Fluorosulphuric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Fluorosulphuric acid hydrolyses in aqueous media to H₂SO₄and HF. In buffered medium as in the standard in vitro test systems the respective salts are formed. The sole resulting ions are fluoride and sulphate. Therefore NaF and Na₂SO₄are adequate and sufficient surrogates for investigation of mutagenicity of fluorosulphuric acid. Sodium fluoride and sodium sulphate were investigated as surrogates for fluorosulphuric acid in the Salmonella microsome test for point mutagenic effects (Herbold, 1987 & 1988). Evidence of mutagenic activity was not found for sodium fluoride and sodium sulphate with and without metabolic activation. A weak bacteriotoxic effect was found for sodium fluoride for doses >= 12500 µg/plate for two strains. Conclusion from these results: there is no evidence for mutagenic activity of fluorosulphuric acid.

Link to relevant study records

Reference

Reference 1

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:

July 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: only 4 strains, no strains with AT base pair at the primary reversion site, culture media used not reported

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: partly deficient in lipopolysaccharide side chains of their cell walls, UV repair ability malfunctions

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

first test: 0, 20, 100, 500, 2500, 12500 µg per plate
repeat test: 0, 375, 750, 1500, 3000, 6000, 12000 µg per plate

Vehicle / solvent:

the solvent used for NaF was demineralized water, and for the positive controls DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylenediamine (TA 1537 + TA 98), +S9: 2-aminoanthracene

Remarks:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h




NUMBER OF REPLICATIONS:
four plates per strain and dose, both with and without S-9 mix, were used for mutant count. Four plates per strain were also used for each positive control.




DETERMINATION OF CYTOTOXICITY
- other: background growth on the plates for the mutant determination; toxic effect assumed when mutant count per plate was clearly lower than the negative control count in dose correlation; determination of titer

Evaluation criteria:

A reproducible and dose-related increase (ie. at least twice the negative control count) in mutant counts for at least one strain was considered positive.
In the case of no reproducible and dose-related increase in mutant counts in at least one strain, the result was evaluated as negative.
In the case of questionable results, the investigations continued, probably by the use of modifications, until a final evaluation was possible.

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:

: Doses up to and including 6000 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had only a weak bacteriotoxic effect in TA 1535 and TA 100, so that this range could nevertheless be used for evaluation purposes.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: S. typhimurium TA1535, TA100, TA1537, TA98

Remarks:

Migrated from field 'Test system'.

None of the four strains used showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S-9 mix and was confirmed by the results of the repeat tests.

 

Table 1. Summary of the results of the Ames test on NaF without S-9 mix

	TA 1535			TA 100			TA 1537			TA 98
Test/doses µg/plate	Means revertantsper plate	Titer per ml 10^8	Quotient	Means revertantsper plate	Titer per ml 10^8	Quotient	Means revertantsper plate	Titer per ml 10^8	Quotient	Means revertantsper plate	Titer per ml 10^8	Quotient
1^sttest
0	11	30.8	1.0	61	6.4	1.0	7	37.6	1.0	16	56.0	1.0
20	11	24.4	1.0	72	7.7	1.2	7	33.7	1.0	16	58.0	1.0
100	9	27.7	0.8	74	7.9	1.2	6	35.1	0.9	23	64.6	1.4
500	10	35.3	0.9	71	12.9	1.2	5	34.1	0.6	16	53.0	1.0
2500	9	35.1	0.8	67	12.6	1.1	6	35.3	0.8	19	54.3	1.2
12500	9	17.9**	0.8	69	7.2	1.1	5	32.9	0.7	17	66.8	1.0
Na-Azide	601	36.4	55.9*
NF				217	9.1	3.6*
4-NPDA							48	34.8	6.6*	93	59.6	5.7*
2-AA (+S9)		38.7			11.9			34.6			49.4
2^ndtest
0	14	48.6	1.0	64	20.9	1.0	7	50.8	1.0	17	62.0	1.0
375	15	45.7	1.1	57	11.3	0.9	6	47.8	0.9	14	65.2	0.9
750	13	49.2	0.9	62	9.9	1.0	5	39.2	0.7	19	63.9	1.1
1500	12	48.2	0.9	56	10.4	0.9	8	48.5	1.3	18	67.6	1.1
3000	16	46.1	1.1	66	10.0	1.0	8	47.6	1.2	20	69.2	1.2
6000	14	51.3	1.0	67	10.1	1.0	5	49.4	0.8	15	73.8	0.9
12000	15	26.1**	1.1	77	7.4	1.2	5	45.7	0.8	15	80.0	0.9
Na-Azide	922	49.9	67.0*
NF				257	13.2	4.0*
4-NPDA							68	57.8	10.4*	130	53.5	7.7*
2-AA (+S9)		54.8			13.3			58.1			58.3

