Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 701-246-8 | CAS number: -
Toxicological information
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
A total of 4 reliable, guideline studies are available for the substance. An initial Ames test providing negative results was repeated as was conducted without the full complement of strains detailed in the test guideline. The latest Ames test also provided negative results in all strains tested.
An in vitro chromosome aberration assay and an in vitro mammalian cell gene mutation assay using the TK gene also provided negative results.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine deficiency
- Species / strain / cell type:
- other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Preliminary test: 5, 50, 500 and 5000 ug/plate
Main test: 0, 50, 150, 500, 1500 and 5000 ug/plate - Vehicle / solvent:
- Dimethyl sulphoxide was used as the solvent
- Untreated negative controls:
- yes
- Remarks:
- no solvent or test substance
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: N-Ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 2-Nitrofluorene and 2-Aminoanthracene
- Details on test system and experimental conditions:
- For the preliminary toxicity test, 4 concentrations were assessed for toxicity using the five strains. An aliquot of bacterial culture (10 hour culture) and S9 mix or phosphate buffer (pH 7.4) were added to glass bottles. An aliquot of the test solution was added immediately followed by 2ml of histidine deficient agar. This mixture was shaken thoroughly and overlaid onto petri dishes containing 25ml minimal agar. One petri dish was used for each dose level. All plate were incubated for 3 days at 37 degrees C. After incubation, revertant colonies were counted using a Seescan Automatic Colony counter.
An aliquot of bacterial culture (10 hour culture) and S9 mix or phosphate buffer (pH 7.4) were added to glass bottles. An aliquot of the test solution was added immediately followed by 2ml of histidine deficient agar. This mixture was shaken thoroughly and overlaid onto petri dishes containing 25ml minimal agar. Three petri dishes were used for each dose level. A set of plates containing only bacterial culture and S9 mix or phosphate buffer were also prepared (to act as control). All plate were incubated for 3 days at 37 degrees C. After incubation, revertant colonies were counted using a Seescan Automatic Colony counter. This test was repeated at a later date using the same procedure and using the same concentrations of test substance. - Evaluation criteria:
- Mean number of revertant colonies from all treatment groups were compared to the number of colonies obtained for the solvent control groups. The mutagenic potential of the test substance was assessed by the following criteria:
a) If treatment with the test substance causes an increase in revertant colony numbers of at least twice that of the concurrent solvent controls and there is evidence of a positive –dose response relationship in two separate experiments, in any bacterial strain with or without S9 mix, then it is considered to showed evidence of mutagenic activity.
b) If treatment with the test substance does not cause a reproducible increase of at least 1.5 times that of the concurrent solvent control at any dose level or bacterial strain, then it is considered to show no evidence of mutagenic activity.
c) If the result do not sufficiently conclude a ‘positive’ or ‘negative’ response, the following approach is used:
i) Repeat tests may be conducted with modifications of the experimental method. These modifications include the use of a narrower dose range (than that already tested), use of different concentrations of liver homogenate in the S9 mix. If an increase in revertant colony numbers are seen, the substance can be considered to have mutagenic activity.
ii) If no positive response can be seen, then statistical analysis can be performed in the test data to determine the significance of observed increase in revertant colony numbers. - Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The preliminary test showed that toxicity was observed in strain TA98 in the presence and absence of S9 mix and also in strain TA100 in the absence of S9 mix at the highest concentration. Based on these results, 5000ug/plate was selected as the highest dose in the main test.
See tables 1, 2 and 3 for revertant colony counts for the preliminary test, main test 1 and main test 2 respectively. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Under the conditions of this study, no mutagenic activity was detected. - Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Endpoint investigated = chromosome damage
- Species / strain / cell type:
- lymphocytes: Human
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S9 mix
- Test concentrations with justification for top dose:
- First test concentrations: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 ug/ml.
Second test concentrations:
- Without S9 mix, 18 hour harvest: 62.5, 125, 250, 375, 500, 750 and 1000 ug/ml
- With S9 mix, 18 hour harvest: 125, 250, 500 and 1000 ug/ml
- Without S9 mix, 32 hour harvest: 15.6, 31.3, 62.5, 125, 250, 375, 500, 750 and 1000 ug/ml
- With S9 mix, 32 hour harvest: 125, 250, 500 and 1000 ug/ml - Vehicle / solvent:
- Nu-Film-17 (Pinolene) was immiscible in DMSO and so was prepared directly in the tissue culture medium.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: ethyl methanesulphonate, cyclophosphamide
- Details on test system and experimental conditions:
- First test: Human lymphocyte cultures (0.9E06 cells/ml) that had been incubated for 48 hours were centrifuged at 200g. The cell pellets were resuspended in appropriate dosing solutions (5ml) to give the desired final concentrations of Nu-Film-17 (Pinolene) (2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250. 500 and 1000 ug/ml). Duplicate cultures in the absence of S9 mix were prepared for each concentration of the test substance and four cultures were left untreated to serve as the negative control group. For the positive control group, ethyl methansulphonate was used at concentrations of 500 and 750 ul/ml in duplicate cultures. The cultures without S9 mix were incubated for 18 hours.
The test was also performed in duplicate cultures in the presence of S9 mix. For the positive control group with S9 mix, cyclophosphamide was added to duplicate cultures at concentrations of 10 and 15 ug/ml. Three hours after dosing cultures with the S9 mix, these were centrifuged and cell pellets resuspended in fresh medium and incubated for an additional 15 hours.
Second test: Human lymphocyte cultures (1E06 cells/ml) that had been incubated for 48 hours were centrifuged at 200g. The cell pellets were resuspended in appropriate dosing solutions (5ml) to give the desired final concentrations of Nu-Film-17 (Pinolene):
- without S9 mix, 18 hour harvest: 62.5, 125, 250, 375, 500, 750 and 1000 ug/ml
- with S9 mix, 18 hour harvest: 125, 250, 500 and 1000 ug/ml
- without S9 mix, 32 hour harvest: 15.6, 31.3, 62.5, 125, 250, 375, 500, 750 and 1000 ug/ml
- with S9 mix, 32 hour harvest: 125, 250, 500 and 1000 ug/ml
Duplicate cultures were prepared and used for each concentration of the test substance and four cultures were left untreated. For the 32 hour harvest ethyl methansulphonate was used at concentrations of 250 and 500 ul/ml in duplicate cultures (without S9 mix) and cyclophosphamide was used at concentrations of 2.5 and 5 ug/ml (with S9 mix). Three hours after dosing cultures with the S9 mix, these were centrifuged and cell pellets resuspended in fresh medium and incubated for an additional 15 or 29 hours. Cultures that were treated without S9 mix were incubated for 18 or 32 hours.
Harvesting and fixation: Colchicine was added to each culture to stop mitotic activity 2 hours before harvesting. Cell suspensions were centrifuged and cell pellets treated with a hypotonic solution (20% Hanks balanced salt solution). After treatment with the hypotonic solution, cell suspensions were again centrifuged and cell pellets fixed with freshly prepared fixative (3 parts methanol:1 part glacial acetic acid). These pellets were allowed to fixed for 2 hours upon which were centrifuged and resuspended in fresh fixative. These cell suspensions were placed onto microscope slides and allowed to air-dry and then stained with Giemsa and mounted in DPX. - Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Only at highest concentration - not used for metaphase analysis
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Toxicity:
- First test: The mitotic indices of Nu-Film-17 (Pinolene) treated human lymphocytes can be found in table 1. A reduction in mitotic index was observed at the 500 ug/ml dose group in the absence of S9 mix. The higher dose group (1000 ug/ml) was too toxic for analysis, thus the concentrations selected for metaphase analysis were 62.5, 250 and 500 ug/ml. In the presence of S9 mix, no significant reduction in mitotic index was observed when compared to the control value, thus the doses selected for metaphase analysis were 125, 500 and 1000 ug/ml.
