Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, rel. 2): non mutagenic up to 5000 µg/plate (+/- S9) in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100:
- CHO/HPRT Mutation Assay (OECD 476, GLP, rel. 1), non mutagenic up to cytotoxicity limit (-S9) or up to limit concentration (+S9)
- Human lymphocyte chromosome aberration test (OECD 473, GLP, rel. 1), no significant increase in the frequency of cells with aberrations in either the presence or absence of metabolic activation: non clastogenic up to limit concentration

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
between 03 November 2010 and 08 February 2011
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: Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990).
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination:yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Cell Line
The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.

Cell Culture
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5% foetal calf serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in air.

Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.



Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared in house from the livers of male Sprague-Dawley CD strain rats. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg), prior to S9 preparation on the fourth day.
Test concentrations with justification for top dose:
Preliminary Cytotoxicity Test: dose ranges of 35.63 to 1140 µg/mL and 35.63 to 2280 µg/mL were used for the 4-hour without and with S9 exposure groups respectively. A dose range of 8.91 to 2280 µg/mL was used for the 24-hour exposure group.

4-hour without S9: 35.63, 71.25, 142.5, 285, 427.5, 570, 712.5, 855 µg/mL
4-hour with S9 (2%): 71.25, 142.5, 285, 570, 1140, 2280 µg/mL (up to 10 mM)
24-hour without S9: 8.91, 17.81, 35.63, 71.25, 142.5, 285, 570, 855 µg/mL
4-hour with S9 (1%): 71.25, 142.5, 285, 570, 1140, 2280 µg/mL (up to 10 mM)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle:The test material formed a solution with the solvent suitable for dosing.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Dimethyl benzanthracene (DMBA) (Sigma Batch Numbers 105K1312 and 040H1231 were used for Experiment 1 and Experiment 2 respectively at 0.5 and 1.0 µg/mL as the positive controls in cultures with S9.
Remarks:
With metabolic activation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation Migrated to IUCLID6: Sigma Batch Number 0001423147 at 500 and 750 µg/mL was used in the 4-hour cultures without S9 and at 200 and 300 µg/mL for the 24-hour cultures without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4 hours with S9 (2%) and without S9
Experiment 2: 4 hours with S9 (1%) and 24 hours without S9
- Expression time (cells in growth medium): 6 to 7 days
- Selection time (if incubation with a selection agent): 14 days

SELECTION AGENT (mutation assays): 10 μg/mL 6-Thioguanine

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 2 x 10E5 cells/75 cm² flask (5 replicates per group) in Ham's F12 growth media (5% serum), supplemented with 6-TG

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (triplicate at 200 cells/25 cm² flask in 5 mL of growth medium)
Evaluation criteria:
Significant or dose-related increases in mutant frequency per survivor (see Calculations in "Any other information on materials and methods incl. Tables"
Statistics:
none
Key result
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant changes
- Effects of osmolality: the osmolality did not increase by more than 50 mOsm
- Evaporation from medium: no data in the report but not expected due to the low vapor pressure of the test material
- Water solubility: NA as the test material was dissolved in DMSO
- Precipitation: none observed