 

Na-Azide: Sodium azide, 10 µg/plate

NF: Nitrofurantoin, 0.2 µg/plate

4-NPDA: 4-nitro-1,2-phenylenediamine, 0.5 µg/plate

2-AA: 2-aminoanthracene, 3 µg/plate

 

* = mutagenic effect

** =bacteriotoxic effect

 

 

Table 2. Summary of the results of the Ames test on NaF with S-9 mix

	TA 1535		TA 100		TA 1537		TA 98
Test/doses µg/plate	Means revertantsper plate	Quotient	Means revertantsper plate	Quotient	Means revertantsper plate	Quotient	Means revertantsper plate	Quotient
1^sttest
0	15	1.0	132	1.0	8	1.0	44	1.0
20	17	1.1	122	0.9	8	1.0	35	0.8
100	16	1.1	107	0.8	8	1.0	33	0.7
500	19	1.3	128	1.0	9	1.1	29	0.7
2500	19	1.3	85	0.6	6	0.8	45	1.0
12500	18	1.2	48**	0.4	4	0.5	35	0.8
2-AA	143	9.7*	395	3.0*	27	3.5*	373	8.6*
2^ndtest
0	23	1.0	104	1.0	9	1.0	49	1.0
375	28	1.2	105	1.0	10	1.1	43	0.9
750	25	1.1	128	1.2	11	1.3	36	0.7
1500	19	0.8	108	1.0	7	0.8	42	0.9
3000	24	1.0	133	1.3	7	0.8	53	1.1
6000	20	0.9	132	1.3	8	0.9	36	0.7
12000	20	0.8	102	1.0	6	0.7	32	0.7
2-AA	235	10.1*	540	5.2*	43	5.0*	634	13.1*

 

2-AA: 2-aminoanthracene,3 µg/plate

 

* = mutagenic effect

** =bacteriotoxic effect

 

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

Executive summary:

Herbold BA (1987)

NaF (as a surrogate for fluorosulphuric acid) was investigated in a Salmonella / microsome test for point-mutagenic effects in doses up to 12500 µg per plate on four Salmonella typhimurium LT2 mutants with and without metabolic activation. Test was performed similar to the OECD guideline 471 with restrictions (Only four strains, no strains with AT base pair as primary reversion site. Different positive control substances used in comparison with OECD guideline. Culture media used: not reported).

For the test the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98 were used.

Doses up to and including 6000 µg per plate did not cause any bacteriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed.

At higher doses, the substance had only a bacteriotoxic effect in TA 1535 and TA 100, so that this range could nevertheless be used for evaluation purposes. Substance precipitation occurred from the dose 500 µg per plate.

Evidence of mutagenic activity for NaF was not found with and without metabolic activation. Neither a dose-related doubling of the mutant count nor a biologically relevant increase in the same, was observed in comparison with the negative controls.

The positive controls sodium azide (Na-azide) , nitrofurantoin (NF), 4-nitro-1,2-phenylene-diamine (4-NPDA),  and 2-aminoanthracene (2 -AA) had a marked mutagenic effect, as can be seen from the biologically relevant increase in mutant colonies, compared to the corresponding negative controls.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

July 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Fluorosulphuric acid (CAS 7789-21-1 EC 232-149-4) [target substance] is known to be a very strong inorganic acid (pH < 1). The substance is hydrolytically unstable and reacts violently with water (self-classification EUH014). When in contact with water it rapidly hydrolyses to it hydrolysis products sulphuric acid (CAS 7664-93-9 EC 231-639-5) and hydrofluoric acid (CAS 7664-39-3 EC 231-634-8).

Fast hydrolysis: HSO3F + H2O <=> H2SO4 + HF
Self-ionisation: HSO3F + H2O <=> H3O+ + SO3F-
Slow hydrolysis: SO3F- + H2O <=> HSO4- + HF

Both sulphuric acid (H2SO4) and hydrofluoric acid (HF) are both known to be strong inorganic acids. In water they dissociate rapidly to form hydrogen (H+) and its respective counter anions sulphate (SO42-) and fluoride (F−) ions

H2SO4 (aq) ⇌ H+ (aq) + HSO4- (aq) ⇌ 2H+ (aq) + SO42- (aq)
HF (aq) ⇌ H+ (aq) + F- (aq)

Sodium sulphate (CAS 7757-82-6 EC 231-820-9) [source substance 1], the highly soluble sodium salt of hydrogen sulphate, will be fully hydrated in water as sodium (Na+) and sulphate (SO42-) ions. In analogue, sodium fluoride (CAS 7681-49-4 EC 231-667-8) [source substance 2], the highly soluble sodium salt of fluoride, is also fully hydrated in water as separate sodium (Na+) and fluoride (F-) ions:

