Reaction mass of aluminium and aluminium oxide and calcium oxide and magnesium oxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

15 Dec 2009 – 7 July 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium hydroxide

EC Number:

244-492-7

EC Name:

Aluminium hydroxide

Cas Number:

21645-51-2

IUPAC Name:

aluminum trihydroxide

Constituent 2

Reference substance name:

Aluminium chloride

EC Number:

231-208-1

EC Name:

Aluminium chloride

Cas Number:

7446-70-0

IUPAC Name:

aluminum trichloride

Details on test material:

- Name of test material (as cited in study report): aluminium hydroxide
- Physical state: white powder
- Analytical purity: 99%
- Lot/batch No.: 40/2009
- Expiration date of the lot/batch: nominal date provided by sponsor (31 Dec, 2010)
- Storage condition of test material: at 15 – 25°C in the dark


- Name of test material (as cited in study report): aluminium chloride
- Physical state: white powder
- Analytical purity: 99.999%
- Lot/batch No.: MKBB8304
- Expiration date of the lot/batch: 4 Dec, 2010
- Storage condition of test material: at 15 – 25°C in the dark

Method

Target gene:

thymidine kinase (tk)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for spontaneous mutant frequency

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S-9) was obtained from Molecular Toxicology Incorporated, USA.

Test concentrations with justification for top dose:

Aluminium hydroxide:
6.094, 12.19, 24.38, 48.75, 97.5, 195, 390 and 780 µg/mL for experiments 1 and 2 in the presence and in the absence of metabolic activation.

Aluminium chloride:
Experiment 1, Concentrations assayed: 3.125, 6.25, 12.5, 25 and 50 µg/mL in the presence and in the absence of metabolic activation.
Experiment 2, Concentrations assayed: 5, 10, 20, 40, 60, 80, 100 and 120 µg/mL in the absence of metabolic activation
5, 10, 15, 20, 25, 30, 40 and 50 µg/mL in the presence of metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 0.5% methylcellulose (MC) for aluminium hydroxide and purified water for aluminium chloride

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (-S9) and benzo(a)pyrene (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
+S9: 3 hours
-S9:
Aluminium chloride
- Experiment 1 – 3 hours
- Experiment 2 – 24 hour
Aluminium hydroxide:
- Experiment 1 – 3 hours
- Experiment 2 – 3 hours
- Expression time (cells in growth medium): For an expression period of 2 days, the cultures were maintained in flasks, subculturing as necessary to try to maintain cell densities below 1 x 10E6 cells/mL and also to keep at least 1 x 10E7 cells per flask.

SELECTION AGENT (mutation assays): 5-trifluorothymidine ( final concentration of 3 µg/mL)

NUMBER OF REPLICATIONS: duplicates in 2 experiments

NUMBER OF CELLS EVALUATED: number of TFT-resistant mutants per 10E6 viable cells 2 days after treatment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Mutant frequency (MF): number of TFT-resistant mutants per 10E6 viable cells 2 days after treatment.

MF = - ln P(0)/(number of cells per well x (viability/100))

- where P(0) = (number of wells with no colony/total number of wells)

*The number of cells per well was 2000 on average on all mutant plates.

Criteria used to Assess Assay Validity:
1. The mean mutant frequencies in the negative (vehicle) control are in the normal range between 50 and 70 mutants per 106 viable cells.
2. At least one positive control should show either an absolute increase in mean total MF of at least 300E10-6 (at least 40% of this increase should be in small colony MF), or an increase in small colony mutant frequency of at least 150E10-6 above the vehicle control.
3. The RTG for the positive controls should be greater than 10%.
4. The mean cloning efficiencies of the negative controls between 65% and 120% on day 2.
5. The mean suspension growth of the negative controls between 8 and 32 following 3 hour treatments or between 32 and 180 following 24 hour treatments.
6. No excessive heterogeneity between replicate cultures.

Positive response definition:
A test article was considered to be mutagenic in this assay if:
1. The MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF [global evaluation factor].
2. The linear trend was positive.

“A test article was considered as positive in this assay if BOTH of the above criteria were met. A test article was considered as negative in this assay if neither of the above criteria were met. Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.”

The GEF for microwell assays is defined as 126 mutants per 10E6 viable cells.

Statistics:

The significance of increases in mutant frequencies in comparison with controls and the global evaluation factor [GEF] was assessed. The data were checked for a linear trend in mutant frequency with increasing treatment concentration using weighted regression. The test for linear trend was one-tailed.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Aluminium hydroxide – Cytotoxicity Range-Finder Experiment
There were no appreciable differences in RTG and MF between the vehicle control and the untreated control.

Slightly greater toxicity (lower RTG value) was observed for controls subjected to the percoll differential centrifugation and cell recovery steps compared to controls not subjected to these steps.

These effects were considered minimal and acceptable, and there were no appreciable effects on the MF.

No significant changes in osmolality or pH were observed in the 3 and 24-hour range-finder experiment at the highest tested concentration (780 µg/mL) compared to the vehicle control.

Aluminium chloride – Cytotoxicity Range-Finder Experiment
No significant changes in osmolality or pH were observed in the 3 and 24-hour range-finder experiment at the highest tested/analysed concentration (83.38 µg/mL) compared to the vehicle control.

RANGE-FINDING/SCREENING STUDIES:
Aluminium hydroxide – Cytotoxicity Range-Finder Experiment:
There were no appreciable differences in RTG and MF between the vehicle control and the untreated control.

Slightly greater toxicity (lower RTG value) was observed for controls subjected to the percoll differential centrifugation and cell recovery steps compared to controls not subjected to these steps.

These effects were considered minimal and acceptable, and there were no appreciable effects on the MF.

