Lithium Nickel Cobalt Aluminium Oxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 13 May 2008 and 04 June 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

aluminium(3+) nickel(2+) λ²-cobalt(2+) lithium(1+) tetraoxidandiide

EC Number:

700-042-6

Cas Number:

177997-13-6

Molecular formula:

LiNiCoAlO2 where stoichiometry of Ni+Co+Al equals 1 and ranges are Li = 0.9 – 1.3, Co = 0.01 – 0.2, Ni = 0.75 – 0.99 and Al = 0.01 – 0.2

IUPAC Name:

aluminium(3+) nickel(2+) λ²-cobalt(2+) lithium(1+) tetraoxidandiide

Method

Target gene:

Histidine for Salmonella
Trytptophan for E Coli

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Main test (Experiments 1 and 2): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water according to information provided by the sponsor. In solubility checks performed in-house, the test material was found to be insoluble in dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml. The best doseable suspension was achieved in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: (ENNG)

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: (4NQO)

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: (9AA)

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix Migrated to IUCLID6: (BP)

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)


DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 - 72 hours
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 - 72 hours


SELECTION AGENT (mutation assays): Not applicable.



NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.


OTHER EXAMINATIONS: None

Evaluation criteria:

Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation Criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

tested up to the maximum recommended dose level of 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

tested up to the maximum recommended dose level of 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: The test material was insoluble in sterile distilled water.
- Precipitation: A dark, particulate precipitate was observed under an inverted microscope at 5000 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: None.


RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.



COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.


ADDITIONAL INFORMATION ON CYTOTOXICITY: none.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results for the negative controls (spontaneous mutation rates) are presented in Table 1and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A dark, particulate precipitate was observed under an inverted microscope at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1               Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
128		25		40		19		15
129	(132)	25	(23)	35	(41)	19	(20)	14	(15)
140		19		47		22		15

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
123		13		23		17		20
113	(120)	17	(15)	35	(31)	23	(23)	17	(19)
124		16		34		30		21

 

Table 2               Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 24 May 2008						To: 27 May 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
-	0	108 115 129	(117) 10.7#	27 26 26	(26) 0.6	24 25 30	(26) 3.2		15 18 22	(18) 3.5	8 10 10	(9) 1.2
-	50	134 126 130	(130) 4.0	27 30 34	(30) 3.5	29 29 29	(29) 0.0		14 20 22	(19) 4.2	13 13 8	(11) 2.9
-	150	118 118 132	(123) 8.1	40 23 22	(28) 10.1	26 27 27	(27) 0.6		12 10 12	(11) 1.2	10 10 10	(10) 0.0
-	500	117 103 117	(112) 8.1	29 21 21	(24) 4.6	29 27 31	(29) 2.0		16 12 12	(13) 2.3	16 16 8	(13) 4.6
-	1500	128 139 125	(131) 7.4	40 25 20	(28) 10.4	26 26 32	(28) 3.5		23 9 16	(16) 7.0	11 10 10	(10) 0.6
-	5000	130 140 120	(130) 10.0	16 22 31	(23) 7.5	22 35 33	(30) 7.0		18 12 19	(16) 3.8	10 12 11	(11) 1.0
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		1513 1127 1049	(1230) 248.5	114 119 120	(118) 3.2	651 660 647	(653) 6.7		115 115 114	(115) 0.6	2572 2606 2770	(2649) 105.9

ENNG   N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO   4-Nitroquinoline-1-oxide