RANGE-FINDING/SCREENING STUDIES:
Preliminary Cytotoxicity Test
Dose ranges of 35.63 to 1140 µg/ml and 35.63 to 2280 µg/ml were used for the 4-hour without and with S9 exposure groups respectively. A dose range of 8.91 to 2280 µg/ml was used for the 24-hour exposure group. A greasy/oily precipitate of the test item was observed at the end of exposure at and above 285 µg/ml in the 24-hour exposure group. In the 4-hour exposure groups a greasy/oily precipitate was seen at and above 570 µg/ml and 285 µg/ml in the absence and presence of S9 respectively at the end of exposure. The results of the individual flask counts and their analysis are presented in the attached Table 1. It can be seen that there was a very steep toxicity curve exhibited in the 4-hour exposure group in the absence of S9 between 285 µg/ml and 570 µg/ml. The 4-hour exposure group in the presence of S9 demonstrated no marked reduction in the cloning efficiency (CE) up to and including the maximum recommended dose. The 24-hour exposure group demonstrated a more gradual dose related reduction in cloning efficiency and was completely toxic at 1140 µg/ml.
Mutagenicity Test - Experiment 1
The Day 0 and Day 7 cloning efficiencies are presented in the attached Table 2 and Table 3. As was seen in the preliminary toxicity test there was a steep toxicity curve in the 4-hour group in the absence of metabolic activation (S9) with the dose levels of 570 µg/ml and above being too toxic for plating and the dose level of 427.5 µg/ml showing a 33% reduction in cloning efficiency at Day 0 only. The Day 7 cloning efficiencies demonstrated a very steep toxicity curve between 427.5 and 570 µg/ml. The 4-hour exposure group in the presence of metabolic activation (S9) demonstrated only a slight reduction in cloning efficiency at the maximum dose tested when compared to the vehicle control.
The Day 7 vehicle control cloning efficiencies for the 4-hour exposure group in the absence of S9 were marginally less than 70% but were considered to be acceptable. In the presence of S9 the Day 0 and Day 7 vehicle control cloning efficiencies achieved acceptable levels.
The mutation frequency counts and mean mutation frequency per survivor values are presented in the attached Table 2 and Table 3. Both of the vehicle control mutant frequency per survivor values were within the maximum upper limit of 25 x 10E6 mutants per viable cell, and all positive control dose levels produced marked increases in mutant frequency per survivor values over the vehicle controls. This was taken to indicate that the test method and metabolic action system were functioning adequately.
There were no marked increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10E-6 in either the absence or presence of S9.
Mutagenicity Test - Experiment 2
The Day 0 and Day 7 cloning efficiencies are presented in the attached Tables 4 and 5. It can be seen that in the 4-hour exposure group in the presence of S9 there was a reduction in the cloning efficiency at the maximum dose of 2280 µg/ml at Day 0 although this was not continued onto Day 7. This increased toxicity when compared to Experiment 1 is in line with the reduction in S9 concentration in Experiment 2 and confirms the increased toxicity of the test item in the absence of S9. The 24-hour exposure group demonstrated a very sharp toxicity curve and was slightly more toxic than in the preliminary toxicity test. The dose levels of 570 µg/ml and 855 µg/ml were both too toxic for plating and the dose levels up to and including 285 µg/ml showed no reduction in cloning efficiency when compared to the vehicle control groups.
The vehicle control cloning efficiencies for the Day 0 and Day 7 4-hour and 24 hour exposure groups were less than 70% but were considered to be acceptable as they achieved over the 50% minimum requirement and there was no indication of an increase in mutation frequency for the test item.
The mutation frequency counts and mean mutation frequency per survivor per 10E6 cells values are presented in Tables 4 and 5. Both of the vehicle control mutant frequency per survivor values were within the maximum upper limit of 25 x 10E6 mutants per viable cell, and all positive control dose levels produced marked increases in mutant frequency per survivor values over the vehicle controls. This was taken to indicate that the test method and metabolic action system were functioning adequately.
In the absence and presence of metabolic activation there were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10E-6. The test item was tested to the maximum recommended dose in the presence of S9. In the absence of S9 the steepness of the toxicity curve made it difficult to achieve a dose response curve, however it is considered that the test item has been adequately tested

All tables are attached.

COMPARISON WITH HISTORICAL CONTROL DATA: All values withing historical range

Due to the quantity and or format of the tables please see attached tables.

 

REFERENCES

Cole J, Fox M, Garner R C, McGregor D B, and Thacker J (1990) Gene Mutation in Cultured Mammalian Cells. In'Basic Mutagenicity Tests: UKEMS Recommended Procedures', (Kirkland D J, Ed),Press, Cambridge University Press, New York.

Hsie A W, Brimer P A, Mitchell T J, and Gosslee D G (1975) The dose response relationship for ethylmethane sulfonate induced mutations at the HPRT locus in Chinese hamster ovary cells,Somatic Cell Genetics,1, 247-261.

Hsie A W, Casciano D A, Couch D B, Krahn D F, O'Neill J P, and Whitfield B L (1981) The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutageniety of chemicals. A report of the Gene-tox program,Mut. Res.,86, 193-214.

Scott, D., Galloway, S.M.,, R.R., Ishidate, M. Jr, Brusick, D., Ashby, J. and Myhr, B.C. (1991). Genotoxicity under Extreme Culture Conditions. A report from ICPEMC task Group 9. Mutation Res.,257, 147 – 205.

