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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA with and without metabolic activation

Cytogenicity in mammalian cells (OECD 473, Chromosome aberration test): negative in Chinese Hamster Lung (CHL/IU) cells with and without metabolic activation

Gene mutation in mammalian cells (OECD 490, Mouse Lymphoma assay): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Apr - 24 Jun 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
yes
Remarks:
2-Aminoanthracene was used as sole positive control for all strains in the presence with S9 mix; standard deviations were not calculated
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 2020
Deviations:
yes
Remarks:
2-Aminoanthracene was used as sole positive control for all strains in the presence with S9 mix; standard deviations were not calculated
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
other: EPA/TSCA Guideline, 40 CFR Part 799, FR Vol.62, No.158 § 799.9510 TSCA bacterial reverse mutation test
Version / remarks:
adopted in 1997
Qualifier:
according to guideline
Guideline:
other: Testing Methods for New Chemical Substances , Kanpoan No.287, Eisei No.127, Heisei 09 • 10 • 31 Kikyoku No.2
Version / remarks:
adopted in 1997
Qualifier:
according to guideline
Guideline:
other: Ministry of Labour, Japan, Notification No.77 dated 1 September, 1988 and Notification No.67 dated 2 June, 1997
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (S. typhimurium strains) and trp operon (E. coli strains)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague Dawley rats treated with phenobarbital / benzoflavone
Source of S9 : Oriental Yeast Co., Ltd. (Tokyo, Japan)
Test concentrations with justification for top dose:
Dose finding experiment: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate with and without S9 mix
Main experiment: 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate without S9 mix and 19.5, 39.1, 78.1, 156 and 313 µg/plate with S9 mix
Dose levels for the main mutation experiment were selected based on the results of the dose-finding experiment, in which precipitation of the test substance in vehicle was observed at 78.1 µg/plate without S9 mix and at 313 µg/plate with S9 mix.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 0.01 µg/plate for TA 100 and WP2 uvrA, 0.1 µg/plate for TA 98 (-S9); 2-aminoanthracene, 0.5 µg/plate for TA 98, 1 µg/plate for TA 100, 2 µg/plate for TA 1535 and TA 1537 and 10 µg/plate for WP2 uvrA (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation (dose-finding and main experiment)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
- Number of independent experiments: Following an initial dose-range finding study, a single experiment was performed in the main mutation study. The performance of the dose-range finding experiment was comparable to those of the main mutation study and can be considered as a valid independent experiment.
- Number of cultures per concentration: triplicates for each experiment (dose-finding and main experiment)

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed
Evaluation criteria:
The test substance was judged to be positive (mutagenic potential) if the following criteria were met:
- the test chemical show a dose-dependent increase in the number of revertant colonies
- the increase is at least twice as many as that of the solvent control
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed at 78.1 µg/plate without S9 mix and at 313 µg/plate with S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed at 78.1 µg/plate without S9 mix and at 313 µg/plate with S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed at 78.1 µg/plate without S9 mix and at 313 µg/plate with S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation was observed at 78.1 µg/plate without S9 mix and at 313 µg/plate with S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation of the test substance in vehicle was observed in the absence of S9 mix at concentrations ≥ 78.1 µg/plate and in the presence of S9 mix at concentrations ≥ 313 µg/plate. The observations were made in the dose finding experiment and in the main experiment.

RANGE-FINDING/SCREENING STUDIES:
Following an initial dose-range finding study, a single experiment was performed in the main mutation study. The performance of the dose-range finding experiment was comparable to those of the main mutation study, therefore the dose-finding experiment can be considered as a valid independent experiment.

HISTORICAL CONTROL DATA
- Positive historical control data: The positive control values were within the range of the historical control data (please refer to table 3 under "any other information on results incl. tables").
- Negative (solvent/vehicle) historical control data: The negative control values were within the range of the historical control data (please refer to table 3 under "any other information on results incl. tables").

Table 1: Results of the range-finding study

Dose-finding experiment: Pre-incubation test
Strain TA 100 TA 1535 WP2 uvrA TA 98 TA 1537
Metabolic activation - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9
Vehicle control DMSO
DMSO 95 109 8 14 21 29 21 36 7 12
Test item [µg/plate]
4.88 mean 95 112 7 10 26 30 19 38 7 11
19.5 mean 101 106 12 9 26 30 25 37 8 13
78.1 mean 88# 106 7# 10 24# 31 28# 40 6# 11
313# mean 88 102 9 9 24 30 20 40 6 14
1250# mean 81 105 7 10 24 33 19 40 7 13
5000#mean 90 97 8 7 23 26 12 31 4 9
Positive control
§Mean 595 764 351 221 125 736 345 289 668 171

§: Positive controls are: 9-aminoacridine (80 µg/plate for TA 1537 (-S9 mix)); sodium azide (0.5 µg/plate for TA 1535 (-S9 mix)); 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 µg/plate for TA 100 and WP2 uvrA, 0.1 µg/plate for TA 98 (-S9)) and 2-aminoanthracene (0.5 µg/plate for TA 98, 1 µg/plate for TA 100, 2 µg/plate for TA 1535 and TA 1537 and 10 µg/plate for WP2 uvrA (+S9 mix))

#: Precipitation observed

Table 2: Results of the main mutagenicity experiment:

Main mutation experiment: Pre-incubation test
Strain TA 100 TA 1535 WP2 uvrA TA 98 TA 1537
Metabolic activation - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9
Vehicle control
DMSO mean 121 84 7 7 29 28 18 32 7 15
Test item
4.88 mean 116 - 8 - 21 - 18 - 5 -
9.77 mean 128 - 11 - 28 - 28 - 5 -
19.5 mean 107 103 11 8 26 28 26 38 8 14
39.1 mean 110 93 9 8 26 32 23 31 9 13
78.1 mean 103# 99 7# 12 24# 25 27# 25 9# 12
156 mean - 99 - 9 - 25 - 30 - 12
313# mean - 96 - 5 - 27 - 32 - 15
Positive control
§mean 703 704 348 220 184 497 321 261 855 136
§: Positive controls are: 9-aminoacridine (80 µg/plate for TA 1537 (-S9 mix)); sodium azide (0.5 µg/plate for TA 1535 (-S9 mix)); 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 µg/plate for TA 100 and WP2 uvrA, 0.1 µg/plate for TA 98 (-S9)) and 2-aminoanthracene (0.5 µg/plate for TA 98, 1 µg/plate for TA 100, 2 µg/plate for TA 1535 and TA 1537 and 10 µg/plate for WP2 uvrA (+S9 mix))
#: Precipitation observed

Table 3: Historical control data generated in the testing laboratory in Jul - Dec 1997

Strain TA 100 TA 1535 WP2 uvrA TA 98 TA 1537
Metabolic activation - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9
Vehicle control
Mean 95.8 103.8 7.8 9.2 19.0 24.0 21.7 35.6 7.3 12.5
SD ± 12.6 ± 14.2 ± 2.9 ± 3.3 ± 4.7 ± 5.2 ± 5.4 ± 7.5 ± 4.0 ± 5.6
n 302 177 254 185 204 168 231 173 225 187
Positive control
Mean 565.2 627.2 315.6 194.8 160.1 556.9 410.5 320.5 713.3 159.6
SD ± 96.5 ± 95.7 ± 27.9 ± 26.9 ± 30.3 ± 64.7 ± 39.9 ± 51.8 ± 149.0 ± 31.2
n 258 183 257 187 205 172 231 177 234 191
n: number of experiments performed
Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Mar - 13 Jun 2005
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)
Version / remarks:
adopted in 1997
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
adopted in 2016
Deviations:
yes
Remarks:
evaluation criteria inconsistent with those specified in the guideline with respect to distribution of historical control data (within 95% Poisson limits)
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4E
Version / remarks:
adopted in 2000
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines - Kanpoan No. 287 - Environmental Protection Agency - Eisei No. 127 - Ministry of Health and Welfare - Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade and Industry
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
tk locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Suitability of cells: The L5178Y cell line has successfully been used in in vitro experiments for many years.
- Absence of Mycoplasma contamination: Before freezing, each batch was screened for mycoplasma contamination.
- Methods for maintenance in cell culture: The cells were subcultured at least 3 times a week. The cell cultures were incubated at 37 °C in a humidified atmosphere with 4.5% carbon dioxide and 95.5% air.
- Doubling time: 10 - 12 h
- Modal number of chromosomes: 40 ± 2
- Periodically checked for karyotype stability: yes

MEDIA USED
- Type and composition of media:
HAT medium: Prior to mutagenicity testing, the amount of spontaneous mutants was reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with 1.0 x 10E-4 M hypoxanthine, 2.0 x 10E-7 M aminopterin and 1.6 x 10E-5 M thymidine.

HT medium: Following incubation in HAT medium, the cells were incubated for a 2-days recovery period in RPMI 1640-HT medium supplemented with 1.0 x 10E-4 M hypoxanthine and 1.6 x 10E-5 M thymidine.

Complete culture medium: RPMI 1640 medium supplemented with 15% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 220 µg/mL sodium pyruvate and 1.25 U/mL amphotericin

Treatment medium: RPMI 1640 medium supplemented with 3% horse serum for 4 h exposure or 15% horse serum for 24 h exposure, 100 U/100 µg/mL penicillin/streptomycin, 220 µg/mL sodium pyruvate and 1.25 U/mL amphotericin

Cloning medium: RPMI 1640 complete culture medium

Selection medium: RPMI 1640 complete culture medium supplemented with 5% TFT
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix)
- method of preparation of S9 mix: S9 mix was prepared from the livers of male Wistar HanIbm rats (220 - 320 g in weight) that were treated with an intraperitoneal injection of 80 mg/kg bw/day phenobarbital and b-naphthoflavone each on three consecutive days. The livers were prepared 24 h after the last treatment.
Test concentrations with justification for top dose:
Pre-test on toxicity
With and without S9 mix: 9.8, 19.6, 39.1, 78.1, 156.3, 312.5, 625 and 1250 µg/mL (4 h)
Without S9 mix: 9.8, 19.6, 39.1, 78.1, 156.3, 312.5, 625 and 1250 µg/mL (24 h)

Experiment I
With and without S9 mix: 9.4*, 18.8, 37.5, 75, 150 and 300 µg/mL µg/mL (4 h)

Experiment II
Without S9 mix: 9.4*, 18.8, 37.5, 75, 150 and 300 µg/mL (24 h)

* The cultures were not continued following the expression phase of 72 h
The highest concentration of the range-finding experiment was chosen based on the solubility of the test item. The concentration range in the main experiment was limited by the solubility of the test item.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
yes
Remarks:
Medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
other:
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1 x 10E7 cells/flask in 80cm² flasks
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 and 24 h
- Expression time (cells in growth medium between treatment and selection): 72 h (first experiment) and 48 h (second experiment)
- Selection time: 10 - 15 days
- Method used: microwell plates
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: After the expression period, 4x10E3 cells from each experimental group were seeded into 2 microtiter plates with selective medium including TFT. The viability (cloning efficiency 2) was determined by seeding about 2.0 cells per well into 2 microtiter plates (same medium without TFT).
- Criteria for small (slow growing) and large (fast growing) colonies: Absolute size of the colony (more than 1/3 of a well for large colonies) and optical density of the colony (the density of the small colonies was considerably higher).

SELECTION AGENT:
5 μg/mL trifluorothymidine

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)

Evaluation criteria:
A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.
A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
- The test item reproducibly induces a mutation frequency that is at least two times higher than the spontaneous mutation frequency (negative or solvent control) in the experiment.
- There is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.

A positive 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 solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.

Results of test groups are rejected if the relative total growth, the relative suspension growth and/or cloning efficiency 1 is less than 10 % of the solvent control or the cloning efficiency 2 after the expression period is less than 20 %. 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.
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
Remarks:
Please refer to "Additional information on results" field below, information on negative historical control data.
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: The pH value and osmolality were determined in the solvent control and the maximum concentration of the pre-test without metabolic activation. The pH was 7.57 for the solvent control and 7.59 for 1250 µg/mL test item. The osmolality was 361 mOsm for the solvent control and 344 for 1250 µg/mL test item.
- Precipitation and time of the determination: Precipitation of the test item, observed by the unaided eye, was noted in all experiments at concentrations of 150 µg/mL and above.

