Boron orthophosphate

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:

25 Feb - 01 Jun 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Boron orthophosphate

EC Number:

236-337-7

EC Name:

Boron orthophosphate

Cas Number:

13308-51-5

Molecular formula:

BPO4

IUPAC Name:

boron(+3) cation phosphate

Test material form:

solid: particulate/powder

Method

Target gene:

his/trp operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity test:
- 156, 313, 625, 1250, 2500 and 5000 µg/plate (TA 98 and TA 1535 with and without metabolic activation)
Experiment I+II:
- 313, 625, 1250, 2500 and 5000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle/solvent used: distilled water
- Justification for choice of solvent: Distilled water was used as solvent, since this medium is not suspected of chemical reaction with the test substance and is compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9-mix: daunomycin (DM), sodium azide (NaN), ICR 191 acridine (ICR), 4-nitroquinoline-1-oxide (4-NQO); +S9-mix: 2-aminoanthracene (2-AA), benzo(a)pyrene (BaP)

Remarks:

DM (6 µg/plate: TA 98); NaN (1.5 µg/plate: TA 100, TA 1535); ICR (1 µg/plate: TA 1537); 4-NQO (2 µg/plate: E.coli WP2 uvrA); 2-AA (10 µg/plate: TA 98, TA 100, TA 1535, TA 1537; 50 µg/plate: E.coli WP2 uvrA); BaP (20 µg/plate: TA 98, TA 100)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 plates for each test concentration and control

DETERMINATION OF CYTOTOXICITY
- A preliminary experiment was conducted with the test material in tester strains TA 98 and TA 1535 either in the presence or in the absence of metabolic activation to determine cytotoxicity and solubility of the test substance using concentrations of 156, 313, 625, 1250, 2500 and 5000 µg/plate.
- Method: Cytotoxicity is detected by reduction in the number of revertant colonies

Evaluation criteria:

Means of individual plate counts (triplicates) were calculated for test solutions and controls. In general, a 2 or 2.5-fold increase in the number of revertant colonies per plate over the background (spontaneous revertant frequency) is used as a criterion to distinguish active mutagens from non-mutagenic materials. The presence of dose-response is a further criterion for mutagenic materials.

Statistics:

Mean values and standard deviation were calculated for each test and control concentration.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitations were seen in all test material concentrations which did not influence the results of the assay.

RANGE-FINDING/SCREENING STUDIES
As no relevant cytotoxicity was observed in the preliminary test (data not shown), the highest test concentration used in the main test was 5000 µg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY
No signs of cytotoxicity are reported.

COMPARISON WITH HISTORICAL CONTROL DATA
The colony counts of negative and positive controls were in the dimensions of historical and literature data.

Any other information on results incl. tables

Table 1: Test results - Experiment I - Preincubation method

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	0	79 ± 2	8 ± 0	13 ± 2	13 ± 4	8 ± 2
–	313	87 ± 7	11 ± 2	10 ± 2	12 ± 1	6 ± 1
–	625	69 ± 8	8 ± 2	13 ± 6	11 ± 4	6 ± 3
–	1250	74 ± 6	7 ± 3	15 ± 2	11 ± 4	3 ± 1
–	2500	89 ± 13	7 ± 5	11 ± 3	12 ± 5	4 ± 2
–	5000	85 ± 8	8 ± 1	11 ± 4	15 ± 6	7 ± 3
Positive controls, –S9	Name	NaN	NaN	4-NQO	DM	ICR
	Concentrations (μg/plate)	1.5	1.5	2	6	1
	Mean No. of colonies/plate (average of 3 ± SD)	499 ± 31	431 ± 26	839 ± 67	435 ± 95	2040 ± 59
+	0	68 ± 11	7 ± 2	10 ± 5	13 ± 3	4 ± 1
+	313	63 ± 6	6 ± 0	12 ± 2	17 ± 3	6 ± 1
+	625	59 ± 16	7 ± 1	12 ± 4	18 ± 4	4 ± 2
+	1250	68 ± 20	8 ± 4	11 ± 2	20 ± 5	5 ± 3
+	2500	70 ± 15	6 ± 2	11 ± 6	16 ± 2	4 ± 2
+	5000	72 ± 10	6 ± 4	10 ± 3	18 ± 2	4 ± 2
Positive controls, +S9 (10%)	Name	2-AA / BaP	2-AA	2-AA	2-AA / BaP	2-AA
	Concentrations (μg/plate)	10 / 20	10	50	10 / 20	10
	Mean No. of colonies/plate (average of 3 ± SD)	1807 ± 11 / 584 ± 19	56 ± 7	316 ± 62	2068 ± 60 / 290 ± 79	182 ± 25