Na2SO4 (aq) ⇌ 2Na+ (aq) + SO42- (aq)
NaF (aq) ⇌ Na+ (aq) + F- (aq)

Since all biological systems are water based, the read-across hypothesis is based on the rational that fluorosulphuric acid (HSO3F) violently reacts with water under the formation of its hydrolysis products sulphuric acid (H2SO4) and hydrofluoric acid (HF). Both sulphuric acid and hydrofluoric acid as strong acids, as well as their sodium salts sodium sulphate and sodium fluoride will, to the same extent, dissociate rapidly to form sulphate and fluoride ions in contact with cells, mucous, blood or other fluids. Any systematic effects observed will be directly attributable to the anions sulphate and fluoride. Furthermore, as all environmental endpoints are aquatic or wet, the same read-across hypothesis applies. On this basis, for specific properties, the hazard profile of fluorosulphuric acid can be derived from its hydrolysis products sulphuric acd and hydrofluoric acid and is comparable to its sodium salts sodium sulphate and sodium fluoride with respect to the environment and human health.

An analogue approach is considered applicable since in the dossier read-across is “…employed between a small number of structurally- similar substances; there is no trend or regular pattern on the properties.” According to the Read-Across Assessment Framework (RAAF) guidance criteria (ECHA, 2017), an analogue approach is considered applicable so for the current read-across covered, scenario 1 is considered applicable as the registered substance fluorosulphuric acid, via its hydrolysis products sulphuric acid and hydrofluoric acid, and sodium fluoride and potassium fluoride are transformed to common compounds: the sulphate and fluoride anions.
The different cations – sodium, potassium and hydrogen – are not considered relevant for read-across justification because, following rapid dissociation within biological and aqueous environments, the hydrogen ions are responsible for localised effects only (corrosiveness), with no systemic availability. Sodium and potassium ions, as a ubiquitous essential metal ion, are naturally present in organisms and the environment. Consequently, neither cations are considered of any toxicological or ecotoxicological concern.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance fluorosulphuric acid is a mono-constituent substance with a high degree of purity typical concentration ca. 99.5 % (w/w) (range from 99-100 % (w/w)). Impurities are contained in low concentrations not relevant for classification and labelling.
Both source substances, sodium sulphate and sodium fluoride, are also well-defined mono-constituent substances with a high degree of purity and no impurities considered relevant for classification.
Based on the high degree of purities no impact from potential impurities on the read-across hypothesis applies neither from the target nor from the source substances.

3. ANALOGUE APPROACH JUSTIFICATION
In accordance with regulation EC No 1907/2006 (REACH) Annex XI.2. testing on the registered substance itself is technically not feasible due to intrinsic substance properties and may be omitted. Conducting studies is technically not possible because fluorosulphuric acid reacts violently with water. The substance is hydrolytically unstable and reacts violently with water (self-classification EUH014). When in contact with water it rapidly hydrolyses to it hydrolysis products sulphuric acid and hydrofluoric acid.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: only 4 strains, no strains with AT base pair at the primary reversion site, culture media used not reported

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: partly deficient in lipopolysaccharide side chains of their cell walls, UV repair ability malfunctions

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

first test: 0, 20, 100, 500, 2500, 12500 µg per plate
repeat test: 0, 375, 750, 1500, 3000, 6000, 12000 µg per plate

Vehicle / solvent:

the solvent used for NaF was demineralized water, and for the positive controls DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylenediamine (TA 1537 + TA 98), +S9: 2-aminoanthracene

Remarks:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h




NUMBER OF REPLICATIONS:
four plates per strain and dose, both with and without S-9 mix, were used for mutant count. Four plates per strain were also used for each positive control.




DETERMINATION OF CYTOTOXICITY
- other: background growth on the plates for the mutant determination; toxic effect assumed when mutant count per plate was clearly lower than the negative control count in dose correlation; determination of titer

Evaluation criteria:

A reproducible and dose-related increase (ie. at least twice the negative control count) in mutant counts for at least one strain was considered positive.
In the case of no reproducible and dose-related increase in mutant counts in at least one strain, the result was evaluated as negative.
In the case of questionable results, the investigations continued, probably by the use of modifications, until a final evaluation was possible.

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:

: Doses up to and including 6000 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had only a weak bacteriotoxic effect in TA 1535 and TA 100, so that this range could nevertheless be used for evaluation purposes.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: S. typhimurium TA1535, TA100, TA1537, TA98

Remarks:

Migrated from field 'Test system'.

None of the four strains used showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S-9 mix and was confirmed by the results of the repeat tests.