RANGE-FINDING/SCREENING STUDY
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolality were determined at the maximal concentration of the test item and in the solvent control without metabolic activation. 1x10E7 cells were exposed to each concentration of the test item for 4 and 24 hours without and 4 hours with metabolic activation. Following treatment the cells were incubated for a 2-day growth period. Afterwards, the relative suspension growth (RSG) of the treated cell cultures was calculated.
There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item in the pre-experiment. After 4 h of treatment, precipitation visible to the unaided eye was observed at ≥ 156.3 µg/mL in the presence and absence of metabolic activation. Following continuous treatment, precipitation also occurred at ≥ 156.3 µg/mL. Based on these observations, the concentration range in the main experiment was limited by the solubility of the test item.

HISTORICAL CONTROL DATA
- Positive historical control data: The mutation frequencies of both positive controls were within the range of the historical control data (please refer to Table 1 under 'any other information on materials and methods incl. tables).
- Solvent historical control data: The mutation frequency of the solvent control was within the range of the historical control data (please refer to Table 1 under 'any other information on materials and methods incl. tables).
- Negative historical control data: The mutation frequency of the negative controls of the second culture of the first and second experiment without S9 mix was slightly outside the range of the historical control data (please refer to Table 1 under 'any other information on materials and methods incl. tables). The finding was judged as biologically irrelevant since the corresponding solvent controls as well as the mean values of both cultures remained within the range of historical control data.



Table 2: Summary of results

Concentration (µg/mL) S9 mix rel CE1 RTG Mutant colonies/ 106ells IF rel CE1 RTG Mutant colonies/ 106ells IF
Experiment I (4 h treatment) Culture 1 Culture 2
Negative control (Medium) - 100.0 100.0 60 - 100.0 100.0 39 -
Solvent control (DMSO) - 100.0 100.0 53 1.0 100.0 100.0 58 1.0
Positive control (MMS) 19.5 - 68.8 38.7 281 4.7 78.7 46.2 255 6.5
Test item 9.4 - 90.1 # 58.5 #
18.8 - 151.3 105.8 33 0.6 54.9 80.8 47 0.8
37.7 - 101.9 97.3 45 0.8 42.5 100.1 39 0.7
75.0 - 101.9 93.4 53 1.0 75.8 76.9 52 0.9
150.0p - 73.3 85.9 29 0.5 61.6 90.8 40 0.7
300.0p - 79.2 87.8 25 0.5 47.8 83.5 29 0.5
Negative control (Medium) + 100.0 100.0 56 - 100.0 100.0 55 -
Solvent control (DMSO) + 100.0 100.0 47 1.0 100.0 100.0 55 1.0
Positive control (CPA) 4.5 + 52.2 64.3 307 5.4 50.6 75.0 338 6.1
Test item 9.4 + # #
18.8 + 79.6 102.6 58 1.3 70.0 90.2 33 0.6
37.7 + 71.6 79.2 62 1.3 83.3 83.5 46 0.8
75.0 + 64.7 100.9 47 1.0 79.7 51.9 56 1.0
150.0p + 74.9 90.7 37 0.8 82.1 69.3 33 0.6
300.0p + 78.4 100.6 43 0.9 79.7 78.0 57 1.1
Experiment II (24 h treatment) Culture 1 Culture 2
Negative control (Medium) - 100.0 100.0 59 - 100.0 100.0 37 -
Solvent control (DMSO) - 100.0 100.0 50 1.0 100.0 100.0 49 1.0
Positive control (MMS) 13.0 - 14.5 20.4 516 8.7 11.5 43.6 346 9.3
Test item 9.4 - 88.6 # 87.3 #
18.8 - 76.8 75.1 43 0.9 81.9 107.0 44 0.9
37.7 - 87.1 79.8 47 1.0 90.2 124.2 29 0.6
75.0 - 94.8 83.6 44 0.9 100.0 98.0 36 0.7
150.0p - 103.8 68.7 36 0.7 101.8 106.0 41 0.8
300.0p - 80.4 91.0 57 1.2 85.9 114.3 54 1.1
rel CE: relative cloning efficiency; RTG: relative total growth; IF: induction factor; p: precipitation; #: culture was not continued

For further details on results please refer to attached document under "Attached background material".