DM: daunomycin

NaN: sodium azide

ICR: ICR 191 acridine

4-NQO: 4-nitroquinoline-1-oxide

2-AA: 2-aminoanthracene

BaP: benzo(a)pyrene

Table 2: Test results - Experiment II - Plate incorporation method

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	0	70 ± 12	8 ± 3	10 ± 5	29 ± 4	4 ± 1
–	313	81 ± 6	6 ± 3	12 ± 6	18 ± 8	4 ± 3
–	625	88 ± 13	8 ± 3	11 ± 1	17 ± 3	5 ± 1
–	1250	81 ± 9	7 ± 2	13 ± 2	10 ± 3	6 ± 1
–	2500	97 ± 12	6 ± 3	12 ± 3	17 ± 5	5 ± 2
–	5000	79 ± 7	6 ± 3	14 ± 2	16 ± 4	4 ± 2
Positive controls, –S9	Name	NaN	NaN	4-NQO	DM	ICR
	Concentrations (μg/plate)	1.5	1.5	2	6	1
	Mean No. of colonies/plate (average of 3 ± SD)	468 ± 37	385 ± 12	1185 ± 291	576 ± 99	82 ± 13
+	0	86 ± 12	4 ± 0	12 ± 6	12 ± 4	5 ± 2
+	313	72 ± 3	6 ± 3	12 ± 5	11 ± 6	4 ± 1
+	625	77 ± 4	7 ± 2	8 ± 3	22 ± 8	5 ± 2
+	1250	73 ± 10	5 ± 2	11 ± 2	19 ± 3	5 ± 1
+	2500	73 ± 16	7 ± 3	13 ± 2	14 ± 3	4 ± 2
+	5000	70 ± 5	5 ± 2	14 ± 1	12 ± 7	4 ± 2
Positive controls, +S9 (10%)	Name	2-AA / BaP	2-AA	2-AA	2-AA / BaP	2-AA
	Concentrations (μg/plate)	10 / 20	10	50	10 / 20	10
	Mean No. of colonies/plate (average of 3 ± SD)	856 ± 154 / 383 ± 9	45 ± 2	378 ± 48	1782 ± 201 / 136 ± 23	175 ± 49

DM: daunomycin

NaN: sodium azide

ICR: ICR 191 acridine

4-NQO: 4-nitroquinoline-1-oxide

2-AA: 2-aminoanthracene

BaP: benzo(a)pyrene

Table 3: Historical control data (negative control)

Strain	MIN		MAX		MEAN		SD		N
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
TA 98	13	21	54	65	29	38	16	19	5	5
TA 100	62	63	246	232	112	108	78	70	5	5
TA 1535	7	8	28	20	14	11	8	5	5	5
TA 1537	3	3	4	6	4	5	n.a.	n.a.	2	2
WP2 uvrA	13	13	17	20	15	18	2	3	4	4

SD: standard deviation

n.a.: not applicable

N: number of studies

Table 4: Historical control data (positive control)

Strain	Chemical	MIN	MAX	N
TA 98	2-Aminoanthracene	135	>1000	5
	Daunomycin	198	>500	5
TA 100	2-Aminoanthracene	326	>500	5
	Sodium azide	274	>500	5
TA 1535	2-Aminoanthracene	67	>300	5
	Sodium azide	83	>300	5
TA 1537	2-Aminoanthracene	107	210	2
	ICR 191 Acridine	126	283	2
WP2 uvrA	2-Aminoanthracene	251	407	4
	4-Nitroquinoline-1-oxide	756	1532	4

N: number of studies

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative