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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:
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: Comparable to guideline study with acceptable restrictions
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Genotoxic effects of aluminum chloride in cultured human lymphocytes treated in different phases of the cell cycle.
Author:
Lima et al.
Year:
2007
Bibliographic source:
Food Chem Toxicol 2007; 45: 1154-1159.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
: deviation with respect to the number of metaphases scored; lack of details on test substance
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): aluminium chloride (test material was obtained from Sigma-Aldrich, St. Louis, MO, USA.)- Analytical purity: no data

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
The human lymphocytes were obtained from heparinized whole blood samples provided by four “ normal, healthy donors”, two men and two women, aged 21 to 26 years with no history of smoking/drinking or chronic drug use. Medium:5mL HAM-F10 (78%, heat-inactivated fetal calf serum (20%), phytohemagglutinin-M (2%) and antibiotics 0.01mg/mL of penicillin, 0.005 mg/mL streptomycin.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
0, 5, 10, 15 and 25 μM AlCl3.
Vehicle / solvent:
Methanol (CAS#: 67-56-1, Merck-Schuchardt Co.)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Doxorubicin (0.01 µg/mL)
Details on test system and experimental conditions:
Cell culture processing & conditions:Short-term lymphocyte cultures were initiated according to a standard protocol (reference provided: Preston et al., 1987) using 5 mL HAM-F10 (78%, heat-inactivated fetal calf serum (20%), phytohemagglutinin-M (2%) and antibiotics 0.01 mg/mL of penicillin, 0.005 mg/mL streptomycin. Cultures were maintained at 37 ºC in a humidifed atmosphere composed of 5% CO2 and 95% humidity.Preparation of test solutions:Limited information. The article states that the test solutions were prepared by dissolution in methanol. The authors state (but do not provide the actual data to support the statement) that methanol did not reduce the mitotic index when compared to cultures without its presence and did not induce chromosomal aberrations.Administration of test solutions:Volume: NR; Method not reported.Duration of exposure, schedule/duration of incubations:For the G1 phase: lymphocytes were treated with a combination of 0.2 mL PHA and AlCl3 and then incubated for 52 hours at 37 ºC until fixation.For the transition G1-S phase: cultures were treated with AlCl3 24 hours after stimulation with PHA and then incubated for 52 hours at 37 ºC until fixation.For the S phase:Pulse treatments of AlCl3 were administered for 1 hour and 6 hours, 24 hours after PHA stimulation. After each pulse treatment, cells were washed once in serum-free medium, re-incubated in the complete medium for 52 hours prior to fixation.For the G2 phase:69 hour cultures were treated with AlCl3 for 3 hours and then fixed immediately, giving a total incubation time of 72 hours.Analytical verification of dose levels:Not carried out.Incubations per dose/time point:Unclear; appears to be single cultures but one from each donor? No measures of variability were provided.Further details on study design:Cells were fixed using colchicine (final concentration 0.0016%) at 50 hours and then harvested at 52 hours. The cells were harvested by centrifugation, treated with KCl at 37ºC for 20 minutes. The cells were then centrifuged again and fixed in 1:3 (v/v) acetic acid: methanol. The slides were then prepared, air-dried and stained with 3% Giemsa (ph=6.8) for 8 minutes.No metabolic activation was applied.Measurement of study outcomes:Metaphases were examined using an optical microscope to enumerate the number of structural and mureical CAs. The frequency of CAs was determined in 100 metaphases per culture. The mitotic index was also determined (number of metaphases per 2000 lymphoblasts per culture).Ancillary endpoints examined:-cytotoxicity: The mitotic index was determined (number of metaphases per 2000 lymphoblasts per culture). Polyploidy and endoreduplication.
Evaluation criteria:
CA assayThe statistical significance of differences between treatment and control appears to be the criteria used to define a positive or negative response. Gaps and breaks were included in the total chromosome aberration index. There is uncertainty concerning the actual types of structural aberrations included in the “total” index.
Statistics:
The student’s t-test was used to compare the frequencies of CAs observed in cells exposed to AlCl3 with the control.One-way ANOVA (F-test)was used to test for significant differences in the mitotic index.

Results and discussion

Test results
Species / strain:
lymphocytes:
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Ancillary Data:The MI in treated cultures was 32 to 61% of the negative control MI in the cells assessed in the G1 phase.The MI was also significantly reduced relative to the negative control in all treated cultures in the G1/S phase. At 25 μM AlCl3, the MI was only 21% of the control.The largest decreases in MI were observed in the S-phase cells exposed to pulse treatment of AlCl3 for 6 hours. The MI (expressed as a percentage of the negative control MI) were 25%, 18%, 9%, and 4% for the 5, 10, 15 and 25 μM AlCl3 concentrations, respectively.
Remarks on result:
other: strain/cell type: human lymphocytes
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

(*p<0.05, ** p<0.01)

G1 phase: chromosome aberrations per cell

Dose

(μM)

MI

(%)

Gaps

Breaks

Total

Poly

Endo

Contr

5.6

1

0

1

0

0

5

3.4*

4

6

10*

6

10*

10

2.4*

8

8

16*

10*

9*

15

1.9*

8

9

17*

9*

9*

25

1.8*

8

14

22*

14*

8*

DOX

3.1*

5

4

9*

2

4

 