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Jul - 21 Dec 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 1997
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
adopted in 2016
Deviations:
yes
Remarks:
Please refer to "Any other information on materials and methods incl. tables".
Qualifier:
according to guideline
Guideline:
other: Testing guidelines for Japanese Chemical Substances Control Law, Testing Methods for New Chemical Substances, III. Mutagenicity Test.
Version / remarks:
amended in 1997
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHL/IU cells were obtained from Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan)
- Absence of Mycoplasma contamination: The stored cells had been confirmed not to be contaminated with mycoplasma.
- Number of passages if applicable: 18 - 29
- Methods for maintenance in cell culture: The cells were grown as monolayer in plastic dishes. Approximately 2 x 10E4 cells were seeded in 60 mm diameter plastic dishes and cultured for 3 days prior to each experiment.
- Doubling time: 15 h (most recent measurement in the testing facility: 16 h)
- Modal number of chromosomes: 25

MEDIA USED
- Type and composition of media: Eagle’s MEM medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% calf serum under humidified atmosphere of 5% CO2 in air at 37 °C.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix)
- source of S9: Oriental Yeast Co., Ltd. (Tokyo, Japan)
- method of preparation of S9 mix: S9 mix was prepared from the livers of 7 week old male Sprague Dawley rats treated with phenobarbital (30 mg/kg bw on Day 1, 60 mg/kg bw on Days 2, 3 and 4) and 5,6-benzoflavone (80 mg/kg bw on Day 3).
- concentration or volume of S9 mix and S9 in the final culture medium: 16.5% (60 µL test material solution and 1 mL S9 mix were added to 5.0 mL cultures)

Test concentrations with justification for top dose:
Experiment I:
6 h treatment with and without metabolic activation: 200, 400 and 800 µg/mL

Experiment II:
24 h treatment without metabolic activation: 200, 400 and 800 µg/mL
48 h treatment without metabolic activation: 100, 200 and 400 µg/mL
6 h treatment with metabolic activation: 200, 400 and 600 µg/mL

In each experiment, a cytotoxicity test was conducted to determine the doses to be used in the chromosome aberration test. The concentrations were the same for all treatment periods with and without S9 mix: 3.13, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/mL. The highest concentration for slide preparation was selected to have a growth rate ≤ 50%.
Vehicle / solvent:
- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: Because the test material was oil soluble and suscpetible to hydrolysis, it was dissolved in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 2 x 10E4 cells were seeded in 60 mm-diameter dishes
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 6, 24 and 48 h
- Harvest time after the end of treatment: 6 h treatment: 24 h fixation time (Experiment I) and 30 h fixation time (Experiment II); 24 h treatment: fixation time 24 h; 48 h treatment: fixation time 48 h

SPINDLE INHIBITOR:
0.1 µg/mL colcemid (Life Technologies, Inc., New York, USA) was added 1.5 h prior to harvest.

SLIDE PREPRATION AND METAPHASE ANALYSIS:
- Methods of slide preparation and staining technique used including the stain used: The cells were collected by centrifugation, swollen in 75 mM potassium chloride at room temperature for 20 min and fixed in absolute methanol : glacial acetic acid, 3 : 1, v/v). The fixative was changed 3 times by centrifugation. The cells were pelleted, resuspended in a minimal amount of fixative and spread on glass slides. After aging for at least one day, the slides were stained with 3% Giemsa solution in 1/15 M phosphate buffer for 30 min.
- Number of cells spread and analysed per concentration: 200 (100 cells per culture)
- Criteria for scoring chromosome aberrations: Metaphase cells with standard karyotype were screened out by an automatic metaphase finding system (Magiscan, Applied Imaging International Ltd., England) and scored for chromosomal aberrations. Types of chromosomal aberrations were recorded as chromatid gaps, chromosome gaps, chromatid breaks, chromatid exchanges, chromosome breaks, chromosome exchanges (including chromosome rings and dicentric chromosomes) and fragmentation. Gap was defined as an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the chromatids. Microscope co-ordinates were also recorded for aberrant cells. The incidences of cells with structural aberrations (including and excluding gaps) were obtained.
- Determination of polyploidy: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: growth rate (%)
Evaluation criteria:
The incidence of structurally aberrant cells (excluding gaps) and that of polyploid cells at each experimental point were classified as follows:
Negative: < 5%
Marginal: ± From 5% to less than 10%
Positive: ≥ 10%

The test material was concluded to induce chromosomal aberrations when both of the following criteria were fulfilled
1) The incidence of cells with structural aberrations excluding gaps and/or the incidence of polyploid cells are marginal or positive.
2) Dose response relationships or reproducibility is observed.
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 800 µg/mL after 6 h exposure + S9 mix (1st experiment), at 800 µg/mL after 24 h exposure - S9 mix (2nd experiment), at 600 µg/mL after 48 h exposure - S9 mix (2nd experiment) and at 800 µg/mL after 6 h exposure + S9 mix (2nd experiment)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation of the test substance in medium was observed at all concentrations of both experiments in the presence and absence of metabolic activation.