 

Table 1. Summary of the results of the Ames test on NaF without S-9 mix

	TA 1535			TA 100			TA 1537			TA 98
Test/doses µg/plate	Means revertantsper plate	Titer per ml 10^8	Quotient	Means revertantsper plate	Titer per ml 10^8	Quotient	Means revertantsper plate	Titer per ml 10^8	Quotient	Means revertantsper plate	Titer per ml 10^8	Quotient
1^sttest
0	11	30.8	1.0	61	6.4	1.0	7	37.6	1.0	16	56.0	1.0
20	11	24.4	1.0	72	7.7	1.2	7	33.7	1.0	16	58.0	1.0
100	9	27.7	0.8	74	7.9	1.2	6	35.1	0.9	23	64.6	1.4
500	10	35.3	0.9	71	12.9	1.2	5	34.1	0.6	16	53.0	1.0
2500	9	35.1	0.8	67	12.6	1.1	6	35.3	0.8	19	54.3	1.2
12500	9	17.9**	0.8	69	7.2	1.1	5	32.9	0.7	17	66.8	1.0
Na-Azide	601	36.4	55.9*
NF				217	9.1	3.6*
4-NPDA							48	34.8	6.6*	93	59.6	5.7*
2-AA (+S9)		38.7			11.9			34.6			49.4
2^ndtest
0	14	48.6	1.0	64	20.9	1.0	7	50.8	1.0	17	62.0	1.0
375	15	45.7	1.1	57	11.3	0.9	6	47.8	0.9	14	65.2	0.9
750	13	49.2	0.9	62	9.9	1.0	5	39.2	0.7	19	63.9	1.1
1500	12	48.2	0.9	56	10.4	0.9	8	48.5	1.3	18	67.6	1.1
3000	16	46.1	1.1	66	10.0	1.0	8	47.6	1.2	20	69.2	1.2
6000	14	51.3	1.0	67	10.1	1.0	5	49.4	0.8	15	73.8	0.9
12000	15	26.1**	1.1	77	7.4	1.2	5	45.7	0.8	15	80.0	0.9
Na-Azide	922	49.9	67.0*
NF				257	13.2	4.0*
4-NPDA							68	57.8	10.4*	130	53.5	7.7*
2-AA (+S9)		54.8			13.3			58.1			58.3

 

Na-Azide: Sodium azide, 10 µg/plate

NF: Nitrofurantoin, 0.2 µg/plate

4-NPDA: 4-nitro-1,2-phenylenediamine, 0.5 µg/plate

2-AA: 2-aminoanthracene, 3 µg/plate

 

* = mutagenic effect

** =bacteriotoxic effect

 

 

Table 2. Summary of the results of the Ames test on NaF with S-9 mix

	TA 1535		TA 100		TA 1537		TA 98
Test/doses µg/plate	Means revertantsper plate	Quotient	Means revertantsper plate	Quotient	Means revertantsper plate	Quotient	Means revertantsper plate	Quotient
1^sttest
0	15	1.0	132	1.0	8	1.0	44	1.0
20	17	1.1	122	0.9	8	1.0	35	0.8
100	16	1.1	107	0.8	8	1.0	33	0.7
500	19	1.3	128	1.0	9	1.1	29	0.7
2500	19	1.3	85	0.6	6	0.8	45	1.0
12500	18	1.2	48**	0.4	4	0.5	35	0.8
2-AA	143	9.7*	395	3.0*	27	3.5*	373	8.6*
2^ndtest
0	23	1.0	104	1.0	9	1.0	49	1.0
375	28	1.2	105	1.0	10	1.1	43	0.9
750	25	1.1	128	1.2	11	1.3	36	0.7
1500	19	0.8	108	1.0	7	0.8	42	0.9
3000	24	1.0	133	1.3	7	0.8	53	1.1
6000	20	0.9	132	1.3	8	0.9	36	0.7
12000	20	0.8	102	1.0	6	0.7	32	0.7
2-AA	235	10.1*	540	5.2*	43	5.0*	634	13.1*

 

2-AA: 2-aminoanthracene,3 µg/plate

 

* = mutagenic effect

** =bacteriotoxic effect

 

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

Executive summary:

Herbold BA (1987)

NaF (as a surrogate for fluorosulphuric acid) was investigated in a Salmonella / microsome test for point-mutagenic effects in doses up to 12500 µg per plate on four Salmonella typhimurium LT2 mutants with and without metabolic activation. Test was performed similar to the OECD guideline 471 with restrictions (Only four strains, no strains with AT base pair as primary reversion site. Different positive control substances used in comparison with OECD guideline. Culture media used: not reported).

For the test the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98 were used.

Doses up to and including 6000 µg per plate did not cause any bacteriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed.

