Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Green liquor is highly alkaline solution (pH > 11.5) and it cannot be tested as such without significant modification (lowering the pH) of the composition and the speciation of the constituents of the substance.

Two Green liquor samples (GL1 & GL2) were non-mutagenic based on the negative results in the in vitro gene mutation study in bacteria (REACH Annex VII, section 8.4.1)(EU Method B.13/14/OECD 471.

One GL sample was studied in In vitro cytogenicity study in mammalian cells test system (OECD 473, ver.21-July 1997/EU B10)(REACH Annex VIII, section 8.4.2).

No statistically significant differences in the number of metaphases with numerical aberrations were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

In this 473 key experiment with the use of a metabolic activation system caused marked cytotoxic effects at the highest concentration tested (5 μL/mL at pH 8.7). No marked cytotoxicity was noted in any of the other concentrations tested (5 concentrations, in total 10 results). The followed OECD 473 (year 1997) study guideline did not require measurement/reporting of osmolality of the test solution likewise the more recent version (2016) of the guideline do. High osmolality may have resulted to an artefact one positive result observed.

Link to relevant study records

Referenceopen allclose all

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:
February-March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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
Target gene:
his- (s. typhimurium)
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
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 (substance wet weight, 82.3 % water)
Vehicle / solvent:
deionised water
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.

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
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
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
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
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:
No precipitation of the test substance was seen in any of the concentration groups.
Conclusions:
Interpretation of results (migrated information):
negative

According to these results, "Green liquor 1" 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 1" 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.

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:
February-March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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
Target gene:
his- (s. typhimurium)
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
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:
deionised water
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.

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
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
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
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
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:
No precipitation of the test substance was seen in any of the concentration groups.
Conclusions:
Interpretation of results (migrated information):
negative

According to these results, "Green liquor 2" 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 2" 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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 January 2010 to .... July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully Guideline- and GLP-compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: primary culture
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: not applicable
Metabolic activation:
with and without
Metabolic activation system:
Lyophilized Post-Mitochondrial Supernatant (Moltox, Art. No. 11-01L.2, Lot.: 2266) from male Sprague-Dawley rat livers, induced with Aroclor-1254, in 0.154M KCL
Test concentrations with justification for top dose:
Experiment A (3 hours incubation, no metabolic activation system): 0.062, 0.185, 0.556, 1.667, 5.000 µL/mL
Experiment A (3 hours incubation, with metabolic activation system): 0.062, 0.185, 0.556, 1.667, 5.000 µL/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: The negative control substance was RPMI 1640 medium with L-glutamine (Gibco BRL Life Technologies, UK, article number 21875-034). For each experiment a stock solution was prepared by diluting appropriate amounts of "Green liquor 1" with RPMI.These stock solutions were then diluted with RPMI to achieve the intended final concentrations of the test substance.
All preparations were made freshly before adding them to the cell cultures.

- Justification for choice of solvent/vehicle: Common vehicle for water soluble substances
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
see below for details

Migrated to IUCLID6: and cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Cultures were kept at 37 °C and 5% CO2 for ca. 48 hours before further processing
- Exposure duration: 3 hours (experiment without and experiment with metabolic activation)
- Expression time (cells in growth medium): 15 hours for the cultures treated with test substance for 3 hours (plus 2 hours with spindle inhibitor added)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): 0.1 mL of Colcemid (Gibco BRL Life Technologies, UK, article no 15210-057, 10 µg/mL in balanced salt solution) per culture.
STAIN (for cytogenetic assays): 10 minutes with Giemsa solution (10% v/v in a buffer of 0.067 M KH2PO4 and 0.067 M Na2HPO4 x 2 H2O in deionised water), rinsed in tap-water and then in deionised water.

