Inorganic residual from kraft or soda pulping separated from green liquor in the chemical recovery cycle.

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:

March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Inorganic residual from kraft or soda pulping separated from green liquor in the chemical recovery cycle.

EC Number:

923-511-9

IUPAC Name:

Inorganic residual from kraft or soda pulping separated from green liquor in the chemical recovery cycle.

Details on test material:

Name: "Green liquor sludge".Molecular formula: UVCB.Batch No.: Not stated.EC No.: 923-511-9.Appearance: Solid, light grey when dried.Solubility: In water: washed substance is not solubleIn other solvents: not available.pH: Not available.Conditions of storage: Room temperature. Storage in the dark but may be used under light.Stability at conditions of storage: Not available.Expiry date: Not available.

Method

Target gene:

his- (s. typhimurium)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from male rat livers

Test concentrations with justification for top dose:

5000, 1667, 556, 185, 62 µg/plate

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

Exposure technique:The exposure for the first experiment was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state.For each sample the following solutions were combined:•0.1 mL of the overnight culture of the bacteria, •0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),•0.1 mL of the appropriate test- or reference substance solution and •2 mL of top agar.The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days). The exposure for the second experiment was performed according to the 'Preincubation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate with preceding incubation in the liquid state.For each sample the following solutions were combined:•0.1 mL of the overnight culture of the bacteria, •0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),•0.1 mL of the appropriate test- or reference substance solution.The solutions were preincubated for 20 minutes at 37 °C using a shaker, afterwards combined with 2 mL of top agar and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

Evaluation criteria:

Determination of the toxicity:Additionally to the counting of colonies the bacterial background of the plates was inspected visually. The following signs of toxicity, if present, were recorded:•A reduced bacterial background lawn (mottled instead of homogeneous).•Microcolonies of bacteria instead of a homogeneous background lawn.•No background lawn.•Clearly reduced numbers of revertant colonies.Calculations, criteria for a positive result:Means and standard deviations were calculated for the number of mutants in every concentration group.The criteria for a positive result are: A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:•For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2½ fold of the amount of the spontaneous revertants. •For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 12/3 fold of the amount of the spontaneous revertants.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A precipitate was visible when the test substance was mixed with the agar at the 5000 µg/plate samples. The precipitate was still visible when the colonies were counted but did not impede the counting.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negativeAccording to these results, "Green liquor sludge" is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 5000 µg/plate, which is the limit concentration for this kind of test.

Executive summary:

This study was performed to determine a possible mutagenic action of "Green liquor sludge" with the Salmonella typhimurium reverse mutation test (Ames test). This test is sensitive to frameshift mutations as well as to base pair mutations. The performance of the test with and without an external metabolising system (a lyophilised post-mitochondrial supernatant of homogenised livers of male Sprague Dawley rats, induced with Aroclor 1254, S9 -Mix) enables the detection of the mutagenic action of the test substance itself as well as of its metabolites.

Bacterial strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. The exposure technique was the plate incorporation method in the first experiment and the preincubation method in the second experiment. The following concentrations were tested: 5000, 1667, 556, 185 and 62 µg/plate.

No toxicity of the test substance to the bacteria was observed up to 5000 µg per plate in both experiments.

In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.

According to the results obtained in this study the test item is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 5000 µg/plate, which is the limit concentration for this kind of test.