At higher doses, the substance had only a bacteriotoxic effect in TA 1535 and TA 100, so that this range could nevertheless be used for evaluation purposes. Substance precipitation occurred from the dose 500 µg per plate.

Evidence of mutagenic activity for NaF was not found with and without metabolic activation. Neither a dose-related doubling of the mutant count nor a biologically relevant increase in the same, was observed in comparison with the negative controls.

The positive controls sodium azide (Na-azide) , nitrofurantoin (NF), 4-nitro-1,2-phenylene-diamine (4-NPDA),  and 2-aminoanthracene (2 -AA) had a marked mutagenic effect, as can be seen from the biologically relevant increase in mutant colonies, compared to the corresponding negative controls.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: only 4 strains, no strains with AT base pair at the primary reversion site, culture media used not reported

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Reversion to histidine independence in histidine-deficient mutant Salmonella typhimurium strains.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: partly deficient in lipopolysaccharide side chains of their cell walls, UV repair ability malfunctions

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver S9 fraction

Test concentrations with justification for top dose:

0 (solvent control), 8, 40, 200, 1000 and 5000 ug/plate; initial assay.
0 (solvent control), 312.5, 625, 1250, 2500 and 5000 ug/plate; confirmatory assay

Vehicle / solvent:

the solvent used for sodium sulphate was demineralized water, and for the positive controls DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylenediamine (TA 1537 + TA 98), +S9: 2-aminoanthracene

Remarks:

no

Details on test system and experimental conditions:

Plate incorporation method: 48-hour exposure time

DURATION
- Exposure duration: 48 h




NUMBER OF REPLICATIONS:
four plates per strain and dose, both with and without S-9 mix, were used for mutant count. Four plates per strain were also used for each positive control.




DETERMINATION OF CYTOTOXICITY
- other: background growth on the plates for the mutant determination; toxic effect assumed when mutant count per plate was clearly lower than the negative control count in dose correlation; determination of titer

Evaluation criteria:

A reproducible and dose-related increase (ie. at least twice the negative control count) in mutant counts for at least one strain was considered positive.
In the case of no reproducible and dose-related increase in mutant counts in at least one strain, the result was evaluated as negative.
In the case of questionable results, the investigations continued, probably by the use of modifications, until a final evaluation was possible.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: S. typhimurium TA1535, TA100, TA1537, TA98

Remarks:

Migrated from field 'Test system'.

None of the four strains used showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S-9 mix and was confirmed by the results of the repeat tests.

 

 

Table 1. Summary of the results of the Ames test on Na2SO4 without S-9 mix

	TA 1535			TA 100			TA 1537			TA 98
Test/doses µg/plate	Means revertantsper plate	Titer per ml 10^8	Quotient	Means revertantsper plate	Titer per ml 10^8	Quotient	Means revertantsper plate	Titer per ml 10^8	Quotient	Means revertantsper plate	Titer per ml 10^8	Quotient
1^sttest
0	13	56.0	1.0	102	29.6	1.0	13	50.8	1.0	17	82.0	1.0
8	13	59.5	1.0	112	28.1	1.1	11	56.1	0.9	21	76.3	1.2
40	16	53.4	1.2	99	28.1	1.0	17	59.7	1.4	16	80.5	0.9
200	14	58.4	1.1	98	29.1	1.0	14	59.7	1.1	22	79.5	1.3
1000	13	52.6	1.0	110	29.4	1.1	18	58.9	1.4	22	77.7	1.3
5000	14	93.6	1.1	126	27.4	1.2	15	57.7	1.2	31	84.8	1.8
Na-Azide	1080	52.2	84.7*
NF				385	27.6	3.8*
4-NPDA							183	59.6	14.6*	202	75.0	11.9*
2-AA (+S9)		45.6			30.0			57.1			68.4
2^ndtest
0	13	34.2	1.0	65	35.4	1.0	9	33.4	1.0	15	48.0	1.0
312.5	9	33.3	0.7	83	30.0	1.3	8	30.7	0.8	14	52.9	0.9
625	13	34.1	1.0	74	28.9	1.1	9	34.1	1.0	17	49.0	1.1
1250	13	32.3	1.0	60	33.5	0.9	12	31.4	1.2	13	55.4	0.9
2500	13	39.4	1.0	79	36.2	1.2	13	32.3	1.4	15	54.0	1.0
5000	12	36.7	0.9	82	32.0	1.3	12	34.5	1.3	12	54.2	0.8
Na-Azide	1021	37.4	80.1*					32.4
NF				241	34.7	3.7*
4-NPDA							107	32.4	11.6*	88	50.2	5.8*
2-AA (+S9)		29.0			24.2			28.7			45.3

 