No significant changes in osmolality or pH were observed in the 3 and 24-hour range-finder experiment at the highest tested concentration (780 µg/ml) compared to the vehicle control.

Aluminium chloride – Cytotoxicity Range-Finder Experiment:
No significant changes in osmolality or pH were observed in the 3 and 24-hour range-finder experiment at the highest tested/analysed concentration (83.38 µg/ml) compared to the vehicle control.

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Aluminium hydroxide: mutant frequencies (5-TFT resistant mutants/10⁶viable cells, 2 days after treatment)

Dose (µg/ml)	Experiment 1		Experiment 2
	-S9	+S9	-S9	+S9
0	64.78	59.07	53.44	43.62
UTC*	64.83	59.47	61.05	56.05
6.094	56.02	45.44	59.14	57.14
12.19	61.34	48.94	63.23	42.23
24.38	59.34	58.03	55.94	45.21
48.75	54.81	52.67	61.32	53.57
97.5	61.54	47.28	57.20	42.24
195	54.94	56.35	54.48	77.32
390	49.17	50.76	55.21	57.90
780	58.15	53.51	70.52	65.18
Linear trend	NS**	NS	NS	P<0.01
MMS/ B[a]P***
--/0.5	--	131.34		126.19
--/1	--	317.47		228.41
15/2	659.14	1047.7	441.10	525.12
20/3	755.10	878.38	593.79	883.47

*Untreated control

**Not significant

***MMS dose for -S9- or B[a]P dose for +S9

 

Mutant frequencies in both experiments at all tested concentrations were below the sum of the mean control mutant frequency plus the global evaluation factor (GEF). The observation of a significant linear trend in experiment 2 in the presence of S-9 without any corresponding increases in mutant frequencies approaching GEF was not considered a biologically relevant observation and the study was considered as providing a negative result for Al hydroxide.

 

The proportion of small colony mutants for the negative controls in the absence and presence of S-9 was 56% in experiment 1 and from 44% to 50% in experiment 2. Increased colony mutants were observed for the positive control substances.

 

Aluminium chloride

Experiment 1 (3-hour treatment). Mutant frequencies (5-TFT resistant mutants/10⁶viable cells 2 days after treatment)

Dose (µg/ml)	-S9	+S9
0	91.54	70.46
3.125	70.68	64.03
6.25	56.47	59.90
12.5	68.44	58.97
25	74.50	56.35
50	82.67	69.97
Linear trend	NS*	NS
MMS/ B[a]P**
15/2	699.96	934.97
20/3	1095.71	1650.41

*Not significant

**MMS dose for -S9 or B[a]P dose for +S9

 

Experiment 2 (24-hour treatment in the absence and 3-hour treatment in the presence of S-9). Mutant frequencies (5-TFT resistant mutants/10⁶viable cells 2 days after treatment)

Dose (µg/ml)	-S9	Dose (µg/ml)	+S9
0	52.63*	0	99.69
5	48.60	5	78.60
10	41.47	10	94.77
20	42.54	15	91.54
40	48.29	20	110.31
60	38.88	25	75.33
80	33.66	30	113.59
100	36.18	40	81.95
120	52.27	50	97.03
Linear trend	NS**		NS
MMS/ B[a]P***
5/2	962.16		1077.54
7.5/3	1246.55		887.11

*Based on one replicate only

**Not significant

***MMS dose for -S9 or B[a]P dose for +S9

 

In experiments 1 and 2 mutation frequencies at all tested concentrations were less than the sum of the mean control mutation frequency plus the GEF with a negative linear trend, indicating a negative result.

 

The proportion of small colony mutants for the negative controls in the absence and presence of S-9 were from 44% to 60% in experiment 1 and from 51% to 58% in experiment 2. Marked increases in the number of small and large colony mutants were observed after treatment with positive control chemicals.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Al hydroxide and Al chloride did not induce mutation at the tk locus of L5178Y mouse lymphoma cells under the conditions of these experiments.

Executive summary:

Covance (2010b) reported negative findings from their assay of forward mutations at the tk locus of L5178Y mouse lymphoma cells. They conducted two experiments with Al(OH)₃, each with a 3 hour treatment duration. Two experiments were also conducted with AlCl₃, one with a 3-hour and the other with a 24-hour treatment duration. Each experiment included incubations with and without metabolic activation. Concentrations of the test items were selected based on results of range-finding studies and observation of precipitation in the incubations. In the Al(OH)₃ experiments, eight concentrations ranging from 6.094 µg/mL to 780 µg/mL were used for determination of mutation frequencies. For AlCl₃, results from five concentrations (from 3.125 to 50 µg/mL) were used in the experiments with 3-hour treatment duration both in the presence and absence of metabolic activation. In the experiments with 24 hour duration treatments, 8 concentrations were used: from 5 to 120 µg/mL in the absence of metabolic activation and from 5 to 50 µg/mL in the presence of metabolic activation.Negative (vehicle) and positive (methyl methane sulphonate with S9 activation; benzo-[a]-pyrene without S9 activation) controls were included in each experiment. The results of the experiments with AlCl₃were negative: the mutant frequencies at all the tested concentrations were less than the sum of the mean control mutant frequency plus the global evaluation factor (GEF). A negative linear trend was also observed. In the experiments with Al(OH)₃, the mutation frequencies at all the tested concentrations were less than the sum of the mean control mutant frequency plus GEF. Although a significant positive linear trend in mutation frequencies was observed in the presence of S-9 in one of the experiments with Al(OH)₃, no corresponding increase in mutant frequencies approaching the GEF was observed, and the effect was not observed in the other experiment; therefore, this observation was not considered biologically relevant. This study was conducted in accordance with negative. This study is well described, was conducted in accordance with OECD TG #476 (1997), and with other guidelines, and complied with principles of Good Laboratory Practice.