
Species / strain:
lymphocytes: peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
DOSE RANGE FINDING TEST:
At 1000 μg/ml the test substance precipitated in the culture medium. See Table 1 in section "Any other information on results incl. tables" for cytokinesis-block proliferation index of cultures treated with various test substance concentrations or with negative control substance.
FIRST CYTOGENETIC ASSAY:
In the presence of S9-mix the amount of precipitate on the slides at the concentration of 600 μg/ml interfered with the scoring for the cytokinesis-block proliferation index (not calculated for all concentrations). The experiment was therefore repeated (see section "Test concentrations"). In this repeat experiment no precipitate was observed on the slides and the cytokinesis-block proliferation index could be determined. In the first cytogenetic assay another batch of S9-fraction was used which could have precipitated in combination with the test substance.
Both in the absence and presence of S9-mix, the test substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei (see table 3 in section "Any other information on results incl. tables").
SECOND CYTOGENETIC ASSAY:
All dose levels were selected for the scoring of micronuclei. The test substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei (see table 5 in section "Any other information on results incl. tables").

Any other information on results incl. tables

Table 1. Cytokinesis-block proliferation index of human lymphocyte cultures treated with the test substance in the dose range finding test.

Without metabolic activation (-S9-mix)

3 hours exposure time, 27 hours harvest time:

Concentration

µg/ml

Number of cells with….nuclei

 

CBPI

%

cytostasis

1

2

3 or more

0

332

126

42

1.42

0

10

333

129

38

1.41

2

33

332

132

36

1.41

3

100

343

130

27

1.37

12

333

344

133

23

1.36

15

10001)

358

117

25

1.33

20

 

 

Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time:

Concentration

µg/ml

Number of cells with….nuclei

 

CBPI

%

cytostasis

1

2

3 or more

0

361

122

17

1.31

0

10

359

123

18

1.32

-2

33

365

121

14

1.30

4

100

362

122

16

1.31

1

333

355

130

15

1.32

-3

10001)

371

112

17

1.29

6

11051)

385

104

11

1.25

19

 

 

With metabolic activation (+S9-mix)

3 hours exposure time, 27 hours harvest time:

Concentration

µg/ml

Number of cells with….nuclei

 

CBPI

%

cytostasis

1

2

3 or more

0

314

145

41

1.45

0

10

324

139

37

1.43

6

33

319

138

43

1.45

1

100

333

127

40

1.41

9

333

321

141

38

1.43

4

10001)

369

109

22

1.31

33

Note: All calculations were performed without rounding off.

1)Precipitate present on the slides which could interfere with the scoring for micronuclei.

Table 2. Cytokinesis-block proliferation index of human lymphocytes cultures treated with the test substance in the first cytogenetic assay and in repetition experiment.

Without metabolic activation (-S9-mix)

3 hours exposure time, 27 hours harvest time:

 

Concentration µg/ml

 

CBPI

 

Mean CBPI

%

cytostasis

0

1.50-1.53

1.52

0

100

1.50-1.51

1.51

2

300

1.51-1.54

1.53

-2

6001)

1.50-1.55

1.53

-2

0.25 MMC-C

1.31-1.33

1.32

38

0.1 Colch

1.06-1.08

1.07

87

 

With metabolic activation (+S9-mix)

3 hours exposure time, 27 hours harvest time:

 

Concentration µg/ml

 

CBPI

 

Mean CBPI

%

cytostasis

0

1.57-1.58

1.58

0

100

1.53-1.61

1.57

2

300

1.55-1.56

1.55

4

400

1.54-1.56

1.55

5

500

1.45-1.57

1.51

12

6001)

1.43-1.52

1.48

17

15CP

1.22-1.24

1.23

60

Note: All calculationswere performedwithout rounding off.

1)Test substance precipitated in the culture medium.

 

Table 3. Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with the test substance in the first cytogenetic assay and in repitition experiment.

Without metabolic activation (-S9-mix)

3 hours exposure time, 27 hours harvest time:

 

Concentration(µg/ml)

 

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

1000

1000

2000

1000

1000

2000

A

B

A+B

A

B

A+B

0

0

0

0

0

1

0

1

100

2

0

0

0

1

1

2

300

-2

0

0

0

2

0

2

6002)

-2

0

0

0

0

2

2

0.25 MMC-C

38

2

1

3

32

28

60***

0.1 Colch

87

47

51

98***

10

12

22***

 

With metabolic activation (+S9-mix)

3 hours exposure time, 27 hours harvest time:

 

Concentration(µg/ml)

 

Cytostasis(%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

1000

1000

2000

1000

1000

2000

A

B

A+B

A

B

A+B

0

0

0

0

0

4

2

6

300

4

0

0

0

0

1

1

500

12

0

0

0

2

1

3

6002)

17

0

0

0

0

3

3

15CP

60

0

0

0

23

27

50***

*) Significantly different from control group (Chi-square test),*P <0.05,**P<0.01 or ***P <0.001.

1) Duplicate cultures are indicated by A and B.

2) test substance precipitated in the culture medium.

 

Table 4. Cytokinesis-block proliferation index of human lymphocyte cultures treated with the test substance in the second cytogenetic assay.

Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time: 

 

Concentration (µg/ml)

 

CBPI

 

Mean CBPI

%

Cytostasis

0

1.32-1.32

1.32

0

100

1.32-1.36

1.34

-6

300

1.31-1.32

1.32

2

6001)

1.30-1.33

1.31

2

0.15 MMC-C

1.19-1.20

1.20

38

0.05 Colch

1.00-1.00

1.00

100

Note: All calculationswere performed without rounding off.

 1) Test substance precipitated in the culture medium.

Table 5. Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with the test substance in the second cytogenetic assay.

Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time: 

 

Concentration(µg/ml)

 

Cytotoxicity (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

1000

1000

2000

1000

1000

2000

A

B

A+B

A

B

A+B

0

0

0

1

1

1

4

5

100

-6

0

0

0

1

0

1

300

2

0

0

0

0

2

2

6002)

2

0

0

0

0

3

3

0.15 MMC-C

38

1

2

3

30

25

55***

0.05 Colch

100

47

50

97***

03)

14)

1

 

*) Significantly different from control group (Chi-square test),*P <0.05,**P<0.01 or ***P <0.001.

1) Duplicate cultures are indicated by A and B.

2) Test substance precipitated in the culture medium.

3) Number of micronucleated cells per 104 binucleated cells (see deviation number 3).

4) Number of micronucleated cells per 124 binucleated cells (see deviation number 3).

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that this test is valid and that the test compound is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.
Executive summary:

Aluminum chloride, basic (purity 30 - 40 ± 5%), was tested for its clastogenicity and aneugenicity potential in an in vitro micronucleus assay in cultured peripheral human lymphocytes (with independent repeat). The study was conducted according to OECD Guideline 487 and in compliance with GLP.

After a dose range finding test, two independent main cytogenetic experiments were conducted:

In the first cytogenetic assay, the test compound was tested up to 600 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). At 600 μg/ml, precipitation of the test substance was observed.

In the second cytogenetic assay, the test substance was also tested up to 600 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. The test compound precipitated in the culture medium at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range, except in the presence of S9-mix. Although the number of binucleated cells with miconuclei was above the historical

control data range in the presence of S9-mix, it was less than 10. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test compound did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Finally, it is concluded that this test is valid and that the test compound is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report