RANGE-FINDING/SCREENING STUDIES: Cytotoxicity tests were conducted in parallel to the main experiments. Precipitation of the test substance in medium was observed at ≥ 100 µg/mL in both experiments in the presence and absence of metabolic activation. Moderate decreases in the growth rate were observed, but no growth rate was reduced to ≤ 50%. Based on these findings, the highest dose for the chromosomal aberration test was set at 800 µg/mL.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity, evident as a growth rate reduction of ≥ 50% was observed in the first experiment after 6 h exposure at 800 µg/mL (48.8%) in the presence of metabolic activation. In the second experiment, cytotoxicity was observed after 6 h of exposure at 600 µg/mL (34.2%) in the presence of S9 mix, after 24 h of exposure at 800 µg/mL (40.6%) without S9 mix and after 48 h of exposure (48.1%) without S9 mix.

HISTORICAL CONTROL DATA
- Positive historical control data: Not given in the study report.
- Solvent historical control data: Please refer to Table 1 under “Any other information on material and methods incl. tables”.

Table 2: Results of the chromosomal aberration test, first experiment

Experiment 1: 6 h treatment - S9 mix, 18 h recovery, 24 h harvest
Chemical Concentration (µg/mL) Cells scored Total No. of structural aberrations Cells with aberrations (%) Polyploid cellsc Growth rate (%)d
incl. gaps without gaps incl. gaps without gaps Judge % Judge
DMSO 1% 200 2 0 1.0 0.0 - 1.0 - 100.0
Test item 200p 200 4 3 1.5 1.0 - 0.0 - 99.0
400p 200 6 2 3.0 1.0 - 1.5 - 91.4
800p 200 7 6 3.5 3.0 - 1.0 - 69.9
MMC 0.06 200 141 134 44.0 42.0 + 0.0 - 86.3

Experiment 1: 6 h treatment + S9 mix, 18 h recovery, 24 h harvest

Chemical Concentration (µg/mL) Cells scored Total No. of structural aberrations Cells with aberrations (%) Polyploid cellsc Growth rate (%)d
incl. gaps without gaps incl. gaps without gaps Judge % Judge
DMSO 1% 200 4 3 2.0 1.5 - 0.5 - 100.0
Test item 200p 200 0 0 0.0 0.0 - 0.5 - 101.6
400p 200 4 3 2.0 1.5 - 0.0 - 87.8
800p 200 3 2 1.5 1.0 - 1.5 - 48.8
CP 10 200 87 84 31.5 30.5 + 0.0 - 87.8
CP: Cyclophosphamide, positive control + S9 mix; MMC: Mitomycin C, positive control - S9 mix; p: precipitate observed; -: negative; ±: marginal; +: positive; c: scored from 100 cells/culture; d: percentage of control value

