Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: Two Ames tests were performed with the test substance and were used in weight of evidence. The studies were performed according to a method similar to OECD Guideline 471 in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA (Kato, 1993) and S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA1538 (Godek, 1983). According to the results of these studies, the test substance is not mutagenic in the Ames test with and without metabolic activation.

DNA damage and/or repair test in mammalian cells: this test was performed according to a method equivalent to OECD guideline 482 in a rat hepatocyte primary culture of adult male F344 rats. The results of the hepatocyte primary culture/DNA Repair Assay indicated that, under the conditions of this study, the test substance was not genotoxic.

Gene mutation test in mammalian: The study was performed according to OECD Guideline 476 and ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests of Pharmaceuticals. The results of the CHO/HGPRT Mutation Assay indicated that, under the conditions of this study, the test substance was concluded to be negative. In vitro chromosome aberration test: The study was performed according to OECD Guideline 473 and GLP principles. Under the conditions of the in vitro chromosome aberration assay using CHO, the test substance was concluded to be negative for the induction of structural and numerical chromosome aberrations in both the non-activated and S9- activated test systems.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20 April 1993 - 12 May 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Well-documented GLP study performed according to a method similar to OECD TG 471 without deviations.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1-(bis(3-(dimethylamino)-propyl)amino)-2-propanol
- Substance type: Organic
- Physical state: Liquid at ambient temperature
- Analytical purity: more than 99%
- Lot/batch No.: 184-1-0284
- Stability under test conditions: stable
- Other: Solubility: soluble in water and acetone more than 5%
Target gene:
Histidine locus (S. typhimurium)
Tryptophan locus (E. coli)
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:
Rat liver homogenate S9, treated with phenobarbital and 5,6-benzoflaone to induce drug metabolic enzymes
Test concentrations with justification for top dose:
156, 313, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9 mix for TA1535; dissolved in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
Remarks:
Without S9 mix for TA100, WP2 uvrA, TA98; dissolved in dimethylsulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix for TA1537; dissolved in dimethylsulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix for TA100; dissolved in dimethylsulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9 mix; dissolved in dimethylsulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix; dissolved in dimethylsulfoxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 0.1 mL of test substance was placed in glass tube. Then 0.1 mL of the sub-culture, and 0.5 mL of S9 mix or 0.5 mL of 0.1 M sodium phosphate buffer pH 7.4 was added to the test substance and incubated at 37°C for 20 minutes. The suspension was then thoroughly mixed with 2.0 mL of the top agar and overlaid onto the minimum glucose agar plate. After 48 hours' incubation at 37°C, revertant colonies were counted.

NUMBER OF REPLICATIONS: Test was taken on the two plates per concentration.

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in number of revertant colonies per plate
Evaluation criteria:
The numbers of revertant colonies of all test substance groups were compared with those obtained from both negative control groups. The test substance was considered as positive if a reproducibility produced, a dose related and more than 2 fold increase in the number of revertant colonies occurred at the negative control value.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed at 2500 µg/plate and at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The dose levels of the reverse mutation test were determined from the results of the dose finding test with 6 doses. The test substance showed toxicity at dose of 5000 µg/plate, although, increases in the number of revertant colonies were not observed in any of bacterial strains used. From the above results, reverse mutation test was conducted at 6 doses (156, 313, 625, 1250, 2500, 5000 µg/plate).
Remarks on result:
other: all strains/cell types tested
Conclusions:
It is concluded therefore that the test substance is considered as negative for mutagenic potential with and without metabolic activation under these test conditions employed.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24-08-1983 to 02-09-1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Test article 5601-23-1
- Substance type: Clear light yellow liquid
- Physical state: Liquid
- Stability under test conditions: There was no change in the physical state of the test or control articles during the assay.
Target gene:
histidine locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction of rat liver homogenate obtained from Aroclor 1254-treated Sprague-Dawley rats
Test concentrations with justification for top dose:
100, 333, 1000, 3333, 10000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation, TA1535 and TA100 (10 µg/mL)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation, TA1537 (1500 µg/mL)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation, TA1538 and TA98 (50 µg/mL)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
With metabolic activation (50 µg/mL) in all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in duplicate.

DETERMINATION OF CYTOTOXICITY
- Method: The background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored as by the presence of pindot colonies and the absence of a confluent lawn of bacteria.
Evaluation criteria:
A positive result is defined in a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeate assay. If the solvent control is within one standard deviation of the historical mean for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Strain TA100 showed slight inhibition of bacterial growth, as evidenced by a dense pindot colony lawn, at the 10000 µg/plate level
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Strain TA1538 showed slight inhibition of bacterial growth, as evidenced by a dense pindot colony lawn, at the 10000 µg/plate level
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The test article 5601-23-1 produced slight inhibition of bacterial growth, as evidenced by very dense pindot colony lawn formation in strains TA1538 and TA100 of Salmonella typhimurium, at 10000 µg/plate. The high dose chosen for the plate assay was 10000 µg/plate. Additional doses were 100, 333, 1000, and 3333 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: All solvent and positive controls used in the evaluation of the test article were within the acceptable range of mean historical data.

Remarks on result:
other: all strains/cell types tested
Conclusions:
The results for the test substance were negative in strains TA 1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at doses of 100, 333, 1000, 3333 and 10000 µg/plate.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May 2102 - 19 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests of Pharmaceuticals
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Jeffcat ZR-50
- Substance type: Amber liquid
- Physical state: Liquid
- Analytical purity: 100%
- Lot/batch No.: PFW100119
- Storage condition of test material: Room temperature, stored protected from light
Target gene:
HGPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
CHO cells were cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT).
F12FBS5+Hx medium (seeding and treatment, cultering after treatment)
F12FBS5-Hx medium is Ham's F12 medium without hypoxanthine supplemented with 5% dialyzed FBS, 100 units penicillin/mL, 100 µg streptomycin/mL and 2 mM L-glutamine/mL.
- Properly maintained: yes
- Cells used in each mutation assay were within four subpassages from cleansing in order to assure karyotypic stability
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity assay: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 2000 µg/mL
Initial concurrent cytotoxicity test: 250, 500, 1000, 1500, 1750 and 2250 µg/mL (non-activated cultures) and 250, 500, 1000, 1500 and 2500 µg/mL (S9-activated cultures)
Mutation assay: 250, 500, 1000, 1500 and 2500 µg/mL (activated study)
Repeat concurrent cytotoxicity test: 500, 750, 1000, 1250, 1500, 2000 and 2250 µg/mL (non-activated cultures)
Mutation assay: 500, 750, 1000, 1250, 1500 and 2000 µg/mL (non-activated study)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
4 µg/mL in S9 activated cultures
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
0.2 µg/mL in non-activated cultures
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 2 days (7 to 9)
- Selection time (if incubation with a selection agent): 3 days incubation
- Fixation time (start of exposure up to fixation or harvest of cells): no data

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 2 x 1E06 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
- The test article was considered to induce a positive response if there was a concentration-related increase in mutant frequencies with at least two consecutive concentrations showing mutant frequencies of > 40 mutants per 1E06 clonable cells
- If a single point above 40 mutants per 1E06 clonable cells was observed at the highest concentration, the test article was considered equivocal
- If no culture exhibited a mutant frequency of > 40 mutants per 1E06 mutants per 1E06 clonable cells, the test article was considered negative.
Species / strain:
Chinese hamster Ovary (CHO)
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
- Effects of pH: In order to reach pH 7, the pH of the five to eight highest concentrations was adjusted, using 1N HCl prior to adding S9 or target cells to the treatment medium.
- Effects of osmolality: The osmolality of the solvent control was 280 mmmol/kg and the osmolality of the top concentration, 2500 µg/mL, was 300 mmol/kg (preliminary toxicity assay).
- Precipitation: In the preliminary toxicity assay, no visible precipitate was observed in the treatment medium at the beginning or end of treatment.
- Water solubility: The test article formed a clear solution in water at approximately 25 mg/mL in the solubility test.

RANGE-FINDING/SCREENING STUDIES:
Cloning efficiency relative to the solvent controls (relative cloning efficiency) at 2500 µg/mL was 0% without activation and 100% with S9 activation. Based on the results of the toxicity test, the concentrations chosen for the initial mutagenesis assay ranged from 250 to 2250 µg/mL for the non-activated cultures and from 250 to 2500 µg/mL for the S9-activated cultures.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the solvent and positive controls are situated within the ranges of the historical control data (data from 2009-2011).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the initial mutagenesis assay, no visible precipitate was observed in the treatment medium at the beginning or end of treatment. Relative cloning efficiency was 60% at a concentration of 1500 µg/mL and < 10% at concentrations >= 1750 µg/mL in the non-activated system and 92% at the high concentration in the S9-activated system. None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 1E06 clonable cells. The non-activated portion failed due to a lack of cultures with between 10 and 20% relative cloning efficiency. The mutagenesis assay was repeated in the absence of S9 activation using concentrations from 500 to 1750 µg/mL.

The second and third trials of the mutagenesis assay in the absence of S9 activation failed due to the presence of contamination in the cultures. Based on the toxicity results from the first and second trial of the mutagenesis assay, the concentrations chosen for the third and fourth trials of the mutagenesis assay in the absence of S9 activation ranged from 500 to 2500 µg/mL.

Fourth trial: no visible precipitate was observed in the treatment medium at the beginning or end of treatment. Cultures treated with concentrations of 500, 750, 1000, 1250, 1500, 2000 and 2250 µg/mL were cloned for concurrent cytotoxicity. Relative cloning efficiency was 54% at a concentration of 1500 µg/mL and < 10% at concentrations >= 2000 µg/mL in the non-activated system. None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 1E06 clonable cells.
Remarks on result:
other: all strains/cell types tested
Conclusions:
All criteria for a valid study were met. The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, the test substance was concluded to be negative with or without metabolic activation.
Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
18-10-1983 - 18-01-1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
no
GLP compliance:
not specified
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Specific details on test material used for the study:
- Name of test material (as cited in study report): 5601-23-1
Species / strain / cell type:
hepatocytes: Freshly prepared rat hepatocyte primary cell cultures from adult male F344 rats are used as the target mammalian cell
Details on mammalian cell type (if applicable):
- Type and identity of media: Williams medium E supplemented with 10% calf serum and 50 µg/mL gentamycin (WMES)
- Properly maintained: yes
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
1, E-01, E-02, E-03, E-04, E-05 %v/v
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 1% DMSO, E-04 M pyrene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18 to 20 hours in presence of the test chemical in [3H]-TdR-WME. Each coverslip is removed from its well and rinsed in three succcessive 100 mL washes of WME. Coverslips are then immersed with the cell surface up in 2 mL of 1% sodium citrate (Fisher) in a clean 6 well dish for 10 min to allow the nuclei to swell, which permits better quantification of nuclear grains.
- Fixation time (start of exposure up to fixation or harvest of cells): The cells are fixed in three 30 minute changes of ethanol-glacial acetic acid (3:1), air dried and mounted cell surface up on glass slides with Permount (Fisher). Slides are placed in slide grip holders (Peel-a-way Scientific) and dipped, in total darkness, into NTB-2 emulsion, prewarmed for 1 hr at 45°C. Slides are dried overnight by suspending the slides, in their holders, from a rack in a light-tight box. Slides are placed in cardboard slide boxes which are wrapped in foil and stored at 4°C. After 7 days, autoradiographs are developed in D19 (Eastman Kodak) for 4 min, placed in a stop bath of acidified tap water for 30 seconds, immersed in fixer (Eastman Kodak) for 10 min, and washed in running tap water for 5 min.