NUMBER OF REPLICATIONS: 2 per culture

NUMBER OF CELLS EVALUATED:
Mitotic indices: 2000 lymphocytes per culture
Chromosome aberrations: 100 metaphases per culture (i.e. 200 per concentration, apart from cultures with obviously high numbers of metaphases with aberrations)

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic indices were determined by counting a total of 2000 lymphocytes per cell culture and by recording the number of lymphocytes in any stage of mitosis. This number was then expressed as percentage of mitotic lymphocytes.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
Only well spread cells with 44 to 47 chromosomes, polyploid and endoreduplicated cells were acceptable for analysis. Structural aberrations were scored according to well defined (and reported) criteria.
Statistics:
The Chi2-Test (two-tailed, p=0.05) was used for the comparison between the negative control and the test substance cultures. If the results were positive, comparisons were made separately between the negative control and each concentration. If conditions for the Chi2-Test were not met, Fisher’s Exact Test was used. Chi2-Test or Fisher’s Exact Test were also used for the comparison between the negative and the positive controls.
Species / strain:
lymphocytes: primary cell culture
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: primary cell culture
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
Yes, historical control data (on negative and positive controls) given in the report.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
see "any other information on results incl. tables"
Remarks on result:
other: strain/cell type: lymphocytes
Remarks:
Migrated from field 'Test system'.

For further tables see attachment.

Mitotic index

Experiment without a metabolic activation system:

Almost complete cytotoxicity occurred at the highest concentration of 5 µL/mL medium.
At the test substance concentrations of 0.185 µL/mL, 0.556 µL/mL and 1.667 µL/mL marked cytotoxicity was observed (46.8%, 28.7% and 38.3% respectively).
At 0.062 µL/mL the mitotic indices of the test substance treated cultures were reduced to 58.5% of the corresponding negative controls.

Test substance concentration
(µL/mL)

0.062

0.185

0.556

1.667

5.0

Mitotic index
(% of respective negative control)

58.5

46.8

28.7

38.3

2.1

Bold figures: These concentrations were analysed

Experiment with a metabolic activation system, 3 hours of incubation:

Marked cytotoxicity (32.7%) was observed at the highest concentration of 5 µL/mL.
No marked cytotoxicity was noted in any of the other concentrations tested.

Test substance concentration
(µL/mL)

0.062

0.185

0.556

1.667

5.0

Mitotic index
(% of respective negative control)

103.7

72.9

111.2

68.2

32.7

Bold figures: These concentrations were analysed

Numerical aberrations

No statistically significant differences in the number of metaphases with numerical aberrations were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

 

Gaps

No statistically significant increases in the number of gaps were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

Structural aberrations

In the experiment without a metabolic activation system the number of metaphases with structural aberrations was not statistically significantly increased at any concentration analysed compared to the concurrent negative controls. At the highest concentration evaluated (0.556 µL/mL) the number of metaphases with chromatid aberrations was beyond the range of the historical negative controls and within the range of historical positive controls. The numbers of the other test substance concentrations were within the range of historical negative controls

In the experiment with a metabolic activation system the number of metaphases with structural aberrations was statistically significantly higher at the highest test substance concentration (5 µL/mL) compared to the corresponding negative control. The numbers were also clearly beyond the data of historical negative controls. No such increase was noted at the other lower test concentrations analysed. At a concentration of 1.667 µL/mL one multiple aberration occurred which is also beyond the data of historical controls and thus provides an indication of clastogenic properties of the test substance at this concentration as well.

.

 

Positive controls

The positive control substances caused in each experiment clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system, thus demonstrating that the test systems were adequate and that the metabolic activation system functioned properly.

Additional statistically significant differences to the negative controls like the number of gaps, the number of chromatid-type aberrations, etc., are indicated in the Tables.