9AA     9-Aminoacridine

#            Standard deviation

Table 3               Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 24 May 2008						To: 27 May 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
+	0	114 85 93	(97) 15.0#	15 18 19	(17) 2.1	40 21 35	(32) 9.8		21 27 10	(19) 8.6	22 14 20	(19) 4.2
+	50	91 93 99	(94) 4.2	16 16 21	(18) 2.9	41 25 26	(31) 9.0		36 22 15	(24) 10.7	23 15 8	(15) 7.5
+	150	86 101 112	(100) 13.1	18 15 15	(16) 1.7	33 34 34	(34) 0.6		19 19 21	(20) 1.2	15 23 16	(18) 4.4
+	500	102 100 80	(94) 12.2	14 12 10	(12) 2.0	35 34 33	(34) 1.0		10 24 20	(18) 7.2	27 16 14	(19) 7.0
+	1500	81 85 91	(86) 5.0	11 12 13	(12) 1.0	34 24 29	(29) 5.0		14 24 23	(20) 5.5	12 19 20	(17) 4.4
+	5000	91 93 92	(92) 1.0	19 9 15	(14) 5.0	29 34 26	(30) 4.0		23 19 21	(21) 2.0	22 13 10	(15) 6.2
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		2370 2221 2043	(2211) 163.7	319 354 307	(327) 24.4	443 514 510	(489) 39.9		126 151 114	(130) 18.9	207 158 206	(190) 28.0

2AA     2-Aminoanthracene

BP         Benzo(a)pyrene

#            Standard deviation

Table 4               Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 01 June 2008						To: 04 June 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
-	0	106 80 93	(93) 13.0#	17 10 17	(15) 4.0	43 34 35	(37) 4.9		18 21 34	(24) 8.5	18 17 22	(19) 2.6
-	50	91 101 86	(93) 7.6	23 16 14	(18) 4.7	41 31 36	(36) 5.0		25 27 27	(26) 1.2	21 22 21	(21) 0.6
-	150	93 111 104	(103) 9.1	15 13 20	(16) 3.6	41 39 39	(40) 1.2		16 28 23	(22) 6.0	16 19 21	(19) 2.5
-	500	110 107 108	(108) 1.5	15 10 18	(14) 4.0	43 43 42	(43) 0.6		17 23 23	(21) 3.5	17 19 20	(19) 1.5
-	1500	94 117 108	(106) 11.6	15 10 10	(12) 2.9	33 29 37	(33) 4.0		34 22 21	(26) 7.2	18 23 14	(18) 4.5
-	5000	100 118 112	(110) 9.2	11 9 15	(12) 3.1	44 28 35	(36) 8.0		28 30 21	(26) 4.7	22 21 15	(19) 3.8
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		416 586 568	(523) 93.4	85 147 138	(123) 33.5	758 762 745	(755) 8.9		167 146 141	(151) 13.8	2028 2103 1603	(1911) 269.6

ENNG   N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO   4-Nitroquinoline-1-oxide

9AA     9-Aminoacridine

#            Standard deviation

Table 5               Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 01 June 2008						To: 04 June 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
+	0	93 102 175	(123) 45.0#	14 10 14	(13) 2.3	29 26 31	(29) 2.5		14 19 26	(20) 6.0	15 13 21	(16) 4.2
+	50	109 98 108	(105) 6.1	9 9 9	(9) 0.0	21 23 23	(22) 1.2		18 27 21	(22) 4.6	19 25 18	(21) 3.8
+	150	78 72 79	(76) 3.8	11 9 9	(10) 1.2	23 23 29	(25) 3.5		20 19 25	(21) 3.2	23 13 17	(18) 5.0
+	500	99 68 91	(86) 16.1	9 12 12	(11) 1.7	23 22 29	(25) 3.8		20 28 23	(24) 4.0	21 17 22	(20) 2.6
+	1500	102 90 98	(97) 6.1	10 9 9	(9) 0.6	18 33 30	(27) 7.9		17 26 21	(21) 4.5	14 14 12	(13) 1.2
+	5000	92 109 122	(108) 15.0	12 10 9	(10) 1.5	20 22 23	(22) 1.5		25 34 9	(23) 12.7	13 20 16	(16) 3.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		2195 2014 2025	(2078) 101.5	445 142 275	(287) 151.9	412 487 486	(462) 43.0		113 188 184	(162) 42.2	271 570 510	(450) 158.2

2AA     2-Aminoanthracene

BP         Benzo(a)pyrene

#            Standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction. 

The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA^-were treated with suspensions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A dark, particulate precipitate was observed under an inverted microscope at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.