Na-Azide: Sodium azide,10 µg/plate

NF:Nitrofurantoin,0.2 µg/plate

4-NPDA: 4-nitro-1,2-phenylenediamine, 0.5 µg/plate

2-AA: 2-aminoanthracene,3 µg/plate

 

* = mutagenic effect

** =bacteriotoxic effect

 

 

Table 2. Summary of the results of the Ames test on Na2SO4 with S-9 mix

	TA 1535		TA 100		TA 1537		TA 98
Test/doses µg/plate	Means revertantsper plate	Quotient	Means revertantsper plate	Quotient	Means revertantsper plate	Quotient	Means revertantsper plate	Quotient
1^sttest
0	18	1.0	116	1.0	12	1.0	29	1.0
8	15	0.9	141	1.2	13	1.2	31	1.1
40	14	0.8	144	1.2	13	1.2	30	1.0
200	13	0.7	141	1.2	8	0.7	31	1.1
1000	14	0.8	145	1.2	13	1.1	37	1.3
5000	16	0.9	160	1.4	11	1.0	42	1.4
2-AA	167	9.5*	698	6.0*	76	6.6*	1020	35.2*
2^ndtest
0	13	1.0	98	1.0	13	1.0	22	1.0
312.5	18	1.4	76	0.8	12	1.0	28	1.3
625	13	1.0	85	0.9	15	1.2	29	1.3
1250	9	0.7	83	0.8	15	1.2	33	1.5
2500	15	1.1	121	1.2	13	1.0	31	1.4
5000	16	1.3	105	1.1	20	1.6	28	1.3
2-AA	224	17.3*	696	7.1*	89	7.1*	832	37.8*

 

2-AA: 2-aminoanthracene,3 µg/plate

 

* = mutagenic effect

** =bacteriotoxic effect

 

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

Executive summary:

Herbold BA (1987)

Sodium sulphate (as a surrogate for fluorosulphuric acid) was investigated in a Salmonella / microsome test for point-mutagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants with and without metabolic activation. Test was performed similar to the OECD guideline 471 with restrictions (Only four strains, no strains with AT base pair as primary reversion site. Different positive control substances used in comparison with OECD guideline. Culture media used: not reported).

For the test the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98 were used.

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed.

Evidence of mutagenic activity of sodium sulphate was not found with and without metabolic activation. Neither a dose-related doubling nor a biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

The positive controls sodium azide (Na-azide) , nitrofurantoin (NF), 4-nitro-1,2-phenylene-diamine (4-NPDA),  and 2-aminoanthracene (2 -AA) had a marked mutagenic effect, as was seen by a the biologically relevant increase in mutant colonies compared with the corresponding negative controls.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Study period:

July 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Fluorosulphuric acid (CAS 7789-21-1 EC 232-149-4) [target substance] is known to be a very strong inorganic acid (pH < 1). The substance is hydrolytically unstable and reacts violently with water (self-classification EUH014). When in contact with water it rapidly hydrolyses to it hydrolysis products sulphuric acid (CAS 7664-93-9 EC 231-639-5) and hydrofluoric acid (CAS 7664-39-3 EC 231-634-8).

Fast hydrolysis: HSO3F + H2O <=> H2SO4 + HF
Self-ionisation: HSO3F + H2O <=> H3O+ + SO3F-
Slow hydrolysis: SO3F- + H2O <=> HSO4- + HF

Both sulphuric acid (H2SO4) and hydrofluoric acid (HF) are both known to be strong inorganic acids. In water they dissociate rapidly to form hydrogen (H+) and its respective counter anions sulphate (SO42-) and fluoride (F−) ions

H2SO4 (aq) ⇌ H+ (aq) + HSO4- (aq) ⇌ 2H+ (aq) + SO42- (aq)
HF (aq) ⇌ H+ (aq) + F- (aq)

Sodium sulphate (CAS 7757-82-6 EC 231-820-9) [source substance 1], the highly soluble sodium salt of hydrogen sulphate, will be fully hydrated in water as sodium (Na+) and sulphate (SO42-) ions. In analogue, sodium fluoride (CAS 7681-49-4 EC 231-667-8) [source substance 2], the highly soluble sodium salt of fluoride, is also fully hydrated in water as separate sodium (Na+) and fluoride (F-) ions:

Na2SO4 (aq) ⇌ 2Na+ (aq) + SO42- (aq)
NaF (aq) ⇌ Na+ (aq) + F- (aq)

Since all biological systems are water based, the read-across hypothesis is based on the rational that fluorosulphuric acid (HSO3F) violently reacts with water under the formation of its hydrolysis products sulphuric acid (H2SO4) and hydrofluoric acid (HF). Both sulphuric acid and hydrofluoric acid as strong acids, as well as their sodium salts sodium sulphate and sodium fluoride will, to the same extent, dissociate rapidly to form sulphate and fluoride ions in contact with cells, mucous, blood or other fluids. Any systematic effects observed will be directly attributable to the anions sulphate and fluoride. Furthermore, as all environmental endpoints are aquatic or wet, the same read-across hypothesis applies. On this basis, for specific properties, the hazard profile of fluorosulphuric acid can be derived from its hydrolysis products sulphuric acd and hydrofluoric acid and is comparable to its sodium salts sodium sulphate and sodium fluoride with respect to the environment and human health.