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

Under the conditions of this study "Green liquor 1" induced structural chromosomal aberrations in cultured human lymphocytes after a treatment length of 3 hours with the use of a metabolic activation system. The conclusion is based on a statistically significant increase of metaphases with structural aberrations at a test substance concentration of 5 µL/mL and on the fact that these figures were far beyond the range of historical negative controls.
Executive summary:

Objective

The study was performed to determine possible mutagenic properties of "Green liquor 1" by means of an in vitro mammalian chromosome aberration test in human lymphocytes, according to Regulation (EC) No 440/2008 method B.10. and to OECD Guideline 473.

 

Methods

A total of two experiments was performed and analysed: one of them without and one with the use of a metabolic activation system (liver microsomes from Aroclor 1254 induced rats, with a co-factor solution). A concentration range between nominal 0.062 and 5 µL test substance per mL medium was tested. 5 µL/mL was chosen in accordance with the EC directive and the OECD guideline.

Primary lymphocyte cultures were established from whole blood freshly obtained from one female donor. After 48 hours of incubation,"Green liquor 1"was added. In all experiments the test substance was washed out three hours later and the cultures were cultivated for another 17 hours. In all experiments Colcemid was added two hours before the end of the cultivation period, and then cells were fixed and slides prepared.

The test substance was diluted with RPMI medium. For each concentration of the test substance two cultures were established. One negative control (RPMI) and one positive control (methanesulfonic acid methyl ester, MMS, for cultures without metabolic activation system and cyclophosphamide, CP, for cultures with a metabolic activation system) were set up concurrently in each experiment.

The concentrations of "Green liquor 1" in the experiments performed were 0.062, 0.185, 0.556, 1.667 and 5 µL/mL in each experiment.

In general, apart from cultures with obviously high numbers of metaphases with aberrations, 100 metaphases per culture (i.e. 200 per concentration) were analysed for structural and numerical chromosomal aberrations. The slides were coded before analysis. The mitotic indices were calculated from 2000 lymphocytes per culture for an assessment of cytotoxicity.

 

Results

Cytotoxicity

Experiment without a metabolic activation system:

Almost complete cytotoxicity occurred atthe highest concentrationof 5 µL/mL medium.
At the test substance concentrations of 0.185 µL/mL, 0.556 µL/mL and 1.667 µL/mLmarked cytotoxicity was observed (46.8%, 28.7% and 38.3% respectively).
At 0.062 µL/mL the mitotic indices of the test substance treated cultures were reduced to 58.5% of the corresponding negative controls.

Experiment with a metabolic activation system, 3 hours of incubation:

Marked cytotoxicity (32.7%) was observed at the highest concentration of 5 µL/mL.
No marked cytotoxicity was noted in any of the other concentrations tested.

Numerical aberrations

No statistically significant differences in the number of metaphases with numerical aberrations were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

Gaps

No statistically significant increases in the number of gaps were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

 

Structural aberrations

In the experiment without a metabolic activation system the number of metaphases with structural aberrations was not statistically significantly increased at any concentration analysed compared to the concurrent negative controls. At the highest concentration evaluated (0.556 µL/mL) the number of metaphases with chromatid aberrations was beyond the range of the historical negative controls and within the range of historical positive controls. The numbers of the other test substance concentrations were within the range of historical negative controls

In the experiment with a metabolic activation system the number of metaphases with structural aberrations was statistically significantly higher at the highest test substance concentration (5 µL/mL) compared to the corresponding negative control. The numbers were also clearly beyond the data of historical negative controls. No such increase was noted at the other lower test concentrations analysed. At a concentration of 1.667 µL/mL one multiple aberration occurred which is also beyond the data of historical controls and thus provides an indication of clastogenic properties of the test substance at this concentration as well.

As experiment A with metabolism gave positive results the performance of an experiment B with metabolism (treatment length 3 hours) and without a metabolic activation system with an extended treatment length of 20 hours was not required.

 

Positive controls

The positive control substances caused in each experiment clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system, thus demonstrating that the test systems were adequate and that the metabolic activation system functioned properly.

