Registration Dossier
Registration Dossier
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EC number: 940-223-9 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The in-vitro clastogenicity of the registration susbtance, Hostapon SLG, is investigated according to the OECD Guideline 473. Negative results were obtained.
Further, the in-vitro genotoxicity of the registration substance, Hostapon SLG is derived based on the read-across to Hostapon SG.
The genotoxicity of Hostapon SG was investigated in Ames test (OECD 471) and in mouse lymphoma assay (OECD 476). The results obtained in these studies are indicative of no mutagenic potential. Further, two chromosome aberrations studies are available reporting positive response. However, these studies were re-evaluated according to the currently accepted Guideline OECD 473 (adopted in 2016) by the registrant and the data are evaluated as indicative of no significant clastogenicity.
Likewise, the registration substance, Hostapon SLG is considered as of no significant genotoxic activity.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 homogenate
- Test concentrations with justification for top dose:
- 0.025, 0.05, 0.1 mg/ml
Precipitation at 0.15 mg/ml - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Not clastogenic
- Executive summary:
Hostapon SLG was investigated according to the Guideline OECD 473 (version 1997). CHO cells were treated with the test substance with and without metabolic activation at concentrations of 0.025, 0.05 and 0.1 mg/ml. The concentration of 0.15 mg/l was associated with enhanced precipitation. Negative results were obtained in two indenpendent experiments. Hostapon SLG is not clastogenic in in-vitro assay.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2008
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- induced rat liver S9-mix
- Test concentrations with justification for top dose:
- 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I and experiment II
- Vehicle / solvent:
- Deionised water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar plate incorporation; preincubation
DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours
NUMBER OF REPLICATIONS: 3 plates
- Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed .
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration .
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Hostapon SG is not mutagenic in Ames test.
- Executive summary:
Hostapon SG was investigated for its mutagenicity in bacterial mutation reverse assay according to the Guideline OECD 471. Up to the concentration of 5000 µg/plate no mutagenic property was found without and with metabolic activation in strains TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA in two indepedant experiments. Hostapon SG is not mutagenic in Ames test.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine Kinase Locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- other: Clone 3.7.2C
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- Experiment I:
without S9 mix: 5.9; 11.8; 23.5; 47.0; 94.0; 141.0 µg/mL
with S9 mix: 11.8; 23.5; 47.0; 94.0; 188.0; 282.0 µg/mL
Experiment II:
without S9 mix: 11.8; 23.5; 47.0; 94.0; 188.0; 282.0 µg/mL
with S9 mix: 8.8; 17.5; 35.0; 70.0; 105.0; 140.0 µg/mL
Following the expression phase of 48 hours the cultures at the maximum concentration of both main experiments were not continued due to exceedingly strong toxic effects. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solubility properties - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days
SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Evaluation criteria:
- A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the
corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. - Statistics:
- Linear regression analysis (least squares) using SYSTAT(R) 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected
- Effects of osmolality: Not increased
- Evaporation from medium: Not examined
- Water solubility: Up to 25 %
- Precipitation: pre-experiment: at 750, 1500, 3000 µg/mL; main experiments: no precipitation occurred
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was chosen with regard to the molecular weight of the test item. Test item concentrations between 23.4 and 3000 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation.
Strong toxic effects occurred at 187.5 µg/mL and above with and without metabolic activation following 4 and 24 hours of treatment.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was observed by the unaided eye at 750 µg/mL and above with and without metabolic activation at both treatment intervals.
The dose range of the main experiments was set according to the data generated in the pre-experiment. The experimental part of the second
experiment with metabolic activation was terminated due to exceedingly severe cytotoxicity and repeated at lower concentrations. In both main
experiments the individual concentrations were generally spaced by a factor of 2.0 in the lower range. A narrower spacing was used at the highest concentrations to cover the cytotoxic range more closely.
To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.
COMPARISON WITH HISTORICAL CONTROL DATA: complies
ADDITIONAL INFORMATION ON CYTOTOXICITY: None - Remarks on result:
- other: strain/cell type: in vitro gene mutation assay with L5178Y cells
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The genotoxicity of Hostapon SG was investigated according to the Guideline OECD 476 (MLA). No mutagenic acitivty was found.
- Executive summary:
Hostapon SG was investigated for its mutagenicity according to the Guideline OECD 476 (MLA). In two independent experiments, the mouse lymphoma L5178Y cell cultures were treated with the test item up to the concentrations associated with 20% cell growth reduction with and without metabolic activation. No mutagenic acitivity was found.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- The concentration used for the test was selected only based on the cytotoxicity, but not based on the precipitation.The results obtained at concentrations with precipiations are not considered as valid.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adpted July 21, 1997
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in-vitro chromosome aberration
- Species / strain / cell type:
- lymphocytes:
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9
- Test concentrations with justification for top dose:
- The highest dose selected was the dose causing 50% reduction of mitotic indices of control.
- Vehicle / solvent:
- water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in culture medium. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: about 1.5
NUMBER OF CELLS EVALUATED: At least 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - Species / strain:
- lymphocytes:
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- lymphocytes:
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: The author of the study evaluated the results obtained as "positive". The registrant performed re-evaluation, taking account the precipitation in the culture medium as the potential artifical factor for false-positive responce.
- Conclusions:
- Hostapon SG was investigated according to the Guideline OECD 473. No genotoxic activity was observed up to the concentration not associated with precipitation.
- Executive summary:
Hostapon SG was investigated according to the Guideline OECD 473 (version 1997). Human lymphocytes were treated with the test substance with and without metabolic activation up to the concentration associated with 50% reduction of mitotic indices. In general, reduced cytotoxicity was observed with metabolic activation, leading to the use of higher concentrations with metabolic activation for the genotoxicity investigation. These concentrations were associated with enhanced precipitation.
Total five independent experiments were performed. No increase of aberration frequency was observed without metabolic activation. Significant increase of aberration frequency was obtained with metabolic activation at concentrations associated not only with significant cytotoxicity but also with enhanced precipitation. The author of the study evaluated the results obtained as "positive".
The registrant performed re-evaluation in 2018, taking account the precipitation in the culture medium as potential interfering factor responsible for a false-positve response (according to the current OECD Guideline, version 2016). Based on the re-evaluation, no significant genotoxic activity can be assigned up to the concentrations not associated with remarkable precipitation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1998
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: See 'Remarks'
- Version / remarks:
- Guidelines for Toxicity Studies Required for Approval Application for Manufacturing (Importing) Pharmaceutical Products (Notification No. 24, Evaluation and Licensing Division I, September 11, 1989)
- Principles of method if other than guideline:
- The studa design is consisted of two experiments (reported as "direct method" and "metabolic activation method" in the given report. The first experiment ("direct method") comprises cell treatment of 24h/48h without metabolic activation. The second experiment ("metabolic activation method") comprises cell treatment of 6h with and wihout metabolic acitvation.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- The test material is equivalent to the commercial product of the registration substance.
- Species / strain / cell type:
- other: Chinese hamster lung fibroblasts CHL
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Eagle`s MEM supplemented with 10% calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- induced rat liver S9-mix
- Test concentrations with justification for top dose:
- Direct method:
40, 80, 120 and 160 microgram/mL (24 hour / 48 hour treatments)
Metabolic activation method:
78, 156, 313 and 625 microgram / mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Physiological saline
- Justification for choice of solvent/vehicle: Accepted solvent for this type of study - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-dimethylnitrosamine
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Direct method (in medium) / Metabolic activation method (with and without S9-mix)
A] Direct method
DURATION
- Preincubation period: 3 days
- Exposure duration: 24 hours and 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Following treatment (24 hour or 48 hour treatment)
SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa`s staining solution in Soerensen buffer
NUMBER OF REPLICATIONS: duplicates
NUMBER OF CELLS EVALUATED: 200 in each concentration, 100 cells per dish
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
B] Metabolic activation method
DURATION
- Preincubation period: 3 days
- Exposure duration: 6 hours (with and without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): Following treatment
SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa`s staining solution in Soerensen buffer
NUMBER OF REPLICATIONS: duplicates
NUMBER OF CELLS EVALUATED: 200 in each concentration, 100 cells per dish
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - Evaluation criteria:
- Frequencies of cells with structural aberrations were calculated, both for the case in which dells with gaps were included, and in which cells with gaps were excluded. Assessments were based on the frequency of cells with structural aberrations including gaps.