An analogue approach is considered applicable since in the dossier read-across is “…employed between a small number of structurally- similar substances; there is no trend or regular pattern on the properties.” According to the Read-Across Assessment Framework (RAAF) guidance criteria (ECHA, 2017), an analogue approach is considered applicable so for the current read-across covered, scenario 1 is considered applicable as the registered substance fluorosulphuric acid, via its hydrolysis products sulphuric acid and hydrofluoric acid, and sodium fluoride and potassium fluoride are transformed to common compounds: the sulphate and fluoride anions.
The different cations – sodium, potassium and hydrogen – are not considered relevant for read-across justification because, following rapid dissociation within biological and aqueous environments, the hydrogen ions are responsible for localised effects only (corrosiveness), with no systemic availability. Sodium and potassium ions, as a ubiquitous essential metal ion, are naturally present in organisms and the environment. Consequently, neither cations are considered of any toxicological or ecotoxicological concern.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance fluorosulphuric acid is a mono-constituent substance with a high degree of purity typical concentration ca. 99.5 % (w/w) (range from 99-100 % (w/w)). Impurities are contained in low concentrations not relevant for classification and labelling.
Both source substances, sodium sulphate and sodium fluoride, are also well-defined mono-constituent substances with a high degree of purity and no impurities considered relevant for classification.
Based on the high degree of purities no impact from potential impurities on the read-across hypothesis applies neither from the target nor from the source substances.

3. ANALOGUE APPROACH JUSTIFICATION
In accordance with regulation EC No 1907/2006 (REACH) Annex XI.2. testing on the registered substance itself is technically not feasible due to intrinsic substance properties and may be omitted. Conducting studies is technically not possible because fluorosulphuric acid reacts violently with water. The substance is hydrolytically unstable and reacts violently with water (self-classification EUH014). When in contact with water it rapidly hydrolyses to it hydrolysis products sulphuric acid and hydrofluoric acid.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: only 4 strains, no strains with AT base pair at the primary reversion site, culture media used not reported

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Reversion to histidine independence in histidine-deficient mutant Salmonella typhimurium strains.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: partly deficient in lipopolysaccharide side chains of their cell walls, UV repair ability malfunctions

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver S9 fraction

Test concentrations with justification for top dose:

0 (solvent control), 8, 40, 200, 1000 and 5000 ug/plate; initial assay.
0 (solvent control), 312.5, 625, 1250, 2500 and 5000 ug/plate; confirmatory assay

Vehicle / solvent:

the solvent used for sodium sulphate was demineralized water, and for the positive controls DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylenediamine (TA 1537 + TA 98), +S9: 2-aminoanthracene

Remarks:

no

Details on test system and experimental conditions:

Plate incorporation method: 48-hour exposure time

DURATION
- Exposure duration: 48 h




NUMBER OF REPLICATIONS:
four plates per strain and dose, both with and without S-9 mix, were used for mutant count. Four plates per strain were also used for each positive control.




DETERMINATION OF CYTOTOXICITY
- other: background growth on the plates for the mutant determination; toxic effect assumed when mutant count per plate was clearly lower than the negative control count in dose correlation; determination of titer

Evaluation criteria:

A reproducible and dose-related increase (ie. at least twice the negative control count) in mutant counts for at least one strain was considered positive.
In the case of no reproducible and dose-related increase in mutant counts in at least one strain, the result was evaluated as negative.
In the case of questionable results, the investigations continued, probably by the use of modifications, until a final evaluation was possible.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: S. typhimurium TA1535, TA100, TA1537, TA98

Remarks:

Migrated from field 'Test system'.

None of the four strains used showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S-9 mix and was confirmed by the results of the repeat tests.