 

Conclusion

In the experiment without the use of a metabolic activation system "Green liquor 1" caused marked cytotoxic effects (reduction of mitotic indices by more than 50 %, compared to concurrent negative controls) atconcentrations between 5 µL/mL and 0.185 µL/mL medium, reducing the mitotic indices to between 2.1 % and 46.8 %. Cytotoxic effects (reduction of mitotic indices to 58.5 %) were still noted at a test substance concentration of 0.062 µL/mL.
In the experiment with
the use of a metabolic activation system "Green liquor 1" caused marked cytotoxic effects at the highest concentration tested (5 µL/mL).No marked cytotoxicity was noted in any of the other concentrations tested.
The highest tested concentration was 5 µL/mL and thus in accordance with the EC directive and the OECD guideline.

Under the conditions of this study "Green liquor 1" induced structural chromosomal aberrations in cultured human lymphocytes after a treatment length of 3 hours with the use of a metabolic activation system. The conclusion is based on a statistically significant increase of metaphases with structural aberrations at a test substance concentration of 5 µL/mL and on the fact that these figures were far beyond the range of historical negative controls.

There was also relevant evidence that "Green liquor 1" did induce structural chromosomal aberrations in cultured human lymphocytes in the experiment without the use of a metabolic activation system, although there was no statistically significant increase compared to the controls. The conclusion is mainly based on an important increase of metaphases with chromatid aberrations at the highest evaluated test substance concentration of 0.556 µL/mL and on the fact that this figure was beyond the range of historical negative controls and within the range of historical positive controls.

The overall results of the study indicate clastogenic properties of the test substance at a treatment length of 3 hours with the use of a metabolic activation system. There is also relevant evidence that the test substance has mutagenic properties at a treatment length of 3 hours without the use of a metabolic activation system.

Genetic toxicity in vivo

Description of key information

Green liquor is highly alkaline solution (pH > 11) and it cannot be tested as such without significant modification (lowering the pH) of the composition and the speciation of the constituents of the substance.

A somatic cell, an in vivo test a mammalian erythrocyte micronucleus test (OECD Guideline 474) was carried out for a sample of green liquor. The test result was negative, with no signs of genotoxicity. The existing dataset is regarded complete enough to draw a conclusion  that green liquor do not show genotoxic activity at the tested concentrations.  

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: Formation of micronuclei in erythrocytes of treated mice
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: NMRI -BomTac (Taconic Co. Denmark)
- Age at study initiation: 5 weeks
- Weight at study initiation:24 to 31 g
- Assigned to test groups randomly: [no/yes, under following basis: ]The animals were assigned to the treatment groups using randomisation schemes
- Fasting period before study: no
- Housing: The animals were housed in an animal room provided with filtered air at a temperature of 21°C ± 3°C and relative humidity of 55% ± 15%.
The mice were kept in single-sex groups of one or two in transparent polycarbonate (macrolone type III) cages (floor area: 810 cm2).

The bedding was softwood sawdust “Jeluxyl” from Jelu Werk GmbH, Josef Ehrler GmbH & Co KG, Ludwigsmühle, D-73494 Rosenberg, Germany. Regular analyses for relevant possible contaminants are performed. Certificates of analysis are retained.

The animals were given Aspen Wood Wool from Tapvei Estonia OÜ, Estonia for environmental enrichment at each change of bedding. An autoclaved brick of aspen wood from the same supplier was also placed in each cage. Regular analyses for relevant possible contaminants are performed. Certificates of analysis for the wood wool and the bricks are retained.
Furthermore, each cage contained a Mouse Igloo™ (Bio-serv). The mouse igloo allowed the animals to show a wide range of natural behaviours.

The cages were identified by cards marked with the study number, group number, animal number, and sex of the animals. The cards were also marked with the dose concentration, dose volume and cage number. The animals were identified using subcutaneously-implanted microchips with unique numbers. The microchips for two mice (numbers 36 and 42) could not be read on the day of dosing, but as they were caged individually, they were identified by the cage labels until euthanasia on the following day.