Slides are stained in Harris' alum hematoxylin and counterstained with eosin. The cells are then dehydrated through 100% ethanol, air dried and coverslipped with Permount.

Results of the HPC/DNA repair test are quantified by determining the net increase in nuclear grains induced by a test chemical. An Artek Model 880 electronic counter with microscopic attachment is used for grain counting. This counter is able to make counts in either a grain mode or an area mode. The area mode is used because it allows aggregates to be distinguished as discrete grains.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: Only those cells which were viable at the time of fixation, indicated by swollen nuclei (as a result of incubation in the hypotonic sodium citrate), and those evenly coated with emulsion are scored. Between 5 to 20 cells randomly selected from each quadrant of the coverslip are counted. The numbers of cells to be scored depends on the nuclear/cyto-plasmic ratio obtained. Counts are obtained for each nucleus; background is determined by counting 3 nuclear sized areas adjacent to the nucleus. Net nuclear grain counts are calculated by subtracting the highest cytoplasmic count from the nuclear count.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of the test compound is identified by the absence of S phase cells in the autoradiograph and by general morphology.
Evaluation criteria:
The compound is reported negative in this assay if the net nuclear count is less than 5 at the highest non-toxic dose.
Species / strain:
hepatocytes: Freshly prepared rat hepatocyte primary cell cultures from adult male F344 rats are used as the target mammalian cell
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed in the slides exposed to the highest concentration of the compound assayed (1%)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
As the mean net nuclear grain counts of the slides exposed at E-01% concentration and at lower concentrations did not exceed 5, it was concluded that compound 5601-23-1 was not genotoxic to the hepatocytes in this HPC/DNA repair assay. Parallel run positive and negative controls gave the expected values. This concluded the HPC/DNA repair assay of compound 5601-23-1.
Remarks on result:
other: all strains/cell types tested
Conclusions:
In summary, the test substance, when assayed at the highest non-toxic concentration of E-01% (v/v) and at the lower concentrations, was not genotoxic to the hepatocytes in the HPC/DNA repair assay.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-05-12 - 2014-06-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Name of test material (as cited in study report): JEFFCAT ZR-50
- Physical state: Clear yellow liquid
- Analytical purity: Approximately 100%
- Purity test date: 04/11/2013
- Lot/batch No.: 2G508
- Expiration date of the lot/batch: considered stable through 10 July 2014
- Storage condition of test material: Storage at room temperature, protected from light
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO-K1 cells (repository number CCL 61), obtained from American Type Culture Collection, Manassas, VA.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity testing: 0.25, 0.75, 2.5, 7.5, 25, 75, 250, 750 and 2500 µg/mL
Chromosome Aberration test, with and without activation, 4 or 20 h treatment time: 100, 300, 1000, 2500 µg/mL
Vehicle / solvent:
Water was used based on the solubility of the test article and compatibility with the target cells.
The test article was soluble in water at concentration of approximately 25 mg/mL, the maximum concentration tested for solubility.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9: 0.1 and 0.2 µg/mL for 4 h exposure and 0.1, and 0.2 µg/mL for 20 h exposure
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9, 2.5, 5 and 7.5 µg/mL for 4h exposure
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preparation of target cells: Incubation at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 for 16-24h.

- Exposure duration: 4h in the S9-non-activated and S9-activated test system or 20h in the S9-non-activated test system

- Expression time (cells in growth medium): 16h for the 4h-exposure in the S9-non-activated test system and S9-activated test system and no expression time for the 20h-exposition in the S9-non-activated test system

- Fixation step (start of exposure up to fixation or harvest of cells): After the four hour treatment period in the non-activated and the S9-activated studies, the treatment medium was aspirated, the cells washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium and returned to the incubator under standard conditions. Two hours prior to cell harvest, Colcemid® was added to all cultures at a final concentration of 0.1 µg/mL. Cells were collected 20 hours (+/- 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analysed in the first division metaphase. Just prior to harvest, the cell cultures were visually inspected for the degree of monolayer confluency relative to the vehicle control. The cells were trypsinised and counted and the cell viability was assessed using trypan blue dye exclusion.
Cells were then collected by centrifugation, treated with 0.075 M KCl, washed with fixative (methanol: glacial acetic acid, 3:1 v/v), capped and stored overnight or longer at 2 or 8°C. To prepare slides, the cells were collected by centrifugation and resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with Giemsa, permanently mounted and identified by the Bioreliance study number, dose level, treatment condition, harvest date, activation system, test phase and replicate tube design.