Table 3: Results of the chromosomal aberration test, second experiment

Experiment 2: 24 h treatment - S9 mix, 24 h harvest
Chemical Concentration (µg/mL) Cells scored Total No. of structural aberrations Cells with aberrations (%) Polyploid cellsc Growth rate (%)d
incl. gaps without gaps incl. gaps without gaps Judge % Judge
DMSO 1% 200 4 3 2.0 1.5 - 1.0 - 100.0
Test item 200p 200 2 1 1.0 0.5 - 0.5 - 89.3
400p 200 1 1 0.5 0.5 - 0.5 - 70.2
800p 200 3 3 1.5 1.5 - 2.0 - 40.6
MMC 0.02 200 46 43 19.0 18.5 + 0.0 - 83.6
Experiment 2: 48 h treatment - S9 mix, 48 h harvest
Chemical Concentration (µg/mL) Cells scored Total No. of structural aberrations Cells with aberrations (%) Polyploid cellsc Growth rate (%)d
incl. gaps without gaps incl. gaps without gaps Judge % Judge
DMSO 1% 200 5 3 2.5 1.5 - 0.0 - 100.0
Test item 100p 200 6 5 3.0 2.5 - 0.5 - 96.7
200p 200 1 1 0.5 0.5 - 1.0 - 82.5
400p 200 3 2 1.5 1.0 - 1.0 - 48.1
MMC 0.02 200 52 52 21.5 21.5 + 0.5 - 98.5
Experiment 2: 6 h treatment + S9 mix, 24 h recovery, 30 h harvest
Chemical Concentration (µg/mL) Cells scored Total No. of structural aberrations Cells with aberrations (%) Polyploid cellsc Growth rate (%)d
incl. gaps without gaps incl. gaps without gaps Judge % Judge
DMSO 1% 200 4 4 2.0 2.0 - 2.0 - 100.0
Test item 200p 200 4 4 1.0 1.0 - 1.0 - 89.3
400p 200 2 2 1.0 1.0 - 1.0 - 80.5
600p 200 1 1 0.5 0.5 - 3.5 - 34.2
CP 10 200 151 150 37.5 37.5 + 0.0 - 64.3
CP: Cyclophosphamide, positive control + S9 mix; MMC: Mitomycin C, positive control - S9 mix; p: precipitate observed; -: negative; ±: marginal; +: positive; c: scored from 100 cells/culture; d: percentage of control value
Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro gene mutation in bacteria:

The test substance was investigated for gene mutation in bacteria (Ames test) according to OECD guideline 471 and in compliance with GLP. Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli strain WP2 uvrA were exposed to the test item in the presence and absence of metabolic activation ((phenobarbital / benzoflavone-induced rat liver S9 fraction).

An initial dose-range finding experiment and the main mutagenicity experiment were both performed according to the pre-incubation method using three plates per condition. Corresponding solvent (DMSO) and appropriate positive controls were included in both experiments. In the dose-range finding experiment, test item concentrations in the range of 4.88 to 5000 µg/plate were applied in the presence and absence of S9 mix. Precipitation of the test item was observed at ≥78.1 µg/plate without S9 mix and at ≥313 µg/plate with S9 mix. Based on these findings, the test item concentrations in the main mutation assay were 4.88 to 78.1 µg/plate without S9 mix and 19.5 to 313 µg/plate with S9 mix. After an incubation period of 48 hours, the plates were inspected for a possible reduction in the bacterial background lawn and the number of revertant colonies were counted.

Cytotoxicity was not observed in any experiment up to and including the highest concentration, neither in the presence, nor in the absence of S9 mix. Precipitation in the main experiment was observed at concentrations ≥ 78.1 µg/plate in the absence of S9 mix at concentrations of ≥ 313 µg/plate in the presence of S9 mix.

There was no statistically significant increase in the frequency of revertant colonies noted for any of the bacterial strains at any dose level, either with or without S9 mix. The number of revertant colonies for the solvent and positive controls were within the range of the laboratory’s historical control data. All of the positive controls induced marked increases in the frequency of revertant colonies, thus confirming the activity of the metabolic activation system and the sensitivity of the test itself.

Based on the experimental findings, the test item is not mutagenic in bacteria with and without metabolic activation.

 

In vitro cytogenicity in mammalian cells:

In vitro cytogenicity in mammalian cells was investigated in a chromosome aberration test performed in accordance with OECD guideline 473 and in compliance with GLP. Two independent experiments were performed in Chinese Hamster Lung (CHL/IU) cells. In parallel to the chromosomal aberration tests, cytotoxicity tests were performed for all experimental conditions using test item concentrations in the range of 3.13 to 800 µg/mL. The highest concentration for cytogenicity evaluation was selected to have a growth rate ≤ 50%.

In the first cytogenicity experiment, the cells were exposed to test item concentrations in the range of 100 – 800 µg/plate for 6 h in the presence and absence of metabolic activation (S9 mix) and harvested 24 h after start of exposure. In the second cytogenicity experiment, the cells were exposed to 200 – 800 µg/mL for 24 h in the absence of S9 mix, to 100 – 400 µg/mL for 48 h in the absence of S9 mix and for 6 h at 200 – 600 µg/mL in the presence of S9 mix. In the second experiment, the cells were sampled directly after continuous treatment in the absence of S9 mix (24 and 48 h) and for the 6 h exposure duration 30 h after start of exposure. Corresponding solvent (DMSO) and positive controls (mitomycin C (MMC), 0.02 µg/mL for 24 and 48 h exposure and 0.06 µg/mL for 6 h exposure without S9 mix and cyclophosphamide (CP), 10 µg/mL with S9 mix) were included in both experiments.

