Reaction mass of sulphuric acid, hydrogen peroxide and peroxomonosulphuric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Sulphuric acid, peroxomonosulphuric acid and hydrogen peroxide are each expected to contribute to the physico-chemical properties of the multi-constituent substance. However, genetic toxicity is likely to be masked in the multi-constituent substance by its corrosive nature. It is therefore considered appropriate to read-across to the main constituent, sulphuric acid, when considering in vitro gene mutation in bacteria and chromosome aberration in in vitro.

in vitro genotoxicity studies on the main component (sulphuric acid) of the multi-constituent test material concluded that any positive results were caused by pH effects. As a result, and in accordance with Regulation (EC) 1907/2006, Annex VIII, section 8.4, column 2, investigation of cytogenetic damage in vivo is considered unnecessary.

OECD guideline 476 cites Scott et al (1991) and notes that positive results, which do not reflect intrinsic mutagenicity, may arise from changes in pH, osmolarity, or high levels of cytotoxicity. Such conditions cannot be avoided with a multi-constituent test substance of pH < 1 and, in accordance with Regulation (EC) 1907/2006, Annex XI, section 2,i n vitro investigation of gene mutation in mammalian cells is not technically possible.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The reaction mass of sulphuric acid, hydrogen peroxide and peroxomonosulphuric acid is predominantly sulphuric acid (>80%). Although all constituents of the reaction mass contribute towards and are essential for the desired technical effects of the range, it is considered acceptable to read-across to data on sulphuric acid. This because significant toxicological effects are likely to be masked in the multi-constituent substance by its corrosive nature and so it considered appropriate to read across to the mean constituent, sulphuric acid, when considering in vitro gene mutation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across data matrix under 'Attached background material' below.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across data matrix under 'Attached background material' below.

4. DATA MATRIX
See read-across data matrix under 'Attached background material' below.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

no

Remarks:

Study pre-dates GLP

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

other: TA 97; TA 98; TA100; TA102; TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from liver homogenates of Sprague-Dawley rats induced with Aroclor 1254 (data not reported)

Test concentrations with justification for top dose:

- Effect was measured in terms of pH (4.0, 5.0, 5.5, 6.0, 6.3, 6.8, 7.0, 7.4, 7.8, 8.0, 9.0)

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunomycin

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

- Salmonella typhimurium tester strains were obtained from Dr B N Ames (University of California, Berkeley, CA)
- The standard plate incorporation assay was carried out according to the procedure developed by Ames and Maron and Ames et al to measure spontaneous reversion rate of tester strains in histidine-free media.
- The effect of pH changes on bacterial reversion rate was evaluated by adoptin two modifications of the standard plate incorporation assay.
- Preincubation of bacteria with buffer solutions at pH values ranging from 4 to 9 was carried out using H3PO4, H2SO4 and their sodium salts.
- Cultures of the tester strains (o.1 mL) were mixed with 0.5 mL of the buffer solutions, incubated at 37 °C for one hour, and then added to the top agar, plated on Vogel-Bonner medium plates and the plates were incubated at 37 °C for 60 hours.
- For agar plate incorporation, a modified Vogel-Bonner medium was prepared using a MgSO4.7H2O (10 g), citric acid.H2O (100 g), K2HPO4 (500 g) and NaNH4HPO4.4H2O (175 g) in 670 mL distilled water, and 10 mL was added to 220 mL of distilled water. The diluted solution was then adjusted to final pH with 10N NaOH.
- After sterilisation, 220 mL agar solution (45 g agar per 1,320 mL water) and 50 mL of 5 % dextrose was added to the 230 mL of pH-adjusted salt solution. Plates were then poured with 25 mL of the above media in each.
- The pH was confirmed for each plate type by using a surface pH electrode (Ingold, Switzerland).
- The pH of the total plate assay system were assumed to be the same as that of the initial base agar.
- All the plate types were prepared with enough NaCl added to the medium to result in an ionic strength identical to that of the pH 7.0 controls.
- Addition of S9 fraction was scheduled for some experiments in order to complete the bioassay protocol as well as to check a previously reported detoxifying action of S9 for some inorganics.

Evaluation criteria:

- The number of revertants per plate were counted

Species / strain:

other: TA 97; TA 98; TA100; TA102; TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at pH 5

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- The reversion properties and specificity of each strain were confirmed by testing methylmethanesulphonate, daunomycin and sodium azide in the standard plate incorporation assay.
- Incubation of S. typhimurium tester strains with different buffer solutions at pH values ranging from 5.5 to 9 had no effect on the bacterial reversion rates.
- Appearance of survivors suggested that acidification of the incubation mixture to pH 5.0 produced toxic effects on bacteria.
- At lower pH values, complete bacterial death was observed.
- The same negative results were obtained by using the base agar plates at different pH values.
- Results were unchanged by addition of S9 fraction (data not reported)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

JUSTIFICATION FOR USE OF READ-ACROSS DATA

 

See comparison of overall physico-chemical and toxicity profiles for target and source chemicals in the data matrix (attached).

