Reaction products of sulphuric acid, rock phosphate and humic acids, potassium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

26 April 2001 -21 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well-documented, GLP-compliant, according to OECD 471 in Salmonella typhimurium and E. coli. The rationale to use data from individual constituents and components of the complex is explained in chapter 1 of the CSR and in the adjacent "read-across document".

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Triple Super Phospate

IUPAC Name:

Triple Super Phospate

Constituent 2

Reference substance name:

Superphosphates, concd.

EC Number:

266-030-3

EC Name:

Superphosphates, concd.

Cas Number:

65996-95-4

Molecular formula:

not applicable (a generic molecular formula cannot be provided for this specific multi-constituent substance)

IUPAC Name:

Superphosphates, concd.

Details on test material:

- Name of test material (as cited in study report): Granular Triple Super Phosphate (GTSP) 45.43% P2O5 equivalent
- Substance type: Brown discrete particles
- Physical state: solid
- Analytical purity: 100% (provided by sponsor)
- Lot/batch No.: June 8, 2000
- Expiration date of the lot/batch: June 8, 2001
- Stability under test conditions: not indicated
- Storage condition of test material: room temperature, protected from light and moisture

Method

Target gene:

Salmonella tester strain cultures: deep rough mutation (ifa) and the deletion in the uvrB gene.
Cultures of tester strains TA98 and TA100: presence of the pKM101 plasmid R-factor.
All WP2 uvrA cultures: deletion in the uvrA gene

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:

other: TA98 + TA1537: reverted from histidine dependence to histidine independence by frameshift mutagens. TA1535: reverted by mutagens that cause basepair substitutions. TA100: reverted by mutagens that cause both frameshift and basepair substitution mutations

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:

other: Specificity of the reversion mechanism in E. coli is sensitive to base-pair substitution mutations, rather than frameshift mutations

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9-mix from male Sprague-Dawley rats

Test concentrations with justification for top dose:

Initial toxicity-mutation assay: 125, 250, 375, 500, 625, 1250, 2500 and 5000 microg/plate
Confirmatory mutagenicity assay: 50, 150, 500, 1250, and 3125 microg/plate for Salmonella and 150, 500, 1250, 3125, and 5000 microg/plate for E. coli

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 1 N HCl (CAS 7647-01-0), obtained from Fischer Scientific. 1 N HCl was heated to 50 °C for approximately 30 minutes
prior to use in test article preparation, All dilutions were held at 50 °C during dosing.
- Justification for choice of solvent/vehicle: Based on compatibility with the target cells and workability of the test article.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene: 1.0 and 10 microg/plate

Remarks:

All tester stains with S9 activation mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 without S9 activation mix

Migrated to IUCLID6: 1.0 microg/plate

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100 and TA 1535 without S9 activation mix

Migrated to IUCLID6: 1.0 microg/plate

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S9 activation mix

Migrated to IUCLID6: 75 microg/plate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA without S9 activation mix

Migrated to IUCLID6: 1000 microg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in top agar (plate incorporation)

DURATION
- Preincubation period: overnight (To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored)
- Exposure duration: approximately 48 to 72 hours at 37+/-2 °C

NUMBER OF REPLICATIONS:
Initial Toxicity-Mutation Assay in duplicate
Confirmatory Mutagenicity Assay in triplicate

DETERMINATION OF CYTOTOXICITY
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope.

OTHER EXAMINATIONS:
- Other: Precipitate was evaluated by visual examination without magnification.

OTHER:
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

Evaluation criteria:

For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated.
Since no positive responses were observed, no statistical analysis of the responses was performed.

Results and discussion

Test results

Test results 1

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:

cytotoxicity

Remarks:

Inital toxicity-mutation assay: toxicity at 5000 micg/plate and with 200 micL of solvent control/plate. Confirmatory mutagenicity assay: slight or moderate reduction in background lawn at 125 micL vehicle controls and corresponding test article aliquots

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Confirmatory mutagenicity assay: slight or moderate reduction in background lawn at 200 microL vehicle controls and corresponding test article aliquots

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 1250-5000 microg/plate

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Initial toxicity-mutation assay:

With (+) or Without (-) S9-mix	Test substance concentration (microg/plate)	Number of colonies/plate^a)
		Base-pair substitution type			Frame shift type
		TA100	TA1535	WP2uvrA		TA98	TA1537
-S9 mix	Solvent control (25 µl)	176	15	13		12	6
	Solvent control (50 µl)	162	11	11		12	7
	Solvent control (100 µl)	106	9	7		5	8
	Solvent control (200 µl)	70	4	8		4	4
	125	183	13	8		7	6
	250	168	18	9		10	6
	375	144	14	7		11	7
	500	168	10	9		8	5
	625	174	13	16		11	7
	1250	154 *	11 *	9 *		13 *	8 *
	2500	112 *	10 *	9 *		6 *	4 *
	5000	87 *	7 *	10 *		6 *	5 *
+S9 mix	Solvent control (25 µl)	204	17	12		19	13
	Solvent control (50 µl)	168	9	11		14	8
	Solvent control (100 µl)	112	12	9		7	3
	Solvent control (200 µl)	16	5	9		7	2
	125	195	7	9		12	6
	250	197	13	9		14	8
	375	199	12	9		15	9
	500	163	12	9		12	6
	625	209	18	8		12	7
	1250	168 *	15 *	9 *		16 *	10 *
	2500	158 *	13 *	10 *		8 *	5 *
	5000	120 *	6 *	11 *		7 *	4 *
Positive control without S9 mix	Name	NaN3	NaN3	MMS		2-NF	9-AA
	Number of colonies/plate	588	196	96		184	891
Positive control with S9 mix	Name	2-AA	2-AA	2-AA		2-AA	2-AA
	Number of colonies/plate	1191	180	289		901	148

a) The average number of revertant colonies from two plates *)Precipitate NaN3= sodium azide; 2-NF= 2-nitrofluorene; 9-AA = 9-aminoacridine; MMS = methyl methanesulfonate; 2 -AA=2 -aminoanthracene Solvent control = 1 N HCl

Confirmatory Mutagenicity assay:

With (+) or Without (-) S9-mix	Test substance concentration (microg/plate)	Number of colonies/plate^a)
		Base-pair substitution type			Frame shift type
		TA100	TA1535	WP2uvrA		TA98	TA1537
-S9 mix	Solvent control (50 µl)	125	13	12		15	4
	Solvent control (125 µl)	63	11	5		8	2
	Solvent control (200 µl)			7
	50	124	12			10	4
	150	117	10	12		14	5
	500	100	13	11		10	4
	1250	99 *	10 *	11		9 *	4 *
	3125	38 *	9 *	9 *		9 *	3 *
	5000			6 *
+S9 mix	Solvent control (50 µl)	138	11	10		14	6
	Solvent control (125 µl)	61	7	9		10	5
	Solvent control (200 µl)			8
	50	127	8			15	6
	150	131	11	14		13	5
	500	123	10	11		11	5
	1250	134 *	9 *	10		13 *	5 *
	3125	95 *	10 *	11 *		8 *	3 *
	5000			9 *
Positive control without S9 mix	Name	NaN3	NaN3	MMS		2-NF	9-AA
	Number of colonies/plate	416	133	87		125	704
Positive control with S9 mix	Name	2-AA	2-AA	2-AA		2-AA	2-AA
	Number of colonies/plate	1410	65	87		583	192

a) The average number of revertant colonies from three plates

*)Precipitate

NaN₃= sodium azide; 2-NF= 2-nitrofluorene; 9-AA = 9-aminoacridine; MMS = methyl methanesulfonate; 2 -AA=2 -aminoanthracene

Solvent control = 1 N HCl

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

All criteria for a valid study were met as described in the protocol.
The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Granular Triple Super Phosphate (GTSP)
did not cause a positive response in the presence and absence of Aroclor-induced rat liver S9.

Executive summary:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test according to OECD TG 471 in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as the Escherichia coli strain WP2 uvrA with and without metabolic activation (Aroclor 1254-induced rat liver S9-mix from male Sprague-Dawley rats) at concentrations of 125, 250, 375, 500, 625, 1250, 2500 and 5000 microg/plate in the initial toxicity-mutation assay and at concentrations of 50, 150, 500, 1250, and 3125 microg/plate for Salmonella and 150, 500, 1250, 3125, and 5000 microg/plate for E. coli in the confirmatory mutagenicity assay. All criteria for a valid study were met as described in the protocol. The results indicate that, under the conditions of this study, the test substance did not cause any positive response in the presence and absence of Aroclor-induced rat liver S9. The test item did not reveal any mutagenic activity. The appropriate reference mutagenes showed distinct positive mutagenic effects.