A total of 200 metaphase cells per condition were scored for the presence of structural and numerical chromosome aberrations. In addition, the growth rate of each culture was assessed to determine cytotoxicity.

A growth rate reduction of ≥ 50% was observed in the first experiment after 6 h exposure at 800 µg/mL with S9 mix, in the second experiment after 6 h of exposure at 600 µg/mL with S9 mix and after 24 and 48 h of exposure at 800 µg/mL without S9 mix. Precipitation of the test substance in medium was observed at all concentrations of both experiments in the presence and absence of metabolic activation.

Treatment with the test item did not induce a biologically relevant increase in the percentage of aberrant metaphases in any of the experiments, neither in the presence nor in the absence of S9 mix. In addition, the frequency of polyploid cells was not affected with or without metabolic activation. Frequencies of aberrant metaphases of solvent control cultures remained within the range of the laboratory’s historical control data. The positive controls MMC and CP showed a clear clastogenic effect and markedly induced the number of aberrant metaphases, demonstrating the sensitivity of the test and the functionality of the S9 mix.

Based on the results of the present study, Sumilizer GP is not clastogenic to CHL/IU cells with and without metabolic activation.

 

In vitro gene mutation in mammalian cells:

Sumilizer GP was tested for its potential to induce gene mutations in mammalian cells according to OECD guideline 490 and in compliance with GLP. The study was performed in L5178Y mouse lymphoma cells (thymidine kinase locus) using duplicate cultures per condition. The test item concentrations were selected based on the results of a preliminary range-finding study, in which precipitation of the test substance in the medium was noted at ≥ 156.3 µg/mL after 4 h of treatment with and without S9 mix and after 24 h of treatment without S9 mix.

Two independent experiments were performed. The cells were exposed to test item concentrations in the range of 9.4 to 300 µg/mL for 4 h with and without metabolic activation (experiment I) and for 24 h continuous treatment without metabolic activation (second experiment). Corresponding negative (culture medium), solvent (DMSO) and positive controls (methylmethane sulfonate (MMS), 19.5 µg/mL for 4 h without S9 mix and 13.0 µg/mL for 24 h without S9 mix and cyclophosphamide (CPA), 4.5 µg/mL for 4 h with S9 mix) were included in each experiment. Following exposure, the cells were incubated for 72 h (first experiment) or 48 h (second experiment) to allow expression of the mutant phenotype. The expression period was followed by a selection period, where cells were incubated in selection medium containing trifluorothymidine (TFT) for 10-15 days.

Precipitation of the test item, observed by the unaided eye, was noted in all experiments at concentrations of 150 µg/mL and above. There was no cytotoxicity (relative total growth < 50%) observed in any experiment at any concentration, neither with nor without S9 mix.

There was no statistically significant or biologically relevant increase in the number of mutant colonies observed in any experiments at any of the tested concentrations, neither in the presence, nor in the absence of metabolic activation. Mutant frequencies of the medium control cultures were slightly outside the range of historical control data, but as the mutant frequencies of the solvent control cultures fitted well into the range of historical control data, the finding was considered irrelevant. The positive control substances CPA (+S9 mix) and MMS (–S9 mix) significantly increased the mutation frequency in L5178Y cells, thus demonstrating the sensitivity and validity of the test system.

Under the conditions of the test, Sumilizer GP did not induce forward mutations in the mouse lymphoma assay with L5178Y TK+/-cells in the absence or presence of metabolic activation. Thus, the test item is considered not mutagenic in mammalian cells in vitro.

 

Conclusion:

In conclusion, assessment of the available experimental data on gene mutation in bacteria, gene mutation in mammalian cells and chromosome aberration in mammalian cells suggests that Sumilizer GP is neither mutagenic nor clastogenic in vitro.

Justification for classification or non-classification

The available data on genetic toxicity in vitro do not meet the criteria for classification according to Regulation No. (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.