- Diet (e.g. ad libitum): A complete pelleted rodent diet “Altromin 1314 fortified” (for growing animals) supplied by Altromin Gesselschaft für Tierenährung mbH, D-32770 Lage, Germany, was available ad libitum. Analyses for major nutritive components and relevant possible contaminants are performed regularly. Certificates of analysis are retained.
- Water (e.g. ad libitum):The animals had free access to bottles with domestic quality drinking water acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth. Analyses for relevant possible contaminants are performed regularly. Certificates of analysis are retained.
- Acclimation period: The mice were allowed to acclimatise for five to six days before the preliminary toxicity test and six days before the main test.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C ± 3°C
- Humidity (%): relative humidity of 55% ± 15%.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): A cycle of 12 hours light and 12 hours darkness. The light was on from 06:00 h to 18:00 h.
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: sterile saline water solution, 0.9% NaCl
- Concentration of test material in vehicle: A solution at the highest concentration required was prepared initially, and then a series of solutions at lower concentrations was prepared by dilution with the vehicle.
- Amount of vehicle (intraperitoneal): The final dose levels were achieved by dosing the mice with a constant dose volume of the solutions (20 mL/kg body weight). The concentrations and dose levels of the test item reported are all expressed in terms of the dry solids content of the test item, calculated by the Sponsor to be 19.6% of the liquid sample received.
- Purity: Not relevant (UVCB)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On each occasion of use, a container of the test item was warmed to room temperature and shaken to dissolve the fine precipitate. A sample of the test item liquid was then taken, weighed, and mixed with sterile saline solution (0.9% NaCl, Fresenius Kabi AG) to prepare a solution at the highest concentrations required. The formulations of Green Liquor were a clear, faint green solution at 20 mg/mL and clear colourless solutions at the lower concentrations prepared. When solutions at several concentrations were required, a solution at the highest concentration required was prepared initially, and then a series of solutions at lower concentrations was prepared by dilution with the vehicle. The final dose levels were achieved by dosing the mice with a constant dose volume of the solutions (20 mL/kg body weight). The concentrations and dose levels of the test item in this report are all expressed in terms of the dry solids content of the test item, calculated by the Sponsor to be 19.6% of the liquid sample received.
Ten containers of the test item were supplied. Once a container of the test item had been opened, it was used on that day only. The test item remaining in that container and the formulations prepared from it were discarded before the end of that day.

In the main test, groups of five male mice were treated with the test item at 10, 20 and 40 mg/kg, the vehicle (0.9% NaCl) or the positive control (Cyclophosphamide) at 20 mg/kg.
Duration of treatment / exposure:
Four exposure groups: low (10 mg/kg), madium (20 mg/kg) and high (40 mg/kg), two vehicle control groups and one positive control group..
Euthanised after 24 hour: one low, one medium and one high dose groups/ and one vehicle control group and one positive control group (5 male animals per each group)
Euthanised after 48 hour: one high dose group and one vehicle control group (5 male animals per group).
Frequency of treatment:
Single treatment
Remarks:
Doses / Concentrations:
10 mg/kg
Basis:
nominal in water
of dry solids content of the test item
Remarks:
Doses / Concentrations:
20 mg/kg
Basis:
nominal in water
of dry solids content of the test item
Remarks:
Doses / Concentrations:
40 mg/kg
Basis:
nominal in water
of dry solids content of the test item
No. of animals per sex per dose:
Groups of five male mice were treated with the test item at 10, 20 and 40 mg/kg, the vehicle (0.9% NaCl) or the positive control (Cyclophosphamide) at 20 mg/kg.
Control animals:
yes
Positive control(s):
Cyclophosphamide;
- Route of administration: Oral gavage,
- Doses / concentrations: 5 male animals 10 ml/kg bodyweight (20 mg/kg) euthanasia after 24 hour
Tissues and cell types examined:
Bone marrow, polychromatic (immature) erythrocytes (PCE)
Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES: The mice from the main test were euthanised by dislocation of the neck at the scheduled time and immediately both femurs from each mouse were dissected free. The bone marrow was flushed out of each pair of femurs into 2.5 mL of foetal calf serum using a syringe and needle.