Negative: < 5%
Equivocal: > 5%, < 10%
Positive: > 10% - Statistics:
- No statistical analysis performed
- Key result
- Species / strain:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Remarks:
- The author of the study evaluated the results obtained as "positive". The registrant performed re-evaluation, taking account the cyotoxicity as the potential interferring factor for false-positive responce.
- Conclusions:
- The genotoxicity of Hostapon SG was investigated in in-vitro chromosome aberration test. No genotoxic activity was found at concentrations associated cyotoxicity of cell growth reduction less than 50%.
- Executive summary:
The genotoxicity of Hostapon SG was investigated in in-vitro chromosome aberration test. Chines hamster lung fibroblasts cell cultures were used as test system in two independent experiments.
In the first experiments the cells were treated for 24h or 48h without metabolic acitivation. The highest concentrations used corresponded to the cell growth reduction down to 15% of control.
In the second experiment the cells were treated for 6h with or without metabolic activation. The highest concentrations used corresponded to the cell growth reduction down to 36% of control
No increase of aberration frequency was observed without metabolic activation in the first and in the second experiment. Significant increase of aberration frequency was obtained with metabolic activation at the highest concentration applied. The author of the study evaluated the results obtained as "positive".
The registrant performed re-evaluation in 2018, taking account the cyototoxicity in the culture medium as potential interfering factor responsible for a false-positve response (according to the current OECD Guideline, version 2016). Based on the re-evaluation, no significant genotoxic activity can be assigned up to the insoluble concentrations for Hostapon SG.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2008
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Justification is provided in Chapter 13.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- induced rat liver S9-mix
- Test concentrations with justification for top dose:
- 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I and experiment II
- Vehicle / solvent:
- Deionised water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar plate incorporation; preincubation
DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours
NUMBER OF REPLICATIONS: 3 plates
- Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed .
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration .
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Not mutagenic in Ames test
- Executive summary:
The genotoxicity of Hostapon SLG is derived based on the read across to Hostapon SG.
Hostapon SG was investigated for its mutagenicity in bacterial mutation reverse assay according to the Guideline OECD 471. Up to the concentration of 5000 µg/plate no mutagenic property was found without and with metabolic activation in strains TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA in two indepedant experiments. Hostapon SG is not mutagenic in Ames test.
Likewise no mutagenic acitivity is assigned to Hostapon SLG.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Justification is provided in Chapter 13.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine Kinase Locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- other: Clone 3.7.2C
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- Experiment I:
without S9 mix: 5.9; 11.8; 23.5; 47.0; 94.0; 141.0 µg/mL
with S9 mix: 11.8; 23.5; 47.0; 94.0; 188.0; 282.0 µg/mL
Experiment II:
without S9 mix: 11.8; 23.5; 47.0; 94.0; 188.0; 282.0 µg/mL
with S9 mix: 8.8; 17.5; 35.0; 70.0; 105.0; 140.0 µg/mL
Following the expression phase of 48 hours the cultures at the maximum concentration of both main experiments were not continued due to exceedingly strong toxic effects. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solubility properties - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days
SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Evaluation criteria:
- A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the
corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. - Statistics:
- Linear regression analysis (least squares) using SYSTAT(R) 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected
- Effects of osmolality: Not increased
- Evaporation from medium: Not examined
- Water solubility: Up to 25 %
- Precipitation: pre-experiment: at 750, 1500, 3000 µg/mL; main experiments: no precipitation occurred
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was chosen with regard to the molecular weight of the test item. Test item concentrations between 23.4 and 3000 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation.
Strong toxic effects occurred at 187.5 µg/mL and above with and without metabolic activation following 4 and 24 hours of treatment.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was observed by the unaided eye at 750 µg/mL and above with and without metabolic activation at both treatment intervals.
The dose range of the main experiments was set according to the data generated in the pre-experiment. The experimental part of the second
experiment with metabolic activation was terminated due to exceedingly severe cytotoxicity and repeated at lower concentrations. In both main
experiments the individual concentrations were generally spaced by a factor of 2.0 in the lower range. A narrower spacing was used at the highest concentrations to cover the cytotoxic range more closely.
To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.
COMPARISON WITH HISTORICAL CONTROL DATA: complies
ADDITIONAL INFORMATION ON CYTOTOXICITY: None - Remarks on result:
- other: strain/cell type: in vitro gene mutation assay with L5178Y cells
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- No mutagenic activity in MLA
- Executive summary:
The genotoxicity of Hostapon SLG is derived based on the read across to Hostapon SG.
Hostapon SG was investigated for its mutagenicity according to the Guideline OECD 476 (MLA). In two independent experiments, the mouse lymphoma L5178Y cell cultures were treated with the test item up to the concentrations associated with 20% cell growth reduction with and without metabolic activation. No mutagenic acitivity was found.