Conclusions:
The test item did not induce significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.
Executive summary:

Introduction.

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The test method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 'In Vitro Mammalian Cell Gene Mutation Tests', Commission Regulation (EC) No 440/2008 and United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Methods.

Chinese hamster ovary (CHO) CHO-K1 cells were treated with the test item at a minimum of six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4‑hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

Exposure Group

Final concentration oftest item(µg/ml)

4-hour without S9

35.63, 71.25, 142.5, 285, 427.5, 570, 712.5, 855

4-hour with S9 (2%)

71.25, 142.5, 285, 570, 1140, 2280

24-hour without S9

8.91, 17.81, 35.63, 71.25, 142.5, 285, 570, 855

4-hour with S9 (1%)

71.25, 142.5, 285, 570, 1140, 2280

 

Results.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion. 

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11th January 1995 to 2nd February 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline study performed according to the previous version of the OECD guideline 471 (1983).
Remarks:
Strain S. typhimurium TA1598 was included along with S. typhimurium strains TA1535, TA1537, TA98 and TA100. No E. coli WP2 or E. coli WP2 (pKM101) strain or S. typhimurium strain TA102 was included, as in the current recommended combination of strains (OECD 471, 1997). However, this deviation from current standards is considered not to have affected the overall assessment of the potential gene mutagenicity of this test material '
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1981
Deviations:
yes
Remarks:
No E. coli WP2 or E. coli WP2 (pKM101) strain or S. typhimurium strain TA102 was included, as in the current recommended combination of strains (OECD 471, 1997).
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Study states Method B14 only.
Deviations:
yes
Remarks:
No E. coli WP2 or E. coli WP2 (pKM101) strain or S. typhimurium strain TA102 was included, as in the current recommended combination of strains (OECD 471, 1997).
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: January 31st 1994 Date of signature: March 16th 1994
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Storage condition of test material: refrigerator in the dark
Target gene:
Histidine operon for Salmonella
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA1537, TA1538, TA98 and TA100
Additional strain / cell type characteristics:
other: TA 100 and TA1535 sensitive to agents inducing base pair mutation. TA1537, TA1538 and TA98 sensitive to agents inducing frame-shift mutations.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver (S9 mix)
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 50, 150, 500, 1500 and 5000 µg/plate.
Experiment 1: 0, 50, 150, 500, 1500 and 5000 µg/plate.
Experiment 2: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: not stated in report
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
3 µg/plate for TA100 and 5 µg/plate for TA1535 (used without S9 mix)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Actone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537 used without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediame (4NOPD)
Remarks:
5 µg/plate for TA1538 used without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 µg/plate for TA98 used without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
2-Aminoanthracene
Positive controls:
no
Positive control substance:
no
Remarks:
2-aminoanthracene non-mutagenic in absence of S9 used at 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, and 0.5 µg/plate for TA1538 and TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 hours
- Expression time (cells in growth medium):48 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: triplicate plating

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: lawn deficiency and colony reduction

OTHER EXAMINATIONS:
- None
Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting reults or equivocal results are obtained, then a third experiment may be used to confirm the response.

To be considered negative the number of induced revertants comapred to spontaneous revertants should be less than two-fold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the recommended dose of 5000 µg/plate.
Statistics:
All data are statistically analysed using the methods recommended by the UKEMS and normally dunnett's method of linear regression is used to evaluate the result.
Key result
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity to the majority of strains exhibited at 5000 µg/plate without S9-mix only.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated in report
- Effects of osmolality: none stated in report
- Evaporation from medium: none stated in report
- Water solubility: none stated in report
- Precipitation: none stated in report


RANGE-FINDING/SCREENING STUDIES: The test material exhibited toxicity at 5000 µg/plate in the strain of Salmonella used (TA100).

COMPARISON WITH HISTORICAL CONTROL DATA: The results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxicity to the majority of strains exhibited at 5000 µg/plate without S9-mix only.

Please refer to the attachment for detailed tables of results

Conclusions:
The test material was not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA1538 TA98 & TA100.
Executive summary:

Method: The method used conformed with the OECD Guidelines for the Testing of Chemicals, Protocol 471 and also with Method B14 in Commission Directive 92/69/EEC, as available at the time of study conduct..