NUMBER OF REPLICATIONS:
The cell count was determined for a minimum of two cultures to determine the number of cells being treated (baseline). The data were presented as cell growth inhibition in the treatment group compared to vehicle control. Cell growth was determined by Relative Increase in Cell Counts (RICC) as a measure of cytotoxicity (Fellows and O'Donovan 2007; Lorge et al., 2008). The cell counts and percent viability were used to determine cell growth inhibition relative to the control vehicle (%cytotoxicity). The cell growth in the treatment froup relative to the vehicle control was calculated based on the following formula:
RICC (%) = 100 ( ( mean viable cells trated - mean viable cells baseline) / (mean viable cells solvent - mean viable cells beaseline) ) x 100

EVALUATION OF CELLS:
To ensure that a sufficient number of metaphase cells are present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each coded treatment group selected for scoring chromosome aberration. Metaphase cells with 20 +/- 2 centromers were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads from each dose level (100 per duplicate culture) were examined and scored for chromatide-type and chromosome-type aberrations (Scott et al., 1990). The number of metaphase spreads that were examined and scored per duplicate culture may be reduced if the percentage of aberrant cells reaches a significant level (at least 10% determined based on historical positive control data) before 100 cells are scored. Chromatide-type aberrations include chromatid isochromatid breaks and exchange figures such as quadriradials (symmetrical and assymetrical interexchanges), triradials, and complex rearrangements. Chromosom-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence if any exchange figure were scored as a break (chromatid or chromosom). Fragments observed with an exchange figure were not scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severly damage cells (counted as 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY vernier for each cell with a structural aberration was recorded. The percentage of cells with numerical aberrations (polyploid and endoreplicated cells) was evaluated for 100 cells per culture (200 cells per dose level).

Evaluation criteria:
The test article was considered to have induced a positive response if it induced a statistically significant and dose-dependant increase of the frequency of aberrant metaphases (p≤ 0.05)). If only Fischer's exact test was statistically significant without dose-dependant increase, the result may be considered equivocal. If neither criterion was met, the results were considered to be negative.
Statistics:
Statistical analysis of the percentage of aberrant cells was performed using the Fischer's exact test. The Fischer's exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Assay:
The test article was soluble in water and in the treatment medium at all dose levels tested at the behinning and conclusion of the treatment period.
The osmolality in treatment medium of the highest dose level tested, 2500 µg/mL, was 284 mmol/kg. The osmolality of the solvent (water) in the treatment medium was 260 mmol/kg. The osmolality of the test article dose level in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%. The pH of the highest dose level of test article in treatment medium was 10.0. In order to maintain neutral pH, the dosing solutions in the medium were adjusted using 1N HCl.
Substantial toxicity (i.e. at least 50% cell growth inhibition, relative to the vehicle control) was not observed at any dose level in all three exposure groups.

Chromosome Aberration Assay:
In the chromosome aberration assay, the test article was soluble in water and in the treatment medium at all dose levels tested at the beginning and conclusion of the treatment period.
The results are reported in "Any other information on results incl. tables".

Controls validation:
The results for the positive and negative controls indicate that all criteria for a valid assay were met. Based on these criteria, the results are justified and do not require a repeat of any portions of the study.

Results:
- Toxicity of JEFFCAT ZR-50 (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the absence of S9 activation was 25% at 2500 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 2500 μg/mL, was 34% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 300, 1000, and 2500 μg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) treatment group (19.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
- Toxicity of JEFFCAT ZR-50 (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the presence of S9 activation was 55% at 2500 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 2500 μg/mL, was 13% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 300, 1000, and 2500 μg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) treatment group (24.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
- Toxicity of JEFFCAT ZR-50 (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 20 hours in the absence of S9 activation was 24% at 2500 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 2500 μg/mL, was 51% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 300, 1000, and 2500 μg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) treatment group (25.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
Remarks on result:
other: all strains/cell types tested

 Treatment (µg/mL)    S9 activation      Treatment time (h)      Mean mitotic index    Cells scored     Aberrations per cell   Cells with  
 Numerical Structural   mean  +/- SD  Numerical (%) Structural (%) 
 Water  -S9 15.8  200  200  0.005   ± 0.071  2.0  0.5
 JEFFCAT ZR-50                  
 300 -S9  4  15.8 200 200   0.010  ±0.100  0.5  1.0
 1000  -S9  4  11.2  200  200  0.000  ±0.000  2.0  0.0
 2500  -S9  4  10.5  200  200  0.015  ±0.158  0.0  1.0
 MMC 0.2  -S9  4  9.1  200  100  0.210  ±0.456  1.5  19.0*
                 
 Water  +S9  4  15.1  200  200  0.015  ±0.122  0.5  1.5
 JEFFCAT ZR-50  +S9  4              
 300  +S9  4  13.1  200 200   0.000  ± 0.000  2.0 0.0 
 1000  +S9  4  12.8 200   200  0.000   ± 0.000 1.0   0.0
 2500  +S9  4  13.1  200  200  0.005   ± 0.071  0.5 0.5 
 CP 2.5  +S9  4  6.7  200  100  0.350   ± 0.716  1.5  24.0*
                   
 Water  -S9 20  15.3  200  200  0.000    ± 0.000  1.5 0.0 
JEFFCAT ZR-50                  
 300 -S9   20  11.8  200  200  0.000  ± 0.000  0.0  0.0
 1000  -S9  20  10.5  200  200  0.000   ±0.000  0.5 0.0 
 2500  -S9  20  7.5  200  200  0.000   ±0.000  0.0  0.0
 MMC 0.1  -S9  20  5.5  200  100  0.290   ±0.537  1.0  25.0*

*p ≤ 0.05

Conclusions:
Under the conditions of this assay, the test substance was concluded to be negative for the induction of structural aberrations in both the non-activated and the S9-activated test systems in the in vitro chromosome aberration assay using CHO.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay:

Two Ames tests (plate incorporation assay) were performed with the test substanceand were used in weight of evidence. The studies were performed according to a method similar to OECD Guideline 471 in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA (Kato, 1993) and S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA1538 (Godek, 1983) with and without metabolic activation.

In the study of Kato (1993), following test concentrations were applied in duplicate: 156, 313, 625, 1250, 2500 and 5000 µg/plate.

Solvent and positive controls were run in parallel and were considered to be valid. According to the results of the study, the substance is not mutagenic in the Ames test with and without metabolic activation.

In the study of Godek (1983), following test concentrations were applied in duplicate: 100, 333, 1000, 3333, 10000 µg/plate. All negative and positive control tubes and plates were done in triplicate. According to the results of the study, the substance is not mutagenic in the Ames test with and without metabolic activation.

CHO/HGPRT Assay:

Clarke (2012) performed an in vitro mammalian cell gene mutation assay in Chinese hamster Ovary (CHO) cells with and without metabolic activation.

Following concentrations were tested in duplicate:

Preliminary toxicity assay: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 2000 µg/mL

Initial concurrent cytotoxicity test: 250, 500, 1000, 1500, 1750 and 2250 µg/mL (non-activated cultures) and 250, 500, 1000, 1500 and 2500 µg/mL (S9-activated cultures)

Mutation assay: 250, 500, 1000, 1500 and 2500 µg/mL (activated study)

Repeat concurrent cytotoxicity test: 500, 750, 1000, 1250, 1500, 2000 and 2250 µg/mL (non-activated cultures)

Mutation assay: 500, 750, 1000, 1250, 1500 and 2000 µg/mL (non-activated study)

A vehicle control (sterile distilled water) and positive control (0.2 µg/mL ethylmethanesulphonate without metabolic activation; 4 µg/mL benzo(a)pyrene with metabolic activation) were scored as well.