 

 

Table 1. Summary of the results of the Ames test on Na2SO4 without S-9 mix

	TA 1535			TA 100			TA 1537			TA 98
Test/doses µg/plate	Means revertantsper plate	Titer per ml 10^8	Quotient	Means revertantsper plate	Titer per ml 10^8	Quotient	Means revertantsper plate	Titer per ml 10^8	Quotient	Means revertantsper plate	Titer per ml 10^8	Quotient
1^sttest
0	13	56.0	1.0	102	29.6	1.0	13	50.8	1.0	17	82.0	1.0
8	13	59.5	1.0	112	28.1	1.1	11	56.1	0.9	21	76.3	1.2
40	16	53.4	1.2	99	28.1	1.0	17	59.7	1.4	16	80.5	0.9
200	14	58.4	1.1	98	29.1	1.0	14	59.7	1.1	22	79.5	1.3
1000	13	52.6	1.0	110	29.4	1.1	18	58.9	1.4	22	77.7	1.3
5000	14	93.6	1.1	126	27.4	1.2	15	57.7	1.2	31	84.8	1.8
Na-Azide	1080	52.2	84.7*
NF				385	27.6	3.8*
4-NPDA							183	59.6	14.6*	202	75.0	11.9*
2-AA (+S9)		45.6			30.0			57.1			68.4
2^ndtest
0	13	34.2	1.0	65	35.4	1.0	9	33.4	1.0	15	48.0	1.0
312.5	9	33.3	0.7	83	30.0	1.3	8	30.7	0.8	14	52.9	0.9
625	13	34.1	1.0	74	28.9	1.1	9	34.1	1.0	17	49.0	1.1
1250	13	32.3	1.0	60	33.5	0.9	12	31.4	1.2	13	55.4	0.9
2500	13	39.4	1.0	79	36.2	1.2	13	32.3	1.4	15	54.0	1.0
5000	12	36.7	0.9	82	32.0	1.3	12	34.5	1.3	12	54.2	0.8
Na-Azide	1021	37.4	80.1*					32.4
NF				241	34.7	3.7*
4-NPDA							107	32.4	11.6*	88	50.2	5.8*
2-AA (+S9)		29.0			24.2			28.7			45.3

 

Na-Azide: Sodium azide,10 µg/plate

NF:Nitrofurantoin,0.2 µg/plate

4-NPDA: 4-nitro-1,2-phenylenediamine, 0.5 µg/plate

2-AA: 2-aminoanthracene,3 µg/plate

 

* = mutagenic effect

** =bacteriotoxic effect

 

 

Table 2. Summary of the results of the Ames test on Na2SO4 with S-9 mix

	TA 1535		TA 100		TA 1537		TA 98
Test/doses µg/plate	Means revertantsper plate	Quotient	Means revertantsper plate	Quotient	Means revertantsper plate	Quotient	Means revertantsper plate	Quotient
1^sttest
0	18	1.0	116	1.0	12	1.0	29	1.0
8	15	0.9	141	1.2	13	1.2	31	1.1
40	14	0.8	144	1.2	13	1.2	30	1.0
200	13	0.7	141	1.2	8	0.7	31	1.1
1000	14	0.8	145	1.2	13	1.1	37	1.3
5000	16	0.9	160	1.4	11	1.0	42	1.4
2-AA	167	9.5*	698	6.0*	76	6.6*	1020	35.2*
2^ndtest
0	13	1.0	98	1.0	13	1.0	22	1.0
312.5	18	1.4	76	0.8	12	1.0	28	1.3
625	13	1.0	85	0.9	15	1.2	29	1.3
1250	9	0.7	83	0.8	15	1.2	33	1.5
2500	15	1.1	121	1.2	13	1.0	31	1.4
5000	16	1.3	105	1.1	20	1.6	28	1.3
2-AA	224	17.3*	696	7.1*	89	7.1*	832	37.8*

 

2-AA: 2-aminoanthracene,3 µg/plate

 

* = mutagenic effect

** =bacteriotoxic effect

 

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

Executive summary:

Herbold BA (1987)

Sodium sulphate (as a surrogate for fluorosulphuric acid) was investigated in a Salmonella / microsome test for point-mutagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants with and without metabolic activation. Test was performed similar to the OECD guideline 471 with restrictions (Only four strains, no strains with AT base pair as primary reversion site. Different positive control substances used in comparison with OECD guideline. Culture media used: not reported).

For the test the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98 were used.

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed.

Evidence of mutagenic activity of sodium sulphate was not found with and without metabolic activation. Neither a dose-related doubling nor a biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

The positive controls sodium azide (Na-azide) , nitrofurantoin (NF), 4-nitro-1,2-phenylene-diamine (4-NPDA),  and 2-aminoanthracene (2 -AA) had a marked mutagenic effect, as was seen by a the biologically relevant increase in mutant colonies compared with the corresponding negative controls.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Justification for non-classification:

The results of the Salmonella microsome tests for point mutagenic effects of the surrogates sodium fluoride and sodium sulphate show that there is no evidence for mutagenic activity of fluorosulphuric acid.

No classification for mutagenicity - genetic toxicity is recommended.