Aluminium tris(dihydrogen phosphate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 22 December 2009 and 05 February 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
(1) Both substances are inorganic salts containing a trivalent aluminium cation and phosphoric acid. Thus, they all share the Al3+ cation and the PO43- anion as common functional groups. The source substance also contains an Na+ ion, this is not expected to influence the toxicological profile of the substance. Therefore the toxicity of the above substances will be predominantly determined by the presence of the Al3+ cation.
(2) Both substances will ultimately dissociate into the common breakdown products of the Al3+ cations and the PO43- anion.
(3) In general, independently of the cation under consideration, the water solubility of phosphates decreases with increasing degree of phosphate condensation (orthophosphate > diphosphate > triphosphate > polyphosphate).
In accordance with the provisions set out in Annex XI, Section 1.5, the results of the studies used for assessment and read-across are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method; cover an exposure duration comparable to or longer than the corresponding test method; and adequate and reliable documentation of the applied method is provided in the technical dossier. Orthophosphates are not considered to be genotoxic and are essential micronutrients. As such the genotoxicity potential of the target substance will be predominantly determined by the presence of the Al3+ cation. On this basis the standard testing requirements, as detailed in Regulation (EC) 1907/2006 (REACH) were conducted on aluminium orthophosphate as this substance contains the greatest amount of aluminium (%w/w) in comparison to the target substance. This approach is considered to be reliable and justified and no further testing for aluminium tris(dihydrogenorthophosphate) is required.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Cross-reference

Reason / purpose for cross-reference:

assessment report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 15 September 2009 Date of Signature on GLP certificate: 26 November 2009

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium orthophosphate (M 13-03)

IUPAC Name:

Aluminium orthophosphate (M 13-03)

Constituent 2

Reference substance name:

22784-12-9

EC Number:

607-158-5

Cas Number:

22784-12-9

IUPAC Name:

22784-12-9

Constituent 3

Reference substance name:

Aluminium orthophosphate

EC Number:

232-056-9

EC Name:

Aluminium orthophosphate

Cas Number:

7784-30-7

Molecular formula:

Al.H3O4P

IUPAC Name:

[phosphato(3-)-kappa~3~O,O',O'']aluminum

Method

Target gene:

Histidine for Salmonella.
Tryptophan 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:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

EXAMPLE
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in–house. The test material formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: at 3 µg/plate

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene at 1 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: at 5µg/plate

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

with S9 mix Migrated to IUCLID6: 2-Aminoanthracene at 2 µg/plate

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: at 2 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene at 10 µg/plate

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: at 0.2 µg/plate

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous muation rate for TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix Migrated to IUCLID6: at 5 µg/plate

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate for TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: at 80 µg/plate

Controls 10

Untreated negative controls:

yes

Remarks:

Sponateous muation rate for TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene at 2 µg/plate

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- 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 hrs


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 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 10E9 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 not be an excessive loss of plates due to 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

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 maximum recommended dose of 5000 micro.g/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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 maximum recommended dose of 5000 micro.g/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 formed the best doseable suspension in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house.
- Precipitation: A pale, fine precipitate was observed at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

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).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

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.

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

Preliminary Toxicity Test

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA^-)

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	82	105	111	111	114	108	109	126	109	103	120P
+	TA100	104	94	99	50	63	83	69	82	90	77	92P
-	WP2uvrA^-	26	26	20	29	26	32	15	25	19	29	28P
+	WP2uvrA^-	28	32	30	25	31	26	29	27	28	24	25P

P         Precipitate

The test material caused a visible reduction in the growth of the bacterial background lawn of Salmonella strain TA100, without S9-mix, at 5000 µg/plate in the range-finding test only. However, this response was not observed in either the preliminary toxicity test or main test and was, therefore, considered spurious and of no biological relevance. No toxicity was noted to any of the remaining bacterial tester strains at any test material dose level in either the absence or presence of S9-mix. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pale, fine precipitate was observed at 5000 µg/plate, this observation 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, at any dose level either with or without metabolic activation or exposure method.

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.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation for the Main test are presented in the tables below:

Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test (Experiment 1)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
93		14		43		13		11
120	(110)	13	(16)	24	(33)	23	(19)	11	(12)
118		22		33		20		14

Main Test (Experiment 2)

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
100		20		21		22		5
102	(101)	18	(19)	16	(20)	22	(22)	7	(7)
100		18		24		21		9

Test Results: Range-Finding Test– Without Metabolic Activation

Test Period		From: 17 January 2010					To: 20 January 2010
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	102 95 99	(99) 3.5#	29 31 19	(26) 6.4	36 35 40		(37) 2.6	20 21 13	(18) 4.4	8 13 16	(12) 4.0
-	50	97 72 91	(87) 13.1	26 28 24	(26) 2.0	35 36 37		(36) 1.0	21 16 15	(17) 3.2	19 19 11	(16) 4.6
-	150	80 88 81	(83) 4.4	21 30 32	(28) 5.9	38 38 38		(38) 0.0	19 17 22	(19) 2.5	21 13 14	(16) 4.4
-	500	87 85 91	(88) 3.1	20 24 16	(20) 4.0	33 36 41		(37) 4.0	15 14 10	(13) 2.6	11 16 14	(14) 2.5
-	1500	91 94 90	(92) 2.1	24 21 16	(20) 4.0	37 36 41		(38) 2.6	22 11 20	(18) 5.9	11 15 13	(13) 2.0
-	5000	62 TP 64 TP 83 TP	(70) 11.6	21 P 30 P 12 P	(21) 9.0	31 P 43 P 34 P		(36) 6.2	18 P 21 P 28 P	(22) 5.1	14 P 16 P 14 P	(15) 1.2
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		530 657 665	(617) 75.7	1521 1622 1317	(1487) 155.4	913 956 898		(922) 30.1	153 156 188	(166) 19.4	595 896 1010	(834) 214.4

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

T        Partial absence of bacterial background lawn

#        Standard deviation

Test Results: Range-Finding Test– With Metabolic Activation

Test Period		From: 17 January 2010					To: 20 January 2010
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	77 90 77	(81) 7.5#	18 16 25	(20) 4.7	46 38 47		(44) 4.9	24 29 21	(25) 4.0	19 17 16	(17) 1.5
+	50	90 63 71	(75) 13.9	9 9 12	(10) 1.7	34 41 49		(41) 7.5	21 25 27	(24) 3.1	10 20 12	(14) 5.3
+	150	97 77 C	(87) 14.1	17 20 26	(21) 4.6	38 48 45		(44) 5.1	30 24 21	(25) 4.6	9 15 17	(14) 4.2
+	500	85 68 77	(77) 8.5	18 29 21	(23) 5.7	30 40 41		(37) 6.1	17 27 21	(22) 5.0	15 15 13	(14) 1.2
+	1500	80 83 94	(86) 7.4	9 19 27	(18) 9.0	47 40 36		(41) 5.6	27 28 24	(26) 2.1	12 14 12	(13) 1.2
+	5000	75 P 77 P 76 P	(76) 1.0	22 P 22 P 21 P	(22) 0.6	37 P 47 P 39 P		(41) 5.3	22 P 16 P 27 P	(22) 5.5	16 P 5 P 11 P	(11) 5.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		846 1120 1065	(1010) 144.9	187 231 228	(215) 24.6	247 276 297		(273) 25.1	97 157 152	(135) 33.3	242 290 307	(280) 33.7

BP      Benzo(a)pyrene

2AA    2-Aminoanthracene

C        Contaminated

P        Precipitate

#        Standard deviation

Test Results: Main Test– Without Metabolic Activation

Test Period		From: 02 February 2010					To: 05 February 2010
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 94 94	(94) 0.6#	13 19 18	(17) 3.2	18 17 17		(17) 0.6	20 19 19	(19) 0.6	15 8 8	(10) 4.0
-	15	89 98 98	(95) 5.2	19 18 16	(18) 1.5	19 19 16		(18) 1.7	15 15 16	(15) 0.6	13 15 13	(14) 1.2
-	50	79 80 81	(80) 1.0	22 22 16	(20) 3.5	16 15 15		(15) 0.6	24 15 22	(20) 4.7	12 15 10	(12) 2.5
-	150	83 87 87	(86) 2.3	19 19 20	(19) 0.6	16 16 15		(16) 0.6	22 18 18	(19) 2.3	12 14 12	(13) 1.2
-	500	84 86 84	(85) 1.2	17 15 17	(16) 1.2	20 18 18		(19) 1.2	15 20 20	(18) 2.9	12 12 11	(12) 0.6
-	1500	77 81 76	(78) 2.6	15 15 16	(15) 0.6	18 20 12		(17) 4.2	21 16 22	(20) 3.2	15 14 13	(14) 1.0
-	5000	98 P 100 P 97 P	(98) 1.5	22 P 17 P 15 P	(18) 3.6	19 P 15 P 22 P		(19) 3.5	22 P 22 P 16 P	(20) 3.5	13 P 14 P 9 P	(12) 2.6
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		552 593 439	(528) 79.8	436 460 470	(455) 17.5	645 681 685		(670) 22.0	152 154 149	(152) 2.5	735 691 703	(710) 22.7

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

#        Standard deviation

FOR REST OF RESULTS SEE OVERALL REMARKS, ATTACHMENTS

Applicant's summary and conclusion

Conclusions:

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

This study is conducted according to an appropriate guideline and under the conditions of GLP, the study is therefore considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint.

Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA^-were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at up to six 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 for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended slightly following the results of the range-finding test and the change in test methodology and was 15 to 5000 µg/plate.

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 a visible reduction in the growth of the bacterial background lawn of Salmonella strain TA100, without S9-mix, at 5000 µg/plate in the range-finding test only. However, this response was not observed in either the preliminary toxicity test or main test and was, therefore, considered spurious and of no biological relevance. No toxicity was noted to any of the remaining bacterial tester strains at any test material dose level in either the absence or presence of S9-mix. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pale, fine precipitate was observed at 5000 µg/plate, this observation 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 or exposure method.

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