G1/S phase: chromosome aberrations per cell

Dose

(μM)

MI

(%)

Gaps

Breaks

Total

Poly

Endo

Contr

5.5

2

0

2

0

1

5

2.8*

12

0

12*

0

0

10

1.9*

15

9

24*

1

0

15

1.5*

14

8

22*

0

1

25

1.2*

18

10

28*

1

0

DOX

3.2*

17

6

23*

5

0

 

S phase: chromosome aberrations per cell

(Pulse treatment for 1 hour)

Dose

(μM)

MI

(%)

Gaps

Breaks

Total

Poly

Endo

Contr

5.4

2

0

2

0

0

5

2.3*

28

8

36**

0

0

10

1.7*

35

11

46**

0

0

15

1.2*

30

14

44**

0

0

25

1.0*

43

19

62**

0

0

DOX

2.4*

10

7

17*

1

1

 

S phase: chromosome aberrations per cell

(Pulse treatment for 6 hours)

Dose

(μM)

MI

(%)

Gaps

Breaks

Total

Poly

Endo

Contr

4.9

2

0

2

0

0

5

1.4*

27

9

36**

0

0

10

1.0*

23

12

35**

0

0

15

0.5**

7

8

15*

0

0

25

0.2**

10

13

23*

0

0

DOX

1.9*

14

10

24*

3

0

 

G2 phase: chromosome aberrations per cell

Dose

(μM)

MI

(%)

Gaps

Breaks

Total

Poly

Endo

Contr

5.8

8

0

8

0

1

5

3.5*

15

4

19*

10*

2

10

3.0*

17

6

23*

14*

0

15

2.9*

23

8

31*

20*

4

25

2.4*

33

14

47**

27*

5

DOX

5.2*

24

8

32*

7

4

 

The authors state that “chromatid gaps and chromatid breaks” were the most frequent chromosome aberrations. However, it is unclear whether the “breaks” in the results tables presented in the article refer to chromatid breaks and/or chromosome breaks. The authors also included gaps in the total damage although gaps are not usually included in the total aberration frequency.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):positive without metabolic activationThe total aberrations (gaps plus breaks) were significantly higher in all treated cultures in cells in the G1 and G1/S phase, the S phase and also the G2 phase. During the G1 phase, the treated cultures also exhibited significantly increased polyploidy and endoreduplication compared with the negative control. The biological relevance of these results is unclear, however due to the high cytotoxicity in the cultures.
Executive summary:

Lima et al. (2007) determined the frequency of structural chromosome aberrations in human peripheral lymphocytes obtained from 4 young (aged 21 to 26 years), healthy, non-smoking donors on exposure to 5, 10, 15 and 25μM aluminium chloride (AlCl3) at different phases of the cell cycle. For the G1 phase, lymphocytes were treated with a combination of 0.2 mL PHA and AlCl3 and then incubated for 52 hours at 37 ºC until fixation. For the transition G1-S phase, cultures were treated with AlCl3 24 hours after stimulation with PHA and then incubated for 52 hours at 37ºC until fixation. For the S phase, pulse treatments of AlCl3 were administered for 1 hour and 6 hours 24 hours after PHA stimulation. After each pulse treatment, cells were washed once in serum-free medium, re-incubated in the complete medium for 52 hours prior to fixation. For the G2 phase, 69 hour cultures were treated with AlCl3 for 3 hours and then fixed immediately, giving a total incubation time of 72 hours. As pointed out by an expert reviewer, the cells would be in exponential growth by 69 hours and not synchronised in G2. The study was generally well-reported but lacked detail in some areas, e.g. the purity of the test compound. It appeared to follow GLP. The number of metaphases assessed was less than that recommended in OECD TG#473 and it is unclear how many cultures were used per dose and time point. The CA results are presented as single values, although results ought to have been available from 4 cultures, i.e. one from each of the 4 donors. No measure of the variability was provided. There is also uncertainty concerning the types of structural aberrations included in the “total” damage index. Both gaps and breaks were included in the index and whether the breaks were chromatid and/or chromosome was not specified. Methanol was used as the vehicle although this does not appear to have compromised the chromosome aberration results from the study. In the presence of metabolic activation, methanol may be metabolised to formaldehye (a DNA cross-linking agent) that can cause a wide range of DNA damage. As metabolic activation was not used in this study, the formation of formaldehyde would be low and the use of methanol as a vehicle is unlikely to have compromised the results of the study. The levels of CA in control cultures were normal, in the range expected for healthy human blood donors, and so it does not appear that the use of methanol compromised the study. Total aberrations (gaps plus breaks) were significantly higher than the control in all treated cultures in the G1 and G1/S phases, the S phase and the G2 phase.

During the G1 phase, the treated cultures also exhibited significantly increased polyploidy and endoreduplication compared with the negative control. Although not reaching statistical significance, breaks alone showed a dose-related increase. The results are positive but require qualification due to reduction of the mitotic index below 50% of the negative control at most of the AlCl3 concentrations tested. The result for chromosome aberrations is positive, but of unclear biological relevance [Klimisch Score=2]. The authors suggested that AlCl3-related adverse effects result from interference with the mitotic apparatus.