Registration Dossier

Administrative data

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)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: Clear greenish or yellowish liquid
Details on test material:
- Name of test material (as cited in study report): Sulfite liquors and Cooking liquors, green
- Substance type:unknown or variable composition (UVCB)
- Physical state: liquid (dissolved dry solids 19.6 et. % in water, (80.4 % water, the substance contains always water 75 % or more )
- Impurities (identity and concentrations): not relevant
- Composition of test material, percentage of components:

Analyte Method Unit Result
Sodium Na SCAN-N 38:10 modif g/l 93
Potassium K SCAN-N 38:10 modif g/l 1,3
Sulfur S SCAN-N 38:10 modif g/l 57
Thiosulfate S2O3= KCL 70:83 modif. g/l 5,7
Sulphate SO42- SCAN-N 6:85 g/l 16,1
Effective alkali NaOH SCAN-N 30:85 g/l 59
Active alkali NaOH SCAN-N 30:85 g/l 120
Total alkali NaOH SCAN-N 30:85 g/l 150
Sodium sulfide Na2S calculated g/l 122
Sodium carbonate Na2CO3 calculated g/l 42,9
Sodium hydroxide NaOH calculated g/l n.d
Sodium thiosulfate Na2S2O3 calculated g/l 7,9
Potassium hydroxide KOH calculated g/l n.d
Potassium carbonate K2CO3 calculated g/l 0,5
Potassium sulfide K2S calculated g/l 1,4
Potassium thiosulfate K2S2O3 calculated g/l 0,08


- Analysis date and laboratorium: 28.8-12.9.2012, Mia Tehomaa, Labtium Ltd, Tekniikantie 2, 02150 Espoo, Finland (lab reporrt 113630)
- Lot/batch No.: 2012-v32 GL1 after sedimentation and filtration
- Stability under test conditions: Stabile
- Storage condition of test material:Stored in a refrigerator at 2 to 8ºC
- Other:

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals 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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
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).

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.