Likewise, no mutagenic acitivity is assigned to Hostapon SLG.
Referenceopen allclose all
APPENDIX 1.INDIVIDUAL DATA OF PERCENTAGE MITOTIC INDEX FOR INITIAL CYTOTOXICITY TEST
Set No. |
Treatment |
Dose (µL/mL) |
Replicate |
Metaphases |
Blast cells |
Mitotic index |
Percentage Mitotic index |
1 (+S9) (3-5hours) |
Negative control |
- |
1 |
37 |
997 |
0.0358 |
3.58 |
2 |
34 |
1006 |
0.0327 |
3.27 |
|||
Vehicle control |
- |
1 |
34 |
1003 |
0.0328 |
3.28 |
|
2 |
35 |
1000 |
0.0338 |
3.38 |
|||
Hostapon SLG
|
0.1 |
1 |
31 |
1004 |
0.0299 |
3.00 |
|
2 |
33 |
1023 |
0.0313 |
3.13 |
|||
0.05 |
1 |
29 |
1011 |
0.0279 |
2.79 |
||
2 |
34 |
1001 |
0.0329 |
3.29 |
|||
0.025 |
1 |
34 |
1020 |
0.0323 |
3.23 |
||
2 |
33 |
1007 |
0.0317 |
3.17 |
|||
2 (-S9) (3-5 hours) |
Negative control |
- |
1 |
37 |
1007 |
0.0354 |
3.54 |
2 |
36 |
991 |
0.0351 |
3.51 |
|||
VC |
- |
1 |
36 |
1007 |
0.0345 |
3.45 |
|
2 |
37 |
1006 |
0.0355 |
3.55 |
|||
Hostapon SLG
|
0.1 |
1 |
33 |
1002 |
0.0319 |
3.19 |
|
2 |
32 |
1002 |
0.0309 |
3.09 |
|||
0.05 |
1 |
33 |
1008 |
0.0317 |
3.17 |
||
2 |
34 |
1035 |
0.0318 |
3.18 |
|||
0.025 |
1 |
43 |
1209 |
0.0343 |
3.43 |
||
2 |
36 |
1039 |
0.0335 |
3.35 |
|||
3 (-S9) (18-20 hours) |
Negative control |
- |
1 |
40 |
1122 |
0.0344 |
3.44 |
2 |
40 |
1023 |
0.0376 |
3.76 |
|||
VC |
- |
1 |
40 |
1048 |
0.0368 |
3.68 |
|
2 |
38 |
1028 |
0.0356 |
3.56 |
|||
Hostapon SLG
|
0.1 |
1 |
37 |
1010 |
0.0353 |
3.53 |
|
2 |
37 |
1035 |
0.0345 |
3.45 |
|||
0.05 |
1 |
38 |
1007 |
0.0364 |
3.64 |
||
2 |
35 |
1015 |
0.0333 |
3.33 |
|||
0.025 |
1 |
37 |
1003 |
0.0356 |
3.56 |
||
2 |
36 |
1003 |
0.0346 |
3.46 |
APPENDIX 2.INDIVIDUAL DATA OFCHROMOSOMAL ABERRATIONAND MITOTIC INDEX
Set No. |
Treatment |
Dose (µL/mL) |
Replicate |
Mitotic Index |
Aberrations |
Total No. of Aberrations |
Total No. of Aberrations without Gaps |
Total % of Aberrated cells |
|||||||||||
GAP |
Break |
Exchange |
Fragments |
Ring |
Deletion |
||||||||||||||
Total No. of MP |
Total No. of Blast cells |
Mitotic Index |
Percentage of MI |
Chromatid |
Chromosome |
Chromatid |
Chromosome |
Chromatid |
Chromosome |
||||||||||
1 (+S9) (3-5 hours) |
Negative control |
- |
R1 |
37 |
968 |
0.0368 |
3.68 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
R2 |
35 |
971 |
0.0348 |
3.48 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|||
Vehicle control |
- |
R1 |
36 |
973 |
0.0357 |
3.57 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
R2 |
36 |
966 |
0.0359 |
3.59 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|||
Positive control (Cyclophosphamide ) |
10 µg/mL |
R1 |
32 |
997 |
0.0311 |
3.11 |
- |
- |
1 |
- |
1 |
0 |
2 |
1 |
5 |
10 |
10 |
9 |
|
R2 |
33 |
972 |
0.0328 |
3.28 |
- |
- |
1 |
3 |
0 |
0 |
1 |
1 |
3 |
9 |
9 |
8 |
|||
Hostapon SLG
|
0.025 |
R1 |
34 |
979 |
0.0336 |
3.36 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
R2 |
34 |
988 |
0.0330 |
3.33 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|||
0.05 |
R1 |
32 |
971 |
0.0319 |
3.19 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
||
R2 |
33 |
1010 |
0.0316 |
3.16 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|||
0.1 |
R1 |
31 |
983 |
0.0306 |
3.06 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
||
R2 |
31 |
993 |
0.0303 |
3.03 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|||
APPENDIX 2(Contd..,). INDIVIDUAL DATA OFCHROMOSOMAL ABERRATIONAND MITOTIC INDEX
Set No. |
Treatment |
Dose (µL/mL) |
Replicate |
Mitotic Index |
Aberrations |
Total No. of Aberrations |
Total No. of Aberrations without Gaps |
Total % of Aberrated cells |
||||||||||||
GAP |
Break |
Exchange |
Fragments |
Ring |
Deletion |
Others* |
||||||||||||||
Total No. of MP |
Total No. of Blast cells |
Mitotic Index |
Percentage of MI |
Chromatid |
Chromosome |
Chromatid |
Chromosome |
Chromatid |
Chromosome |
|||||||||||
2 (-S9) (3-5 hours) |
Negative control |
- |
R1 |
37 |
1012 |
0.0353 |
3.53 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
- |
- |
R2 |
35 |
965 |
0.0350 |
3.50 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
- |
- |
|||
Vehicle control |
- |
R1 |
34 |
981 |
0.0335 |
3.35 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
- |
- |
|
R2 |
33 |
968 |
0.0330 |
3.30 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
- |
- |
|||
Positive Control (Mitomycin C) |
0.05 µg/mL |
R1 |
32 |
982 |
0.0316 |
3.16 |
2 |
- |
1 |
- |
- |
- |
3 |
1 |
3 |
2 |
12 |
10 |
8 |
|
R2 |
33 |
988 |
0.0323 |
3.23 |
2 |
3 |
1 |
2 |
- |
- |
- |
1 |
1 |
3** |
12 |
10 |
6 |
|||
Hostapon SLG
|
0.025 |
R1 |
33 |
993 |
0.0322 |
3.22 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
- |
- |
|
R2 |
34 |
1006 |
0.0327 |
3.27 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
- |
- |
|||
0.05 |
R1 |
31 |
971 |
0.0309 |
3.09 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
- |
- |
||
R2 |
32 |
1002 |
0.0319 |
3.19 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
- |
- |
|||
0.1 |
R1 |
32 |
1023 |
0.0303 |
3.03 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
- |
- |
||
R2 |
32 |
1019 |
0.0304 |
3.04 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
- |
- |
- |
|||
APPENDIX 2(Contd..,). INDIVIDUAL DATA OFCHROMOSOMAL ABERRATIONAND MITOTIC INDEX
Set No. |
Treatment |
Dose (µL/mL) |
Replicate |
Mitotic Index |
|
Aberrations |
Total No. of aberrations |
Total No. of aberrations without gaps |
Total % of aberrated cells |
||||||||||||
GAP |
Break |
Exchange |
Fragment |
Ring |
Deletion |
Endoreduplication |
|||||||||||||||
Total No. of MP |
Total No. of Blast cells |
Mitotic Index |
Percentage of MI |
Chromatid |
Chromosome |
Chromatid |
Chromosome |
Chromatid |
Chromosome |
||||||||||||
3 (-S9) (18-20 hours) |
Negative control |
- |
R1 |
36 |
990 |
0.0351 |
3.51 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
R2 |
37 |
1020 |
0.0350 |
3.50 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
||||
Vehicle control |
- |
R1 |
34 |
975 |
0.0337 |
3.37 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
||
R2 |
35 |
977 |
0.0346 |
3.46 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
||||
Positive Control (Mitomycin C) |
0.05 µg/mL |
R1 |
33 |
974 |
0.0334 |
3.34 |
- |
4 |
2 |
3 |
- |
- |
1 |
1 |
4 |
- |
15 |
11 |
9 |
||
R2 |
34 |
984 |
0.0324 |
3.24 |
4 |
- |
- |
2 |
- |
- |
3 |
1 |
1 |
1 |
12 |
8 |
5 |
||||
Hostapon SLG
|
0.025 |
R1 |
34 |
982 |
0.0335 |
3.35 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
||
R2 |
34 |
973 |
0.0338 |
3.38 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
||||
0.05 |
R1 |
32 |
979 |
0.0317 |
3.17 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|||
R2 |
33 |
982 |
0.0325 |
3.25 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
||||
0.1 |
R1 |
31 |
988 |
0.0304 |
3.04 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|||
R2 |
32 |
984 |
0.0315 |
3.15 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
||||
Summary Tabellen
Summary of Results Pre-Experiment and Experiment I
Study Name: 1211000 |
Study Code: Harlan CCR 1211000 |
Experiment: 1211000 VV Plate |
Date Plated: 17/09/2008 |
Assay Conditions: |
Date Counted: 24/09/2008 |
Metabolic Activation |
Test Group |
Dose Level (µg/plate) |
Revertant Colony Counts (Mean ±SD) |
|||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
||||
Without Activation |
Deionised water |
20 ± 3 |
11 ± 3 |
35 ± 2 |
137 ± 5 |
55 ± 4 |
||
Untreated |
18 ± 3 |
13 ± 3 |
37 ± 10 |
126 ± 4 |
47 ± 3 |
|||
Hostapon SG |
3 µg |
20 ± 3 |
11 ± 4 |
29 ± 1 |
134 ± 12 |
50 ± 4 |
||
10 µg |
19 ± 4 |
14 ± 2 |
33 ± 5 |
124 ± 8 |
52 ± 6 |
|||
33 µg |
18 ± 3 |
11 ± 4 |
35 ± 6 |
132 ± 10 |
55 ± 1 |
|||
100 µg |
19 ± 4 |
8 ± 0 |
37 ± 6 |
139 ± 16 |
52 ± 4 |
|||
333 µg |
13 ± 2 |
10 ± 4 |
32 ± 8 |
122 ± 15 |
63 ± 3 |
|||
1000 µg |
17 ± 2 |
7 ± 1 |
25 ± 5 |
84 ± 11 R |
62 ± 2 |
|||
2500 µg |
8 ± 2 M R |
2 ± 2 M R |
19 ± 2 M R |
36 ± 5 M R |
59 ± 2 |
|||
5000 µg |
2 ± 2 M R |
0 ± 1 M R |
5 ± 2 M R |
27 ± 3 M R |
58 ± 2 |
|||
NaN3 |
10 µg |
2009 ± 31 |
2369 ± 114 |
|||||
4-NOPD |
10 µg |
424 ± 9 |
||||||
4-NOPD |
50 µg |
83 ± 3 |
||||||
MMS |
3.0 µL |
1017 ± 33 |
||||||
With Activation |
Deionised water |
23 ± 3 |
20 ± 5 |
37 ± 4 |
148 ± 5 |
68 ± 4 |
||
Untreated |
24 ± 6 |
19 ± 7 |
42 ± 8 |
148 ± 14 |
64 ± 9 |
|||
Hostapon SG |
3 µg |
24 ± 3 |
19 ± 5 |
39 ± 5 |
129 ± 14 |
64 ± 4 |
||
10 µg |
21 ± 5 |
17 ± 5 |
44 ± 8 |
135 ± 3 |
71 ± 2 |
|||
33 µg |
25 ± 3 |
19 ± 3 |
43 ± 6 |
132 ± 9 |
60 ± 11 |
|||
100 µg |
23 ± 1 |
15 ± 2 |
38 ± 4 |
134 ± 5 |
60 ± 4 |
|||
333 µg |
22 ± 5 |
13 ± 2 |
47 ± 6 |
136 ± 13 |
73 ± 11 |
|||
1000 µg |
15 ± 4 |
16 ± 3 |
35 ± 3 |
116 ± 13 |
69 ± 3 |
|||
2500 µg |
9 ± 3 M R |
8 ± 1 M R |
28 ± 2 M R |
48 ± 7 M R |
66 ± 1 |
|||
5000 µg |
5 ± 1 M R |
1 ± 1 M R |
13 ± 2 M R |
34 ± 5 M R |
59 ± 4 |
|||
2-AA |
2.5 µg |
245 ± 16 |
191 ± 30 |
1267 ± 70 |
1506 ± 43 |
|||
2-AA |
10.0 µg |
232 ± 24 |
||||||
Key to Positive Controls |
Key to Plate Postfix Codes |
||
NaN3 2-AA 4-NOPD MMS |
sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate |
R M |
Reduced background growth Manual count |
Summary of Results Experiment II
Study Name: 1211000 |
Study Code: Harlan CCR 1211000 |
Experiment: 1211000 HV2 Pre |
Date Plated: 01/10/2008 / 14/10/2008* |
Assay Conditions: |
Date Counted: 08/10/2008 / 17/10/2008* |
Metabolic Activation |
Test Group |
Dose Level (µg/plate) |
Revertant Colony Counts (Mean ±SD) |
|||||
TA 1535 |
TA 1537 |
TA 98* |
TA 100 |
WP2 uvrA |
||||
Without Activation |
Deionised water |
19 ± 6 |
12 ± 4 |
22 ± 2 |
131 ± 5 |
51 ± 8 |
||
Untreated |
16 ± 6 |
10 ± 2 |
24 ± 4 |
137 ± 12 |
46 ± 8 |
|||
Hostapon SG |
3 µg |
17 ± 4 |
14 ± 5 |
23 ± 2 |
131 ± 8 |
55 ± 1 |
||
10 µg |
18 ± 4 |
15 ± 2 |
24 ± 2 |
127 ± 9 |
52 ± 8 |
|||
33 µg |
18 ± 3 |
11 ± 6 |
24 ± 4 |
122 ± 1 |
55 ± 6 |
|||
100 µg |
22 ± 6 |
14 ± 3 |
23 ± 1 |
101 ± 15 R |
56 ± 2 |
|||
333 µg |
12 ± 5 |
10 ± 4 |
15 ± 2 |
75 ± 7 R |
47 ± 4 |
|||
1000 µg |
5 ± 2 R M |
2 ± 3 M R |
10 ± 1 M R |
39 ± 5 M R |
49 ± 5 |
|||
2500 µg |
2 ± 2 M R |
0 ± 0 M R |
3 ± 2 M R |
31 ± 10 M R |
46 ± 5 |
|||
5000 µg |
1 ± 1 M R |
0 ± 0 M R |
0 ± 0 M R |
5 ± 2 M R |
37 ± 7 |
|||
NaN3 |
10 µg |
1799 ± 67 |
1998 ± 31 |
|||||
4-NOPD |
10 µg |
1637 ± 116 |
||||||
4-NOPD |
50 µg |
105 ± 5 |
||||||
MMS |
3.0 µL |
418 ± 20 |
||||||
With Activation |
Deionised water |
17 ± 4 |
16 ± 5 |
26 ± 3 |
138 ± 9 |
58 ± 5 |
||
Untreated |
18 ± 4 |
19 ± 7 |
29 ± 3 |
142 ± 37 |
64 ± 7 |
|||
Hostapon SG |
3 µg |
19 ± 4 |
17 ± 4 |
27 ± 0 |
131 ± 11 |
59 ± 3 |
||
10 µg |
22 ± 4 |
17 ± 4 |
25 ± 2 |
132 ± 8 |
58 ± 3 |
|||
33 µg |
20 ± 2 |
19 ± 3 |
24 ± 2 |
141 ± 15 |
59 ± 6 |
|||
100 µg |
21 ± 1 |
15 ± 3 |
22 ± 5 |
140 ± 7 |
57 ± 5 |
|||
333 µg |
18 ± 4 |
14 ± 4 |
19 ± 3 |
110 ± 5 R |
53 ± 2 |
|||
1000 µg |
11 ± 3 M R |
12 ± 2 M R |
10 ± 2 M R |
74 ± 7 R |
54 ± 1 |
|||
2500 µg |
9 ± 3 M R |
8 ± 2 M R |
4 ± 3 M R |
31 ± 2 M R |
59 ± 4 |
|||
5000 µg |
0 ± 0 M R |
0 ± 0 M R |
0 ± 0 M R |
31 ± 4 M R |
61 ± 3 |
|||
2-AA |
2.5 µg |
256 ± 8 |
105 ± 10 |
1616 ± 151 |
1346 ± 157 |
|||
2-AA |
10.0 µg |
334 ± 24 |
||||||
Key to Positive Controls |
Key to Plate Postfix Codes |
||
NaN3 2-AA 4-NOPD MMS |
sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate |
R M |
Reduced background growth Manual count |
* Repeated experiment
Summary Table
| relative | mutant | relative | mutant | |||||
| conc. µg | S9 | total | colonies/ | total | colonies/ | |||
| per mL | mix | growth | 106cells | threshold | growth | 106cells | threshold | |
| Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Experiment I / 4 h treatment | culture I | culture II | ||||||
| Solv. control with water | - | 100.0 | 100 | 226 | 100.0 | 71 | 197 | |
| Pos. control with MMS | 19.5 | - | 40.3 | 292 | 226 | 28.4 | 330 | 197 |
| Test item | 5.9 | - | 116.4 | 109 | 226 | 100.2 | 79 | 197 |
| Test item | 11.8 | - | 102.1 | 88 | 226 | 90.2 | 81 | 197 |
| Test item | 23.5 | - | 114.9 | 101 | 226 | 90.3 | 71 | 197 |
| Test item | 47.0 | - | 94.7 | 89 | 226 | 104.3 | 60 | 197 |
| Test item | 94.0 | - | 22.4 | 111 | 226 | 14.9 | 123 | 197 |
| Test item | 141.0 | - | culture was not continued# | culture was not continued# | ||||
| Solv. control with water | + | 100.0 | 75 | 201 | 100.0 | 115 | 241 | |
| Pos. control with CPA | 3.0 | + | 57.9 | 185 | 201 | 54.0 | 263 | 241 |
| Pos. control with CPA | 4.5 | + | 28.2 | 236 | 201 | 28.0 | 465 | 241 |
| Test item | 11.8 | + | 85.1 | 76 | 201 | 88.5 | 103 | 241 |
| Test item | 23.5 | + | 78.9 | 90 | 201 | 96.3 | 74 | 241 |
| Test item | 47.0 | + | 79.7 | 96 | 201 | 102.9 | 94 | 241 |
| Test item | 94.0 | + | 81.6 | 96 | 201 | 83.7 | 86 | 241 |
| Test item | 188.0 | + | 18.0 | 124 | 201 | 23.3 | 130 | 241 |
| Test item | 282.0 | + | culture was not continued# | culture was not continued# | ||||
| Experiment II / 24 h treatment | culture I | culture II | ||||||
| Solv. control with water | - | 100.0 | 67 | 193 | 100.0 | 129 | 255 | |
| Pos. control with MMS | 13.0 | - | 20.4 | 649 | 193 | 25.9 | 697 | 255 |
| Test item | 11.8 | - | 59.5 | 117 | 193 | 61.8 | 169 | 255 |
| Test item | 23.5 | - | 66.8 | 84 | 193 | 74.8 | 94 | 255 |
| Test item | 47.0 | - | 64.5 | 77 | 193 | 52.2 | 128 | 255 |
| Test item | 94.0 | - | 43.7 | 69 | 193 | 39.1 | 140 | 255 |
| Test item | 188.0 | - | 1.6 | 195 | 193 | 1.6 | 171 | 255 |
| Test item | 282.0 | - | culture was not continued# | culture was not continued# | ||||
| Experiment II / 4 h treatment | culture I | culture II | ||||||
| Solv. control with water | + | 100.0 | 70 | 196 | 100.0 | 98 | 224 | |
| Pos. control with CPA | 3.0 | + | 44.6 | 218 | 196 | 26.4 | 408 | 224 |
| Pos. control with CPA | 4.5 | + | 22.0 | 272 | 196 | 16.4 | 378 | 224 |
| Test item | 8.8 | + | 47.1 | 163 | 196 | 82.9 | 125 | 224 |
| Test item | 17.5 | + | 74.4 | 87 | 196 | 77.8 | 133 | 224 |
| Test item | 35.0 | + | 48.6 | 86 | 196 | 92.9 | 95 | 224 |
| Test item | 70.0 | + | 50.5 | 99 | 196 | 98.6 | 78 | 224 |
| Test item | 105.0 | + | 47.4 | 67 | 196 | 81.7 | 80 | 224 |
| Test item | 140.0 | + | culture was not continued# | culture was not continued# | ||||
threshold = number of mutant colonies per 106cells of each solvent control plus 126
# culture not continued due to exceedingly strong toxic effects
Summary of results of the chromosome aberration study: without metabolic activation |
|||||||
Exp |
Preparation Interval |
Test item concentration [µg/mL] |
Mitotic indices in % of control |
Aberrant cells |
Carrying exchange |
Evaluation based on the Guideline adopted July 29, 2016 |
|
Incl. gaps* |
Excl. gaps* |
||||||
Exposure period 4 hrs without S9 mix |
|||||||
IA |
22 hrs |
Deionized water, 10% (v/v) |
100.0 |
1.5 |
1.5 |
0.0 |
Negative: Precipitation occurred at 99.5 µg/mL, which should be considered as the valid high dose. The dose 304.6 µg/mL is not compliant to the Guideline adopted in 2016. |
|
|
EMS, 770 µg/mL |
51.7 |
8.5 |
8.5S |
0.5 |
|
|
|
56.8 |
82.1 |
0.0 |
0.0 |
0.0 |
|
|
|
99.5P |
70.3 |
0.0 |
0.0 |
0.0 |
|
|
|
304.6P |
61.8 |
1.0 |
1.0 |
0.0 |
|
IB |
22 hrs |
Deionized water, 10% (v/v) |
100.0 |
0.5 |
0.5 |
0.0 |
Negative: Precipitation occurred at 50.0 µg/mL. The doses 200 and 325 µg/mLis not compliant to the Guideline adopted in 2016. |
|
|
EMS, 770 µg/mL |
74.8 |
8.5 |
8.5S |
3.0 |
|
|
|
25.0 |
101.0 |
1.5 |
0.5 |
0.0 |
|
|
|
50.0P |
98.0 |
0.5 |
0.5 |
0.0 |
|
|
|
200.0P |
83.0 |
2.0 |
2.0 |
0.0 |
|
|
|
325.0P |
45.1 |
2.5 |
2.5 |
0.0 |
|
Exposure period 22 hrs without S9 mix |
|||||||
IIA |
22 hrs |
Deionized water, 10% (v/v) |
100.0 |
0.0 |
0.0 |
0.0 |
Negative: No Precipitation observed, but the highest dose induced clear cytotoxicity.
|
|
|
EMS, 660 µg/mL |
38.0 |
21.5 |
20.5S |
9.0 |
|
|
|
30.5 |
96.1 |
1.0 |
1.0 |
0.0 |
|
|
|
53.3 |
82.8 |
1.0 |
1.0 |
0.0 |
|
|
|
93.3 |
62.4 |
1.0 |
1.0 |
0.0 |
|
IIB |
22 hrs |
Deionized water, 10% (v/v) |
100.0 |
2.5 |
2.0 |
0.0 |
Negative: No Precipitation observed, but the highest dose induced clear cytotoxicity.
|
|
|
EMS, 550 µg/mL |
43.1 |
22.0 |
21.5S |
9.0 |
|
|
|
100.0 |
86.3 |
3.0 |
2.0 |
0.0 |
|
|
|
140.0 |
56.5 |
1.5 |
1.5 |
0.0 |
|
|
|
160.0 |
37.8 |
0.5 |
0.5 |
0.0 |
|
* Including cells carrying exchanges P Precipitation occurred at the end of treatment S Aberration frequency statistically significant higher than corresponding control values |
|||||||
Summary of results of the chromosome aberration study: with metabolic activation |
|||||||
Exp |
Preparation Interval |
Test item concentration [µg/mL] |
Mitotic indices in % of control |
Aberrant cells |
Carrying exchange |
Evaluation based on the Guideline adopted July 29, 2016 |
|
Incl. gaps* |
Excl. gaps* |
||||||
Exposure period 4 hrs with S9 mix |
|||||||
IA |
22 hrs |
Deionised water 10.0 % (v/v) |
100.0 |
1.0 |
1.0 |
0.0 |
Negative: Precipitation occurred at 99.5 µg/mL, which should be considered as the valid high dose. The dose 304.6 µg/mL is not compliant to the Guideline adopted in 2016. |
|
|
CPA, 15.0 µg/mL |
79.6 |
12.5 |
12.0S |
1.0 |
|
|
|
56.8 |
100.0 |
0.0 |
0.0 |
0.0 |
|
|
|
99.5P |
98.9 |
0.5 |
0.5 |
0.0 |
|
|
|
304.6P |
88.9 |
1.0 |
1.0 |
0.0 |
|
IIA |
22 hrs |
Deionised water 10.0 % (v/v) |
100.0 |
2.0 |
2.0 |
0.0 |
Negative: Precipitation occurred at 100 µg/mL. The dose 500 µg/mL is not compliant to the Guideline adopted in 2016. |
|
|
CPA, 15.0 µg/mL |
69.9 |
22.5 |
22.0S |
5.5 |
|
|
|
50.0 |
89.3 |
1.0 |
1.0 |
0.0 |
|
|
|
100.0P |
93.2 |
2.0 |
2.0 |
0.0 |
|
|
|
500.0P |
87.4 |
3.5 |
3.5 |
1.0 |
|
IIB |
22 hrs |
Deionized water, 10% (v/v) |
100.0 |
1.5 |
1.5 |
0.0 |
Negative: Precipitation occurred at 100 µg/mL. The dose 520 µg/mL is not compliant to the Guideline adopted in 2016. |
|
|
CPA, 7.5 µg/mL |
78.2 |
9.5 |
8.0S |
2.0 |
|
|
|
50.0 |
72.8 |
2.0 |
1.5 |
0.0 |
|
|
|
100.0P |
87.9 |
1.5 |
1.5 |
0.0 |
|
|
|
520.0P |
56.8 |
8.0 |
8.0S |
2.5 |
|
IIC |
22 hrs |
Deionized water, 10% (v/v) |
100.0 |
0.5 |
0.5 |
0.0 |
Negative: Precipitation occurred at 100 µg/mL, and the aberration cells were statistically increased. Taking account the results obtained in other three independent experiments were negative and the incidence is marginally increased, the overall assessment should be negative. The doses 400 and 440 µg/mL are not compliant to the Guideline adopted in 2016. |
|
|
CPA, 7.5 µg/mL |
54.9 |
19.5 |
19.5S |
6.0 |
|
|
|
50.0 |
91.7 |
2.0 |
1.5 |
0.0 |
|
|
|
100.0P# |
99.5 |
2.8 |
2.5S |
0.3 |
|
|
|
400.0P# |
61.1 |
6.3 |
6.0S |
1.0 |
|
|
|
440.0P# |
37.8 |
9.0 |
8.8S |
1.0 |
|
* Including cells carrying exchanges P Precipitation occurred at the end of treatment S Aberration frequency statistically significant higher than corresponding control values # Evaluation of 200 metaphases per culture |
|||||||
Results of cytotoxicity test |
||||
Concentrations (mg/mL) |
Growth rate in the direct method (%) |
Growth rate in the metabolic activation method (%) |
||
|
Values in duplicates |
Mean |
|
Mean |
0.010 |
94 96 |
95 |
96 101 |
99 |
0.020 |
90 90 |
90 |
102 98 |
100 |
0.039 |
79 73 |
76 |
89 89 |
89 |
0.078 |
72 78 |
74 |
86 83 |
85 |
0.156 |
15 14 |
15 |
89 91 |
90 |
0.313 |
0 0 |
0 |
68 50 |
59 |
0.625 |
0 0 |
0 |
36 35 |
36 |
1.25 |
0 0 |
0 |
0 0 |
0 |
2.5 |
0 0 |
0 |
0 0 |
0 |
5 |
0 0 |
0 |
0 0 |
0 |
Growth rate was calculated by regarding the growth rate in the solvent treatment group as 100%. Approximate IC50: Direct method: 0.105 mg/mL Metabolic activation method: 0.420 mg/mL |
||||
Results of chromosome aberration test (direct method, 24h treatment) |
|||||||||
Concentrations (µg/mL) |
Number of observed cells |
Number of cells with numerical aberrations |
Number of cells with structural aberrations |
||||||
g |
ctb |
cte |
csb |
cse |
TA |
TAG |
|||
Non-treatment control |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Solvent control |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
40 |
200 |
1 |
0 |
1 |
1 |
o |
0 |
2 |
2 |
80 |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
120 |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
160 |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Positive control |
200 |
0 |
0 |
30 |
69 |
0 |
2 |
91 |
91 |
g: chromatid gap or chromosome gap ctb: chromatid break cte: chromatid exchange csb: chromosome break cse: chromosome exchange TA: total number of aberration cells excluding gaps TAG: total number of aberration cells including gaps Solvent control: physiological salne Positive control: MMC (0.05 µg/mL) |
|||||||||
Results of chromosome aberration test (direct method, 48h treatment) |
|||||||||
Concentrations (µg/mL) |
Number of observed cells |
Number of cells with numerical aberrations |
Number of cells with structural aberrations |
||||||
g |
ctb |
cte |
csb |
cse |
TA |
TAG |
|||
Non-treatment control |
200 |
1 |
0 |
1 |
0 |
0 |
0 |
1 |
1 |
Solvent control |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
40 |
200 |
5 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
80 |
200 |
1 |
0 |
2 |
2 |
0 |
0 |
4 |
4 |
120 |
200 |
1 |
0 |
3 |
0 |
0 |
0 |
3 |
3 |
Positive control |
200 |
0 |
1 |
50 |
92 |
0 |
7 |
128 |
128 |
g: chromatid gap or chromosome gap ctb: chromatid break cte: chromatid exchange csb: chromosome break cse: chromosome exchange TA: total number of aberration cells excluding gaps TAG: total number of aberration cells including gaps Solvent control: physiological salne Positive control: MMC (0.05 µg/mL) |
|||||||||
Results of chromosome aberration test (metabolic activation method, without S9 mix) |
|||||||||
Concentrations (µg/mL) |
Number of observed cells |
Number of cells with numerical aberrations |
Number of cells with structural aberrations |
||||||
g |
ctb |
cte |
csb |
cse |
TA |
TAG |
|||
Non-treatment control |
200 |
1 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
Solvent control |
200 |
0 |
0 |
2 |
0 |
0 |
0 |
2 |
2 |
78 |
200 |
2 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
156 |
200 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
313 |
200 |
4 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
625 |
200 |
2 |
0 |
1 |
0 |
0 |
0 |
1 |
1 |
Positive control |
200 |
1 |
1 |
1 |
0 |
0 |
0 |
1 |
2 |
g: chromatid gap or chromosome gap ctb: chromatid break cte: chromatid exchange csb: chromosome break cse: chromosome exchange TA: total number of aberration cells excluding gaps TAG: total number of aberration cells including gaps Solvent control: physiological salne Positive control: N-nitrosodimethylamine (0.4 µg/mL) |
|||||||||
Results of chromosome aberration test (metabolic activation method, with S9 mix) |
|||||||||
Concentrations (µg/mL) |
Number of observed cells |
Number of cells with numerical aberrations |
Number of cells with structural aberrations |
||||||
g |
ctb |
cte |
csb |
cse |
TA |
TAG |
|||
Non-treatment control |
200 |
1 |
1 |
1 |
0 |
0 |
0 |
1 |
2 |
Solvent control |
200 |
3 |
1 |
2 |
0 |
0 |
0 |
2 |
2 |
78 |
200 |
0 |
0 |
2 |
0 |
0 |
0 |
2 |
2 |
156 |
200 |
2 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
313 |
200 |
3 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
625 |
200 |
0 |
0 |
11 |
35 |
0 |
3 |
46 |
46 |
Positive control |
200 |
0 |
1 |
45 |
126 |
0 |
7 |
146 |
147 |
g: chromatid gap or chromosome gap ctb: chromatid break cte: chromatid exchange csb: chromosome break cse: chromosome exchange TA: total number of aberration cells excluding gaps TAG: total number of aberration cells including gaps Solvent control: physiological salne Positive control: N-nitrosodimethylamine (0.4 µg/mL) |
|||||||||
Comments of the registrant: the concentration of 625 µg/mL corresponds to the 35% of cell growth when compared to control and therefore not compliant to the OECD 473 Guideline adopted in 2016. Discarding the result obtained at this concentration, the overall result of the study should be negative. |
|||||||||
Summary Tabellen
Summary of Results Pre-Experiment and Experiment I
Study Name: 1211000 |
Study Code: Harlan CCR 1211000 |
Experiment: 1211000 VV Plate |
Date Plated: 17/09/2008 |
Assay Conditions: |
Date Counted: 24/09/2008 |
Metabolic Activation |
Test Group |
Dose Level (µg/plate) |
Revertant Colony Counts (Mean ±SD) |
|||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
||||
Without Activation |
Deionised water |
20 ± 3 |
11 ± 3 |
35 ± 2 |
137 ± 5 |
55 ± 4 |
||
Untreated |
18 ± 3 |
13 ± 3 |
37 ± 10 |
126 ± 4 |
47 ± 3 |
|||
Hostapon SG |
3 µg |
20 ± 3 |
11 ± 4 |
29 ± 1 |
134 ± 12 |
50 ± 4 |
||
10 µg |
19 ± 4 |
14 ± 2 |
33 ± 5 |
124 ± 8 |
52 ± 6 |
|||
33 µg |
18 ± 3 |
11 ± 4 |
35 ± 6 |
132 ± 10 |
55 ± 1 |
|||
100 µg |
19 ± 4 |
8 ± 0 |
37 ± 6 |
139 ± 16 |
52 ± 4 |
|||
333 µg |
13 ± 2 |
10 ± 4 |
32 ± 8 |
122 ± 15 |
63 ± 3 |
|||
1000 µg |
17 ± 2 |
7 ± 1 |
25 ± 5 |
84 ± 11 R |
62 ± 2 |
|||
2500 µg |
8 ± 2 M R |
2 ± 2 M R |
19 ± 2 M R |
36 ± 5 M R |
59 ± 2 |
|||
5000 µg |
2 ± 2 M R |
0 ± 1 M R |
5 ± 2 M R |
27 ± 3 M R |
58 ± 2 |
|||
NaN3 |
10 µg |
2009 ± 31 |
2369 ± 114 |
|||||
4-NOPD |
10 µg |
424 ± 9 |
||||||
4-NOPD |
50 µg |
83 ± 3 |
||||||
MMS |
3.0 µL |
1017 ± 33 |
||||||
With Activation |
Deionised water |
23 ± 3 |
20 ± 5 |
37 ± 4 |
148 ± 5 |
68 ± 4 |
||
Untreated |
24 ± 6 |
19 ± 7 |
42 ± 8 |
148 ± 14 |
64 ± 9 |
|||
Hostapon SG |
3 µg |
24 ± 3 |
19 ± 5 |
39 ± 5 |
129 ± 14 |
64 ± 4 |
||
10 µg |
21 ± 5 |
17 ± 5 |
44 ± 8 |
135 ± 3 |
71 ± 2 |
|||
33 µg |
25 ± 3 |
19 ± 3 |
43 ± 6 |
132 ± 9 |
60 ± 11 |
|||
100 µg |
23 ± 1 |
15 ± 2 |
38 ± 4 |
134 ± 5 |
60 ± 4 |
|||
333 µg |
22 ± 5 |
13 ± 2 |
47 ± 6 |
136 ± 13 |
73 ± 11 |
|||
1000 µg |
15 ± 4 |
16 ± 3 |
35 ± 3 |
116 ± 13 |
69 ± 3 |
|||
2500 µg |
9 ± 3 M R |
8 ± 1 M R |
28 ± 2 M R |
48 ± 7 M R |
66 ± 1 |
|||
5000 µg |
5 ± 1 M R |
1 ± 1 M R |
13 ± 2 M R |
34 ± 5 M R |
59 ± 4 |
|||
2-AA |
2.5 µg |
245 ± 16 |
191 ± 30 |
1267 ± 70 |
1506 ± 43 |
|||
2-AA |
10.0 µg |
232 ± 24 |
||||||
Key to Positive Controls |
Key to Plate Postfix Codes |
||
NaN3 2-AA 4-NOPD MMS |
sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate |
R M |
Reduced background growth Manual count |
Summary of Results Experiment II
Study Name: 1211000 |
Study Code: Harlan CCR 1211000 |
Experiment: 1211000 HV2 Pre |
Date Plated: 01/10/2008 / 14/10/2008* |
Assay Conditions: |
Date Counted: 08/10/2008 / 17/10/2008* |
Metabolic Activation |
Test Group |
Dose Level (µg/plate) |
Revertant Colony Counts (Mean ±SD) |
|||||
TA 1535 |
TA 1537 |
TA 98* |
TA 100 |
WP2 uvrA |
||||
Without Activation |
Deionised water |
19 ± 6 |
12 ± 4 |
22 ± 2 |
131 ± 5 |
51 ± 8 |
||
Untreated |
16 ± 6 |
10 ± 2 |
24 ± 4 |
137 ± 12 |
46 ± 8 |
|||
Hostapon SG |
3 µg |
17 ± 4 |
14 ± 5 |
23 ± 2 |
131 ± 8 |
55 ± 1 |
||
10 µg |
18 ± 4 |
15 ± 2 |
24 ± 2 |
127 ± 9 |
52 ± 8 |
|||
33 µg |
18 ± 3 |
11 ± 6 |
24 ± 4 |
122 ± 1 |
55 ± 6 |
|||
100 µg |
22 ± 6 |
14 ± 3 |
23 ± 1 |
101 ± 15 R |
56 ± 2 |
|||
333 µg |
12 ± 5 |
10 ± 4 |
15 ± 2 |
75 ± 7 R |
47 ± 4 |
|||
1000 µg |
5 ± 2 R M |
2 ± 3 M R |
10 ± 1 M R |
39 ± 5 M R |
49 ± 5 |
|||
2500 µg |
2 ± 2 M R |
0 ± 0 M R |
3 ± 2 M R |
31 ± 10 M R |
46 ± 5 |
|||
5000 µg |
1 ± 1 M R |
0 ± 0 M R |
0 ± 0 M R |
5 ± 2 M R |
37 ± 7 |
|||
NaN3 |
10 µg |
1799 ± 67 |
1998 ± 31 |
|||||
4-NOPD |
10 µg |
1637 ± 116 |
||||||
4-NOPD |
50 µg |
105 ± 5 |
||||||
MMS |
3.0 µL |
418 ± 20 |
||||||
With Activation |
Deionised water |
17 ± 4 |
16 ± 5 |
26 ± 3 |
138 ± 9 |
58 ± 5 |
||
Untreated |
18 ± 4 |
19 ± 7 |
29 ± 3 |
142 ± 37 |
64 ± 7 |
|||
Hostapon SG |
3 µg |
19 ± 4 |
17 ± 4 |
27 ± 0 |
131 ± 11 |
59 ± 3 |
||
10 µg |
22 ± 4 |
17 ± 4 |
25 ± 2 |
132 ± 8 |
58 ± 3 |
|||
33 µg |
20 ± 2 |
19 ± 3 |
24 ± 2 |
141 ± 15 |
59 ± 6 |
|||
100 µg |
21 ± 1 |
15 ± 3 |
22 ± 5 |
140 ± 7 |
57 ± 5 |
|||
333 µg |
18 ± 4 |
14 ± 4 |
19 ± 3 |
110 ± 5 R |
53 ± 2 |
|||
1000 µg |
11 ± 3 M R |
12 ± 2 M R |
10 ± 2 M R |
74 ± 7 R |
54 ± 1 |
|||
2500 µg |
9 ± 3 M R |
8 ± 2 M R |
4 ± 3 M R |
31 ± 2 M R |
59 ± 4 |
|||
5000 µg |
0 ± 0 M R |
0 ± 0 M R |
0 ± 0 M R |
31 ± 4 M R |
61 ± 3 |
|||
2-AA |
2.5 µg |
256 ± 8 |
105 ± 10 |
1616 ± 151 |
1346 ± 157 |
|||
2-AA |
10.0 µg |
334 ± 24 |
||||||
Key to Positive Controls |
Key to Plate Postfix Codes |
||
NaN3 2-AA 4-NOPD MMS |
sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate |
R M |
Reduced background growth Manual count |
* Repeated experiment
Summary Table
| relative | mutant | relative | mutant | |||||
| conc. µg | S9 | total | colonies/ | total | colonies/ | |||
| per mL | mix | growth | 106cells | threshold | growth | 106cells | threshold | |
| Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Experiment I / 4 h treatment | culture I | culture II | ||||||
| Solv. control with water | - | 100.0 | 100 | 226 | 100.0 | 71 | 197 | |
| Pos. control with MMS | 19.5 | - | 40.3 | 292 | 226 | 28.4 | 330 | 197 |
| Test item | 5.9 | - | 116.4 | 109 | 226 | 100.2 | 79 | 197 |
| Test item | 11.8 | - | 102.1 | 88 | 226 | 90.2 | 81 | 197 |
| Test item | 23.5 | - | 114.9 | 101 | 226 | 90.3 | 71 | 197 |
| Test item | 47.0 | - | 94.7 | 89 | 226 | 104.3 | 60 | 197 |
| Test item | 94.0 | - | 22.4 | 111 | 226 | 14.9 | 123 | 197 |
| Test item | 141.0 | - | culture was not continued# | culture was not continued# | ||||
| Solv. control with water | + | 100.0 | 75 | 201 | 100.0 | 115 | 241 | |
| Pos. control with CPA | 3.0 | + | 57.9 | 185 | 201 | 54.0 | 263 | 241 |
| Pos. control with CPA | 4.5 | + | 28.2 | 236 | 201 | 28.0 | 465 | 241 |
| Test item | 11.8 | + | 85.1 | 76 | 201 | 88.5 | 103 | 241 |
| Test item | 23.5 | + | 78.9 | 90 | 201 | 96.3 | 74 | 241 |
| Test item | 47.0 | + | 79.7 | 96 | 201 | 102.9 | 94 | 241 |
| Test item | 94.0 | + | 81.6 | 96 | 201 | 83.7 | 86 | 241 |
| Test item | 188.0 | + | 18.0 | 124 | 201 | 23.3 | 130 | 241 |
| Test item | 282.0 | + | culture was not continued# | culture was not continued# | ||||
| Experiment II / 24 h treatment | culture I | culture II | ||||||
| Solv. control with water | - | 100.0 | 67 | 193 | 100.0 | 129 | 255 | |
| Pos. control with MMS | 13.0 | - | 20.4 | 649 | 193 | 25.9 | 697 | 255 |
| Test item | 11.8 | - | 59.5 | 117 | 193 | 61.8 | 169 | 255 |
| Test item | 23.5 | - | 66.8 | 84 | 193 | 74.8 | 94 | 255 |
| Test item | 47.0 | - | 64.5 | 77 | 193 | 52.2 | 128 | 255 |
| Test item | 94.0 | - | 43.7 | 69 | 193 | 39.1 | 140 | 255 |
| Test item | 188.0 | - | 1.6 | 195 | 193 | 1.6 | 171 | 255 |
| Test item | 282.0 | - | culture was not continued# | culture was not continued# | ||||
| Experiment II / 4 h treatment | culture I | culture II | ||||||
| Solv. control with water | + | 100.0 | 70 | 196 | 100.0 | 98 | 224 | |
| Pos. control with CPA | 3.0 | + | 44.6 | 218 | 196 | 26.4 | 408 | 224 |
| Pos. control with CPA | 4.5 | + | 22.0 | 272 | 196 | 16.4 | 378 | 224 |
| Test item | 8.8 | + | 47.1 | 163 | 196 | 82.9 | 125 | 224 |
| Test item | 17.5 | + | 74.4 | 87 | 196 | 77.8 | 133 | 224 |
| Test item | 35.0 | + | 48.6 | 86 | 196 | 92.9 | 95 | 224 |
| Test item | 70.0 | + | 50.5 | 99 | 196 | 98.6 | 78 | 224 |
| Test item | 105.0 | + | 47.4 | 67 | 196 | 81.7 | 80 | 224 |
| Test item | 140.0 | + | culture was not continued# | culture was not continued# | ||||
threshold = number of mutant colonies per 106cells of each solvent control plus 126
# culture not continued due to exceedingly strong toxic effects
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
The registration substance is not mutagenic under in-vivo condition based on the read-across to Hostapon SG:
Hostapon SG is not mutagenic in in-vivo micronucleus test.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1998
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- micronucleus assay
- Specific details on test material used for the study:
- The test material is equivalent to the commercial product of the registration substance.