DETAILS OF SLIDE PREPARATION: The cell suspension was centrifuged for 10 minutes at 1000 rpm and most of the supernatant was removed. The cells were resuspended in the remainder and smeared on clean glass slides. The slide preparations were fixed in methanol, stained with Giemsa and coverslips were applied.

METHOD OF ANALYSIS: Prior to microscopic analysis, two slides from each animal were given a code number by a person who was not involved in the microscopic analysis (both slides from each animal were given the same code number). The code labels covered all unique identification marks on the slides to ensure that they were scored without bias. The coded slides were sent to Microptic Cytogenetic Services, 2 Langland Close, Mumbles, Swansea SA3 4LY, United Kingdom to be scored using a microscope. The slide scoring was performed according to OECD GLP.
For each animal, 1000 erythrocytes were examined and the number that were polychromatic (PCE) was recorded. During the examination of 1000 erythrocytes, the number of normochromatic erythrocytes (NCE) with micronuclei was also recorded. The scoring of PCE was then continue until a total of 2000 PCE had been examined, and the number of PCE with micronuclei in 2000 PCE was recorded.


OTHER:
Evaluation criteria:
The test item will be considered to have shown genotoxic activity in this study if all of the following criteria are met:

• increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative controls, for one or both sexes
• the increases are dose-related (when more than one dose level has been tested)
• the increases are reproducible between the animals of each group
• the increases are statistically significant.

The historical control range for the test laboratory will also be considered when evaluating the biological significance of small increases.
The test item will be concluded to have given a negative response if no reproducible, statistically significant increases are observed.
Statistics:
The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups will be compared to the values found in the negative control group. Statistical analysis will be performed using one way Analysis of Variance based on rank values (Blom’s method (2)). The statistical analyses will be performed with SAS® procedures (the version will be stated in the study report) described in SAS/STAT® User’s Guide, SAS OnlineDoc®, 1999, SAS Institute Inc., Cary, North Carolina 27513, USA.
Sex:
male
Genotoxicity:
negative
Remarks:
Green Liquor did not show any genotoxic activity in this study
Toxicity:
no effects
Remarks:
Green Liquor generally caused transient clinical signs including reduced activity, piloerection and closed eyes in the first 3 to 10 minutes after dosing at 20 and 40 mg/kg. No adverse reactions were observed in any of the other main test groups/controls.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY

- Dose range: 20, 40, 70, 100 and 400 mg/kg body weight by intraperitoneal injection using a dose volume of 20 mL/kg body weight.
- Clinical signs of toxicity in test animals: lethal effects. On the basis of observations, the maximum tolerated non lethal dose level (Green liquor dry weight) was determined to be 40 mg/kg and it was selected as the highest dose level for the main test.
- Evidence of cytotoxicity in tissue analyzed: No bone marrow smears were prepared from these range finding study mice.
- Other: Groups of two male and/or two female mice was dosed with the test item at dose levels of 20, 40, 70, 100 and 400 mg/kg body weight by intraperitoneal injection using a dose volume of 20 mL/kg body weight. The first group was dosed at 100 mg/kg and subsequent groups were dosed at higher or lower dose levels for each sex depending on the level of toxicity observed. When a mouse died soon after dosing, the second mouse of the same sex in that group was not dosed, for humane reasons. The mice were examined for visible signs of ill health or reactions to treatment daily during the acclimatisation period, before dosing and at intervals until sacrifice. The mice were weighed just before dosing and approximately 23 and 48 hours later. They were euthanised two days after dosing.


RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay): No biologically or statistically significant increases in the frequency of micronucleated PCE were seen in the groups of mice treated with Green Liquor compared to the vehicle control group at either sampling time.
- Ratio of PCE/NCE in the Micronucleus assay: No marked effect on the frequency of PCE (i.e. PCE/NCE ratio) among total erythrocytes was observed in any group treated with the test item or in the positive control group, compared to the vehicle control group.
- Appropriateness of dose levels and route: The clinical signs, especially the severity and rapidity of onset of the signs observed at the higher dose levels in the preliminary toxicity test, clearly showed that components of the test item were distributed systemically and this was evidence that the target tissue, the bone marrow, was exposed to components of the test item. The intraperitoneal injection route of administration and the doses of the main study are regarded apropriate for the test item (the ip. route was also recommended for this test item by ECHA). Treatment with the test item did not have a marked effect on the body weight of the mice in the main test.
- Statistical evaluation:The increases in the positive control values over the negative control values were large and statistically significant at 1% level (p<0.01), demonstrating the sensitivity of the test. No statistically significant increases in the frequency of micronucleated PCE were seen in the groups of mice treated with Green Liquor compared to the vehicle control group at either sampling time (p< 0.05). Statistical analysis were performed using one way Analysis of Variance based on rank values (Blom’s method).

Summary of results and summary of statistical analysis are given in Table 1. Historical control data of the test laboratory from 2007 to 2012 is given in Table 2.

Table 1. Summary of results and statistical analysis

Euthanasia

Group

Treatment

MnPCE

%PCE

time (h)

 

 

Range

Mean

 

Mean

 

 

 

 

 

 

 

24

1

Vehicle control (0.9% NaCl)

2 – 9

6.4

 

49.9

24

2

Green Liquor(10 mg/kg)

1 – 11

5.2

ns

51.3

24

3

Green Liquor(20 mg/kg)

4 – 8

6.0

ns

53.7

24

4

Green Liquor(40 mg/kg)

5 – 10

7.8

ns

48.8

24

5

Cyclophosphamide (20 mg/kg)

27 – 55

44.0

**

51.4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

48

1

Vehicle control (0.9% NaCl)

2 - 6

4.0

 

43.3

48

4

Green Liquor(40 mg/kg)

4 - 7

5.8

ns

42.3

 

 

 

 

 

 

 

MnPCE    Number of polychromatic (immature) erythrocytes (PCE) with micronuclei (2000 PCE scored/animal)

%PCE      Frequency of PCE among total erythrocytes (%) (1000 erythrocytes scored/animal)

ns            Difference from vehicle control not statistically significant at 5% level (p>0.05)

**            Statistically significant difference from vehicle control at 1% level (p<0.01)

Table 2. Historical control data of the test laboratory from 2007 to 2012

 

Negative control

Positive control

Cyclophosphamide (20 mg/ml)

 

 

 

Number of animals

115

60

Mean value for MnPCE

1.7

46.6

Standard deviation for MnPCE

1.2

23.9

Minimum value for MnPCE

0

10

Maximum value for MnPCE

6

96

 

 

 

MnPCE  Number of polychromatic erythrocytes (PCE) with micronuclei (2000 PCE scored/animal)
Conclusions:
Interpretation of results (migrated information): negative
Green Liquor did not show any genotoxic activity in this mouse micronucleus test.
Executive summary:

The objective of the study was to determine whether the test item, Green Liquor, caused genotoxic effects resulting in the formation of micronuclei in erythrocytes of treated mice. The test item was tested in the Mouse Micronucleus Test performed in accordance with the OECD guideline “Mammalian Erythrocyte Micronucleus Test”, No 474 (1997).