The results of the CHO/HGPRT Mutation Assay indicated that, under the conditions of this study, the test substance was negative with and without metabolic activation. Positive and negative controls were considered valid.

In the initial mutagenesis assay, no visible precipitate was observed in the treatment medium at the beginning or end of treatment. Relative cloning efficiency was 60% at a concentration of 1500 µg/mL and < 10% at concentrations >= 1750 µg/mL in the non-activated system and 92% at the high concentration in the S9-activated system. None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 1E06 clonable cells. The non-activated portion failed due to a lack of cultures with between 10 and 20% relative cloning efficiency. The mutagenesis assay was repeated in the absence of S9 activation using concentrations from 500 to 1750 µg/mL.

The second and third trials of the mutagenesis assay in the absence of S9 activation failed due to the presence of contamination in the cultures. Based on the toxicity results from the first and second trial of the mutagenesis assay, the concentrations chosen for the third and fourth trials of the mutagenesis assay in the absence of S9 activation ranged from 500 to 2500 µg/mL.

Fourth trial: no visible precipitate was observed in the treatment medium at the beginning or end of treatment. Cultures treated with concentrations of 500, 750, 1000, 1250, 1500, 2000 and 2250 µg/mL were cloned for concurrent cytotoxicity. Relative cloning efficiency was 54% at a concentration of 1500 µg/mL and < 10% at concentrations >= 2000 µg/mL in the non-activated system. None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 1E06 clonable cells.

All criteria for a valid study were met. The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, the test substance was concluded to be negative.

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro:

Brat (1984) studied the gene mutation potential in rat hepatocytes (rat hepatocytes in primary culture from fisher 344 rats). Following doses were evaluated in triplicate: 1%, 1E-01%, 1E-02%, 1E-03%, 1E-04% and 1E-05% v/v. Solvent controls (1% DMSO, E-04 M pyrene) and a positive control (benzo(a)pyrene) were scored as well. The substance, when assayed at the highest non-toxic concentration of E-01% (v/v) and at the lower concentrations, was not genotoxic to the hepatocytes in the HPC/DNA repair assay. As the mean net nuclear grain counts of the slides exposed at E-01% concentration and at lower concentrations did not exceed 5, it was concluded that the substance was not genotoxic to the hepatocytes in this HPC/DNA repair assay. Parallel run positive and negative controls gave the expected values.

Chromosome aberration test:

Roy (2014) performed a chromosome aberration assay on the test article the test substance, using Chinese hamster ovary (CHO) cells in both the presence and absence of an Aroclor-induced rat liver S9 metabolic activation system, in order to evaluate the clastogenic potential of the test article. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration test. In both phases, CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9 -activated test system. All cells were harvested 20 hours after treatment initiation.

Based on the findings of the preliminary testing assay, the dose levels ranged from 100 to 2500 µg/mL for all three exposure groups.

In the chromosome aberration assay, substantial toxicity was not abserved at any dose level in the non-activated 4 and 20h exposure groups. Substantial toxicity was observed at dose levels greater than or equal to 1000 µg/mL in the S9 -activated exposure group.

No significant or dose-dependant increases in structural or numerical (polyploid or endoreplicated cells) aberrations were observed in treatment groups with or without S9. All criteria for a valid study were met.

Under the conditions of the assay described in the report, the test item was concluded to be negative for the induction of structural and numerical chromosome aberrations in both the non-activated and the S9-activated test systems in the in vitro chromosome aberration assay using CHO.

 

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation (EC) 1272/2008, the test substance should not be classified for mutagenicity.