EFFECT OF pH ON TESTER STRAINS

Revertants per plate
	pH
Strain	4.0	5.0	5.5	6.0	6.3	6.8	7.0	7.4	7.8	8.0	9.0
TA 97	**	*	75	79	83	82	85	86	82	83	79
TA 98	**	*	29	28	31	30	35	40	32	27	31
TA 100	**	*	102	107	115	117	103	115	131	101	124
TA 102	**	*	182	185	182	178	175	195	197	183	182
TA 1535	**	*	15	11	13	11	15	16	11	17	12
*	Small colonies without bacterial lawn
**	Complete bacterial killing

Conclusions:

Interpretation of results:
negative

No effects were detectable with or without metabolic activation in Salmonella typhimurium strains TA 97, TA 98, TA 100, TA 102 and TA 1535, at concentrations up to those causing cytotoxicity.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The reaction mass of sulphuric acid, hydrogen peroxide and peroxomonosulphuric acid is predominantly sulphuric acid (>80%). Although all constituents of the reaction mass contribute towards and are essential for the desired technical effects of the range, it is considered acceptable to read-across to data on sulphuric acid. This because significant toxicological effects are likely to be masked in the multi-constituent substance by its corrosive nature and so it considered appropriate to read across to the mean constituent, sulphuric acid, when considering chromosome aberration resulting from in vitro studies.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across data matrix under 'Attached background material' below.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across data matrix under 'Attached background material' below.

4. DATA MATRIX
See read-across data matrix under 'Attached background material' below.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The relationship between pH of the medium and clastogenic activity was studied in Chinese hamster ovary K1 cells in vitro.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO K1

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 fraction

Test concentrations with justification for top dose:

- Sulphuric acid: Zero, 2, 4, 6, 8 mM (with S9)
- Sulphuric acid: Zero, 2, 4, 6, 8, 10 mM (without S9)

Untreated negative controls:

yes

Remarks:

acidic phosphate buffered saline

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

CELL CULTURES
- Chinese hamster ovary K1 cells (Flow Laboratories, USA) were maintained in Ham's F12 medium supplemented with sodium bicarbonate (16.7 mM), kanamycin (60 μg/mL) and 10 % fetal calf serum (FCS).
- The cultures were incubated at 37 °C in an atmosphere of 5 % carbon dioxide in air and subcultures were made every 3-4 days.
- The cells were confirmed to be free of mycoplasma.

PREPARATION OF S9 MIX
- S9 (Oriental Yeast Co Ltd, Japan) was derived from the livers of rats pre-treated with phenobarbital and 5,6-benzoflavone.
- The S9 activation system (S9 mix) was prepared just before use and sterilised by filtration, except S9 fraction.

pH ADJUSTMENT OF MEDIUM
- H2SO4 was added to the F12 medium and the pH was measured.
- Dulbecco's phosphate buffered saline (PBS) was pH-adjusted with 0.1 M HCl.
- In organic buffering systems, organic acids (Good's buffers) were used.
- Ham's F12 medium was supplemented with 15 mM MES or Bis-Tris instead of sodium bicarbonate.
- The medium was adjusted to the appropriate pH using either 0.5 M NaOH or 0.5 M HCl.
- Media were filter sterilised prior to use.
- The pH of the medium was measured prior to and after treatment using a pH meter

CHROMOSOMAL ABERRATION TEST
- In the absence of S9 mix, H2SO4 was added to the 3-day-old cultures after a medium change.
- In metabolic activation with S9 mix, cells were treated with S9 mix (prepared to a 5 % final concentration of S9) and sulphuric acid for 6 h.
- In studies with acidic PBS, cells were treated with pH-adjusted PBS for 3 h.
- Recovery times were 18 h in the studies with S9 and 21 h in the studies with acidic PBS.
- In all experiments, chromosome preparations were made 24 h after the start of treatment.
- The number of cells with chromosomal aberrations was recorded on 100 metaphases.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

- Induced aberrations were almost all chromatid breaks without S9 mix.
- Aberrations with S9 mix consisted of chromatid breaks and exchanges.
- Similar results were seen with a second inorganic acid (HCl)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

JUSTIFICATION FOR USE OF READ-ACROSS DATA

See comparison of overall physico-chemical and toxicity profiles for target and source chemicals in the data matrix (attached).

Conclusions:

Interpretation of results:
other: positive results induced by changes in pH

Exposure of CHO-K1 cells to sulphuric acid resulted in low culture medium pH and increased the incidence of chromatid breaks in the presence and absence of metabolic activation. Similar results were seen with a second inorganic acid (HCl) and the positive result was considered to be a consequence of low pH.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

No literature evidence of intrinsic genetic toxicity has been identified for the main constituent (sulphuric acid) and it is considered unnecessary to classify the multi-constituent substance under the terms of Directive 67/548/EEC or GHS as reflected by Regulation (EC) 1272/2008.