In the preliminary toxicity test, male and/or female mice were treated with the test item mixed with sterile saline solution (0.9% NaCl) by intraperitoneal injection at 20, 40, 70, 100 and 400 mg/kg body weight. The dose levels of the test item in this report are expressed in terms of the dry solids content of the test item: calculated by the Sponsor to be 19.6% of the liquid sample received. Clinical signs developed very rapidly after treatment in all mice except the two males dosed at 100 mg/kg. Some males and females died within a few minutes after dosing at dose levels of 70 mg/kg and above. All surviving mice appeared normal within 5 to 58 minutes after dosing and no further clinical signs were observed. No marked effect on body weight was observed. The maximum tolerated dose level was determined to be 40 mg/kg and this was selected as the highest dose level for the main test.

Only male mice were used in the main test because observations in the preliminary test showed that there was not a substantial difference in toxicity of the test item between the sexes.

In the main test, groups of male mice were treated with the vehicle (0.9% NaCl) or the test item at 10, 20 and 40 mg/kg by intraperitoneal injection, or the positive control (Cyclophosphamide) at 20 mg/kg by oral gavage. Five mice from each group were euthanised 24 hours after dosing, and a further five mice dosed with the vehicle or Green Liquor at 40 mg/kg were euthanised 48 hours after dosing.

Bone marrow smears were prepared on glass slides for each of the mice, stained, and scored. Green Liquor generally caused transient clinical signs including reduced activity, piloerection and closed eyes in the first 3 to 10 minutes after dosing at 20 and 40 mg/kg.

No biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes were seen in mice treated with the test item, compared to the negative control values. The positive control treatment caused a large, statistically significant, increase demonstrating the sensitivity of the test system. The clinical signs provided evidence for bone marrow exposure to components of the test item. It is concluded that Green Liquor did not show any genotoxic activity in this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Green Liquor was non-mutagenic based on the negative results in in vitro gene mutation study in bacteria (REACH Annex VII, section 8.4.1), but the results from in vitro cytogenicity study in mammalian cells (REACH Annex VIII, section 8.4.2) were interpreted as positive. The data of both studies was done according to the EU/OECD guidelines being in force at that time and assessed to be of good quality and relevant.

Existing genotoxicity studies in the scientific literature for major GL constituents provided mainly negative results, but hydrogen sulfide that might form in the GL in acidic conditions also gave ambiguous and some positive genotoxicity results.

According to the REACH Annexes VII - X, following a positive result in any of the in vitro studies and in the case that there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study should be proposed (European Chemicals Agency, Guidance on information requirements and chemical safety assessment, Chapter R.7a: Endpoint specific guidance, Table R.7.7 -5). Since the result from the in vitro cytogenicity study in mammalian cells was interpreted as positive, further somatic cell in vivo testing to investigate structural or numerical chromosome aberrations were needed. A mammalian erythrocyte micronucleus test (OECD Guideline 474) was proposed by the Reach registrants in the year 2010.

Genetic toxicity in vivo a mouse micronucleus erythrocyte test was carried out in three test concentrations of green liquor using intraperitoneal administration at the doses of 10, 20, 40 mg/kg substance dry matter, A preliminary toxicity test confirmed that absorption occurs and that appropriate doses/route is used. The test result was negative and green liquor did not show any genotoxic activity in the test.

The existing dataset meets the Reach/CLP information requirements on genetic toxicity and is complete enough to draw conclusions. The existing in vitro data show that the green liquor have no potential to induce both gene and chromosome mutations and the in vivo test has addressed this comprehensively. Therefore, since there is a negative results in adequately conducted, appropriate in vivo test, it is possible to conclude that Green liquor is not an in vivo mutagen.

Justification for selection of genetic toxicity endpoint

The existing in vitro data show that the green liquor have no potential to induce both gene and chromosome mutations and the in vivo test has addressed this comprehensively enough. Therefore, since there is a negative results in adequately conducted, appropriate in vivo test, it is possible to conclude that the green liquor is not an in vivo mutagen.

Justification for classification or non-classification

The genotoxicity potential of Green liquor is low. Information is sufficient to conclude that Green liquor do not meet the CLP/GHS criteria to be classified as a mutagen.