- Species:
- mouse
- Strain:
- ICR
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 33.2 - 40.6 g
- Assigned to test groups randomly: yes
- Housing: aluminium box-type cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26°C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10 - 15 cycles per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / dark cycle - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: physiological saline
- Justification for choice of solvent/vehicle: recommended vehicle
- Concentration of test material in vehicle: 0.5, 1, 2 and 4 % (w/v) - Duration of treatment / exposure:
- 24 hours
- Frequency of treatment:
- two intraperitoneal injections within a 24 hour interval
- Post exposure period:
- no post exposure period
- Dose / conc.:
- 400 mg/kg bw/day
- Dose / conc.:
- 200 mg/kg bw/day
- Dose / conc.:
- 100 mg/kg bw/day
- Dose / conc.:
- 50 mg/kg bw/day
- No. of animals per sex per dose:
- 6
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Mitomycin C
- Tissues and cell types examined:
- bone marrow from right femur
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
Doses were selected on the basis of a preliminary dose-range finder.
TREATMENT AND SAMPLING TIMES:
Treatment period was 24 hours. The test solutions were administered intraperitoneally, twice within this 24 hour time interval.
DETAILS OF SLIDE PREPARATION:
METHOD OF ANALYSIS:
OTHER: - Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000, 2000 mg/kg body weight
- Solubility: yes
- Clinical signs of toxicity in test animals: yes
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction of micronuclei
- Appropriateness of dose levels and route: dose levels based on pre-test, intraperitoneal administration is a recommended route
- Statistical evaluation: yes - Conclusions:
- The in-vivo clastogenicity of Hostapon SG was investigated in micronucleus study in bone marrow in mice. No significant clastogenicity was found.
- Executive summary:
The in-vivo clastogenicity of Hostapon SG was investigated in micronucleus study in bone marrow in mice. Male mice were treated intraperitoneally at concentrations of 400, 200, 100 and 50 mg/kg bw, twice with a 24 hours interval. One death was observed in the 200 mg/kg body weight group (1/6 cases), and four deaths were observed in the 400 mg/kg body weight group (4/6 cases). The surviving animals were killed at 24 hours after the final treatment and the bone-marrow smears were prepared. The proportion of polychromatic erythrocytes (PCE) to total erythrocytes was lower in the 100 and 200 mg/kg bw groups, indicating toxic effect in the bone marrow tissue. The percentage of micronucleated polychromatic ethythrocytes (MNPCE) in the treated group was not significantly higher than that in the negative control group.
Hostapon SG is not clastogenic in in-vivo test system.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 1998
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Justification for type of information:
- Justification is provided in Chapter 13.
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- micronucleus assay
- Specific details on test material used for the study:
- The test material is equivalent to the commercial product of the registration substance.
- Species:
- mouse
- Strain:
- ICR
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 33.2 - 40.6 g
- Assigned to test groups randomly: yes
- Housing: aluminium box-type cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26°C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10 - 15 cycles per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / dark cycle - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: physiological saline
- Justification for choice of solvent/vehicle: recommended vehicle
- Concentration of test material in vehicle: 0.5, 1, 2 and 4 % (w/v) - Duration of treatment / exposure:
- 24 hours
- Frequency of treatment:
- two intraperitoneal injections within a 24 hour interval
- Post exposure period:
- no post exposure period
- Dose / conc.:
- 400 mg/kg bw/day
- Dose / conc.:
- 200 mg/kg bw/day
- Dose / conc.:
- 100 mg/kg bw/day
- Dose / conc.:
- 50 mg/kg bw/day
- No. of animals per sex per dose:
- 6
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Mitomycin C
- Tissues and cell types examined:
- bone marrow from right femur
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
Doses were selected on the basis of a preliminary dose-range finder.
TREATMENT AND SAMPLING TIMES:
Treatment period was 24 hours. The test solutions were administered intraperitoneally, twice within this 24 hour time interval.
DETAILS OF SLIDE PREPARATION:
METHOD OF ANALYSIS:
OTHER: - Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000, 2000 mg/kg body weight
- Solubility: yes
- Clinical signs of toxicity in test animals: yes
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction of micronuclei
- Appropriateness of dose levels and route: dose levels based on pre-test, intraperitoneal administration is a recommended route
- Statistical evaluation: yes - Conclusions:
- The in-vivo genotoxicity of the registration substance Hostapon SLG is derived based on the read-across to Hostapon SG. Hostapon SG was found to be not clastogenic in micronucleus study in bone marrow in mice. No significant in-vivo clastogenicity can be derived for the registration substance.
- Executive summary:
The in-vivo genotoxicity of the registration substance Hostapon SLG is derived based on the read-across to Hostapon SG.
The in-vivo clastogenicity of Hostapon SG was investigated in micronucleus study in bone marrow in mice. Male mice were treated intraperitoneally at concentrations of 400, 200, 100 and 50 mg/kg bw, twice with a 24 hours interval. One death was observed in the 200 mg/kg body weight group (1/6 cases), and four deaths were observed in the 400 mg/kg body weight group (4/6 cases). The surviving animals were killed at 24 hours after the final treatment and the bone-marrow smears were prepared. The proportion of polychromatic erythrocytes (PCE) to total erythrocytes was lower in the 100 and 200 mg/kg bw groups, indicating toxic effect in the bone marrow tissue. The percentage of micronucleated polychromatic ethythrocytes (MNPCE) in the treated group was not significantly higher than that in the negative control group.
Hostapon SG is not clastogenic in in-vivo test system.
Likewise, the registration substance, Hostapon SLG is considered as of no significant clstogenic activity.
Referenceopen allclose all
Results of micronucleus test in male CD-1 mice after introperitoneal administration of sodium N-cocoyl glycinate |
|||
Concentrations (mg/mL) |
No. of animals survived / No, of animals treated |
Frequency of MNPCE (%) |
PCE/RBC |
Solvent control |
6/6 |
0.07 ± 0.08 |
58.8 ± 2.68 |
50 x 2 |
6/6 |
0.18 ± 0.10 |
59.5 ± 7.45 |
100 x 2 |
6/6 |
0.07 ± 0.10 |
** 48.8 ± 5.83 |
200 x 2 |
5/6 |
0.08 ± 0.11 |
** 36.8 ± 4.61 |
400 x 2 |
2/6 |
0.10 |
* 50.6 ± 2.69 |
Positive control (MMC) |
6/6 |
4.77 ± 1.02 |
* 50.6 ± 2.69 |
PCE: polychromatic erythrocytes RBC: total erythrocytes MNPCE: micronucleated PCE MMC: mitomycin C * significantly different from solvent control (P< 0.05) ** significantly different from solvent control (P < 0.01) |
|||
Results of micronucleus test in male CD-1 mice after introperitoneal administration of sodium N-cocoyl glycinate |
|||
Concentrations (mg/mL) |
No. of animals survived / No, of animals treated |
Frequency of MNPCE (%) |
PCE/RBC |
Solvent control |
6/6 |
0.07 ± 0.08 |
58.8 ± 2.68 |
50 x 2 |
6/6 |
0.18 ± 0.10 |
59.5 ± 7.45 |
100 x 2 |
6/6 |
0.07 ± 0.10 |
** 48.8 ± 5.83 |
200 x 2 |
5/6 |
0.08 ± 0.11 |
** 36.8 ± 4.61 |
400 x 2 |
2/6 |
0.10 |
* 50.6 ± 2.69 |
Positive control (MMC) |
6/6 |
4.77 ± 1.02 |
* 50.6 ± 2.69 |
PCE: polychromatic erythrocytes RBC: total erythrocytes MNPCE: micronucleated PCE MMC: mitomycin C * significantly different from solvent control (P< 0.05) ** significantly different from solvent control (P < 0.01) |
|||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
No classification is assigned to the registration substance based on the read-across to Hostapon SG.
For Hostapon SG, negative results were obtained in five in-vitro studies (bacterial reverse mutation assay, mouse lymphoma assay, chromosome aberration test) and in one vivo study (micronucleust test).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
