Phosphorus

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:

December 11 2008 - January 19, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to the OECD Guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phosphorus

EC Number:

231-768-7

EC Name:

Phosphorus

Cas Number:

7723-14-0

IUPAC Name:

phosphine

Constituent 2

Reference substance name:

phosphorus

EC Number:

918-594-3

Cas Number:

7723-14-0

Molecular formula:

(P)n (Phosphorus red modification)

IUPAC Name:

phosphorus

Details on test material:

- Name of test material (as cited in study report): Red Phosphorus

- Physical state: Solid
- Analytical purity: 99.5 % (w/w)
- Lot/batch No.: Fra 07-23 (Red Phosphorus untreated)
- Expiration date of the lot/batch: April 24, 2009
- Storage condition of test material: At room temperature under nitrogen

Method

Target gene:

histidine and tryptophan reversion

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

Deionised Water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

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


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

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

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Tables

The assay was performed in two independent experiments. Each concentration, including the controls, was tested in triplicate.

      Summary of Results Pre-Experiment and Experiment I

Study Name: 1227400	Study Code: Harlan - CCR 1227400
Experiment: 1227400 VV Plate	Date Plated: 11/12/2008
Assay Conditions:	Date Counted: 17/12/2008

Metabolic Activation	Test Group	Dose Level (µg/plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			15 ± 2	27 ± 7	32 ± 8	121 ± 12	44 ± 10
	Untreated			21 ± 4	28 ± 5	26 ± 10	138 ± 13	46 ± 11
	Red	3 µg		15 ± 2	33 ± 4	32 ± 5	115 ± 23	56 ± 18
	Phosphorus	10 µg		21 ± 4	27 ± 8	35 ± 7	129 ± 26	42 ± 3
		33 µg		16 ± 1	32 ± 3	33 ± 9	156 ± 21	42 ± 3
		100 µg		13 ± 1	27 ± 3	34 ± 7	132 ± 6	38 ± 3
		333 µg		17 ± 1	27 ± 6	29 ± 6	153 ± 58	56 ± 9
		1000 µg		19 ± 2^P	28 ± 4^P	31 ± 3^P	118 ± 19^P	46 ± 7^P
		2500 µg		14 ± 1^P	37 ± 6^P	34 ± 6^P	132 ± 8^P	40 ± 8^P
		5000 µg		17 ± 4^P M	17 ± 1^P M	18 ± 3^P M	121 ± 4^P M	50 ± 3^P M
	NaN3	10 µg		1821 ± 91			2122 ± 152
	4-NOPD	10 µg				559 ± 12
	4-NOPD	50 µg			137 ± 55
	MMS	3.0 µL						734 ± 70
With Activation	Deionised water			16 ± 5	35 ± 7	40 ± 9	111 ± 9	36 ± 1
	Untreated			14 ± 6	22 ± 2	46 ± 9	102 ± 10	39 ± 9
	Red	3 µg		14 ± 4	26 ± 6	37 ± 9	93 ± 6	44 ± 11
	Phosphorus	10 µg		17 ± 3	27 ± 4	50 ± 8	113 ± 14	51 ± 5
		33 µg		18 ± 2	40 ± 11	40 ± 8	117 ± 9	49 ± 1
		100 µg		17 ± 2	30 ± 3	39 ± 3	120 ± 9	43 ± 7
		333 µg		14 ± 1	35 ± 7	51 ± 5	92 ± 21	58 ± 4
		1000 µg		17 ± 7^P	31 ± 6^P	45 ± 10^P	92 ± 15^P	40 ± 2^P
		2500 µg		17 ± 2^P	33 ± 4^P	39 ± 11^P	118 ± 11^P	45 ± 5^P
		5000 µg		10 ± 2^P M	20 ± 4^P M	25 ± 3^P M	110 ± 7^P M	51 ± 7^P M
	2-AA	2.5 µg		216 ± 14	155 ± 12	1182 ± 32	1033 ± 55
	2-AA	10.0 µg						242 ± 7

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

     Summary of Results Experiment II

Study Name: 1227400	Study Code: Harlan - CCR 1227400
Experiment: 1227400 HV2 Pre	Date Plated: 14/01/2009
Assay Conditions:	Date Counted: 19/01/2009

Metabolic Activation	Test Group	Dose Level (µg/plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			19 ± 3	12 ± 5	30 ± 9	131 ± 5	47 ± 8
	Untreated			20 ± 3	11 ± 5	39 ± 8	120 ± 13	43 ± 3
	Red	33 µg		18 ± 7	9 ± 2	30 ± 7	121 ± 8	40 ± 2
	Phosphorus	100 µg		18 ± 3	14 ± 4	37 ± 6	120 ± 13	48 ± 8
		333 µg		21 ± 1	16 ± 4	29 ± 7	112 ± 5	48 ± 13
		1000 µg		19 ± 6	9 ± 4	32 ± 7	128 ± 2	47 ± 8
		2500 µg		19 ± 5^P	7 ± 1^P	33 ± 10^P	119 ± 21^P	40 ± 6^P
		5000 µg		14 ± 2^P	12 ± 3^P	36 ± 3^P	114 ± 2^P	38 ± 2^P
	NaN3	10 µg		2112 ± 140			2351 ± 238
	4-NOPD	10 µg				897 ± 23
	4-NOPD	50 µg			101 ± 10
	MMS	3.0 µL						297 ± 46
With Activation	Deionised water			19 ± 6	12 ± 2	39 ± 5	125 ± 22	61 ± 9
	Untreated			17 ± 5	14 ± 2	36 ± 1	126 ± 13	56 ± 18
	Red	33 µg		17 ± 8	13 ± 2	47 ± 4	119 ± 8	58 ± 3
	Phosphorus	100 µg		18 ± 4	10 ± 2	41 ± 7	116 ± 31	54 ± 10
		333 µg		21 ± 7	13 ± 5	37 ± 6	121 ± 20	55 ± 9
		1000 µg		17 ± 3	12 ± 4	30 ± 10	135 ± 18	55 ± 4
		2500 µg		22 ± 5^P	13 ± 6^P	35 ± 2^P	126 ± 18^P	54 ± 14^P
		5000 µg		14 ± 3^P	8 ± 1^P	42 ± 8^P	132 ± 10^P	56 ± 8^P
	2-AA	2.5 µg		283 ± 34	203 ± 21	1491 ± 154	1733 ± 283
	2-AA	10.0 µg						285 ± 20

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P	Precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Red Phosphorus did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item Red Phosphorus was assessed for its potential to induce gene mutations in the plate incorporation test (experi­ment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escheri­chia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:            3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                      33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Red Phosphorus at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher muta­tion rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.