Salmonella typhimurium strains TS1535, TA1537, TA1538, TA98 and TA100 were treated with the test material by the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system 910% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh chemical formulations. An extra dose was added to the second experiment dose range for bacterial strains without S9 -mix to allow for slight toxicity observed in the first experiment.

Discussion:

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range.

All positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolisng system.

The test material casued a reduction in the growth of the bacterial background lawn with the majority of Salmonella strains testsed without S9 -mix only at the maximum recommended dose level.

No significant increases in the frequency of revertant colonies was recoded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation.

Result: Under the test condition, the test material was not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA,1538, TA98 & TA100.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Note: This study was performed according to the previous version of the OECD guideline 471 (1983). Strain S.typhimuriumTA1598 was included along with S. typhimuriumstrains TA1535, TA1537, TA98 and TA100. No E. coli WP2 or E. coli WP2 (pKM101) strain or S.typhimuriumstrain TA102 was included, as in the current recommended combination of strains (OECD 471, 1997).

However, this deviation from current standards is considered not to have affected the overall assessment of the potential gene mutagenicity of this test material.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23rd February 1999 to 10th June 1999
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
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
date of inspection: March 23rd 1998 Date of signature: July 21st 1998
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Donor: healthy volunteer not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
The average generation time (AGT) for the regular donors has been determined to be approximately 14 hours under optimal growth conditions.
- Type and identity of media: Eagle's MEM, supplemented with sodiumbicarbonate, HEPES buffer, L-glutamine, penicillin,/streptomycin, amphotericin B and 15% FCS. Cell division was simulated by addition of phytohaemagglutinin.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone and beta-naphthoflavone induced rat liver (S9 mix)
Test concentrations with justification for top dose:
Experiment 1 (with and without metabolic activation): 17.81, 35.63, 71.25, 142.5, 285, 570*, 1140* and 2280* µg/mL (up to 10 mM).
Experiment 2 (with and without metabolic activation): 17.81, 35.63, 71.25, 142.5, 285, 570*, 1140* and 2280* µg/mL (up to 10 mM).
* = dose levels selected for metaphase analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO - dimethyl sulphoxide

- Justification for choice of solvent/vehicle: not stated in report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
solvent treatment groups were used as the vehicle control
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
750 and 500 µg/mL in Exp. 1 and 2 respectively, in absence of S9.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
solvent treatment groups were used as the vehicle control
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
25 µg/mL for cultures with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration:
Experiment 1: 4 hours with S9, 4 hours without S9
Experiment 2: 4 hours with S9, 20 hours without S9 (continuous exposure)
- Expression time (cells in growth medium): 16 hours in Experiment 1 (with and without S9) and in Experiment 2 (with S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): demicolcine (Colcemid 0.1 µg/mL)
STAIN (for cytogenetic assays): 5% Gurrs Giemsa R66

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: Where possible, the first 100 consecutive well-spread metaphases from each culture were counted.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

OTHER EXAMINATIONS:
- Frequency of polyploid cells

Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary with the concurrent vehicle control value using Fisher's Exact test or Chi-squared test.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1140 and 2280 µg/mL -S9 (Experiment 1)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant change in pH when the test material was dosed into the media
- Effects of osmolality: did not increase by more than 50mOSM
- Evaporation from medium: not stated in report
- Water solubility: not stated in report
- Precipitation: In Experiment 2, in the parallel cultures without blood a precipitate of the test material was observed at the upper two dose levels. No precipitate could be detected in the blood cultures.
- Other factors: Experiment 2: The test material etched the culture flasks at the position of first contact.

RANGE-FINDING/SCREENING STUDIES: not conducted

COMPARISON WITH HISTORICAL CONTROL DATA: All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes.

ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Experiment 1

The qualitative assessment of the slides determined that there were scorable metaphases at all dose levels, although at the upper two dose levels in the absence of metabolic activation, there was some evidence of a reduction in the number of scorable cells.

Greater than 50% mitotic inhibition was achieved at 1140 and 2250 µg/mL in the absence of S9 but there was no clear evidence of toxicity in the presence of S9.

The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the treatment cases.

Experiment 2

The qualitative assesment of the slides determined that there were scorable metaphases in all dose levels in the presence of metabolic activation. however, the extended exposure period used in the absence of activation had caused complete mitotic inhibition at dose levels greater than 570 µg/mL.

Mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed in the absence of activation and approximately 50% mitotic inhibition was achieved at 285 µg/mL, In the presence of S9 the test material was less toxic, but there was a dose-related reduction in mitotic index giving a value that was 33% lower than the control value at 2280 µg/mL.

The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the treatment cases.

Please refer to Attachments 1 and 2 for table of Mitotic Index and Detailed Results respectively

Conclusions:
The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the presence or absence of a liver enzyme metabolising system in either of two separate experiments. ST 03 C 99 was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Method: The method used followed that described in the OECD guidelines for the Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission directive 92/69/EEC.

Human primary lymphocyte cultures were exposed to the test material diluted in DMSO. Four treatment conditions were used for the study, ie. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system (S9) with cell harvest after a 16-hour expression period and a 4-hour exposure in the absence of activation with a 16-hour expression period, this was Experiment 1. In Experiment 2 the 4-hour exposure with addition of 59 was repeated, whilst in the absence of activation the exposure time was increased to 20 hours. The test material was tested up to limit concentration of 2280 µg/mL (10 mM).

Discussion: All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control treatments gave statistically significant increases in the freqency of cells with aberrations indicating the satisfactory performance of the test and the activity of the metabolising system.

Result: The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the presence or absence of a liver enzyme metabolising system in either of two separate experiments. ST 03 C 99 was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Conclusion: Under the test conditions, the test material was considered to be non-clastogenic to human lymphocytes in vitro. 

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay

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

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration (µg/plate/mL)

Statement

1

 

Safepharm, 1995

Ames Test

(OECD 471)

K, rel. 2

Gene mutation

TA 1535,

TA 1537

TA1538

TA 98

TA 100

 

-S9

+S9

Up to 5000

-S9 : non mutagenic

+S9 : non mutagenic

2

 

Harlan, 2011

CHO/HPRT test (OECD 476)

K, rel. 1

Gene mutation

Chinese hamster CHO cells

-S9

+S9

-S9: Up to 855

+S9: Up to 2280 (10 mM)

-S9 : non mutagenic

+S9 : non mutagenic

3

 

Safepharm, 1999

HL CAT

(OECD 473)

K, rel. 1

Chromosomal aberration

Human Lymphocytes

 

-S9

+S9

Up to 2280 (10 mM)

-S9 : non clastogenic

+S9 : non clastogenic

 

Gene mutation Assays (Tests n° 1-2):

The Bacterial Reverse mutation Assay (Ames test), performed according to OECD 471 test guidelines (1983) with the test material (See Table 1), was selected as the key study. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains in both tests, with any dose of the test material, either with or without metabolic activation. Both tests indicate that the test material does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies.The test material is therefore considered as non-mutagenic according to the Ames test.

Note: Test No.1 was performed according to the previous version of the OECD guideline 471 (1983). Strain S.typhimuriumTA1598 was included along with S. typhimuriumstrains TA1535, TA1537, TA98 and TA100. No E. coli WP2 or E. coli WP2 (pKM101) strain or S.typhimuriumstrain TA102 was included, as in the current recommended combination of strains (OECD 471, 1997). However, this deviation from current standards is considered not to have affected the overall assessment of the potential gene mutagenicity of this test material.

Inability to produce gene mutation was confirmed in mammals using an in vitro forward mutation assay in Chinese hamster HPRT CHO (Test n°2). None of the dose levels up to the limit concentration (10 mM) with S9 and up to cytotoxicity limit without S9, either with or without metabolic activation, induced significant mutant frequency increases in the initial, or repeat tests. The test material does not induce forward mutations at the HPRT locus in CHO Chinese hamster cells under activation and non activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases.The test material is therefore considered as negative for inducing forward mutations at the HPRT locus in CHO Chinese hamster cells under activation and non-activation conditions used in this assay. This result confirms the results of Ames test and extends the non-mutagenic effect of the test material to mammalian cells.

 

Chromosomal aberration (Test n°3)

The clastogenic potential of the test material was determined using an in vitro chromosome aberration test in human lymphocytes, which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the limit concentration (10 mM) with the test material, either with or without metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. The test material does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells.The test material is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay.


Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification is proposed according to the Regulation (EC) No. 1272/2008 (CLP).