- Second test, 18 hour harvest: The mitotic indices of Nu-Film-17 (Pinolene) treated human lymphocytes can be found in table 1. A reduction in mitotic index was observed at the 500 ug/ml dose group in the absence of S9 mix. The higher dose group (1000 ug/ml) was too toxic for analysis, thus the concentrations selected for metaphase analysis were 62.5, 250 and 500 ug/ml. In the presence of S9 mix, no significant reduction in mitotic index was observed when compared to the control value, thus the doses selected for metaphase analysis were 125, 500 and 1000 ug/ml.
- Second test, 32 hour harvest: The mitotic indices of Nu-Film-17 (Pinolene) treated human lymphocytes can be found in table 1. A reduction in mitotic index was observed at the 250 and 375 ug/ml dose groups in the absence of S9 mix, thus the concentrations selected for metaphase analysis were 31.3, 125 and 250 ug/ml. In the presence of S9 mix, a slight reduction in mitotic index was observed in the 1000 ug/ml dose group when compared to the control value. The doses selected for metaphase analysis were 125, 500 and 1000 ug/ml.
Metaphase analysis:
- First test: See table 2 for metaphasis analysis data. No statistically significant increases of chromosome aberrations were observed at any concentration either in the presence or absence of S9 mix. The positive control groups (with ethyl methanesulphonate or cyclophosphamide) caused statistically significant increases of aberrant cells.
- Second test, 18 hour harvest: See table 2 for metaphasis analysis data. No statistically significant increases of chromosome aberrations were observed at any concentration either in the presence or absence of S9 mix. The positive control groups (with ethyl methanesulphonate or cyclophosphamide) caused statistically significant increases of aberrant cells.
- Second test, 32 hour harvest: See table 2 for metaphasis analysis data. There were no statistically significant increases in aberrant cells in any dose group in the presence of S9 mix. However, in the absence of S9 mix there was a statistically significant increase in chromosomal damage in the 250 ug/ml dose group, but this was associated with high cytotoxicity and was not considered to be of biological significance. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Under the conditions of this study, no evidence of clastogenic activity was detected. - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- The objective of this study was to evaluate the mutagenic potential of the substance by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: L5178Y/TK^+/- -3.7.2C mouse lymphoma cells. American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD).
- Normal cell cycle time (negative control):
For cell lines:
- Absence of Mycoplasma contamination: Stock cultures of the cells were stored in the freezer (-150°C). The cultures were checked for mycoplasma contamination. Cell density was kept below 1 x 10^6 cells/mL.
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Horse serum: Horse serum was inactivated by incubation at 56°C for at least 30 minutes.
Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Environmental conditions: All incubations were carried out in a humid atmosphere (80 - 100%, actual range 49 - 98.%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 32.3 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data. - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver microsomal enzymes (S9 homogenate) were prepared from male Sprague Dawley rats that had been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES. The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. The concentration of the S9-fraction in the exposure medium was 4% (v/v). - Test concentrations with justification for top dose:
- Dose range finder: 2, 4, 8, 15 and 20 µg/mL (with and without S9 mix, 3 hour treatment).
Dose range finder: 4, 8, 16, 30 and 40 µg/mL (without S9 mix, 24 hour treatment)
Experiment 1: 0.13, 0.25, 0.5, 1, 2, 4, 8 and 15 µg/mL (with and without S9 mix, 3 hour treatment)
Experiment 2: 0.1, 0.3, 0.5, 1, 2, 4 and 8 µg/mL (without S9 mix, 24 hour treatment) - Vehicle / solvent:
- Tetrahydrofuran
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- Preparation of test item: No correction was made for the purity/composition of the test compound. A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in tetra hydrofuran. Test item concentrations were used within 2 hours after preparation. The final concentration of the solvent in the exposure medium was 0.25% (v/v).
Cell cleansing: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10-4 M hypoxanthine, 2 x 10-7 M aminopterine and 1.6 x 10-5 M thymidine (HAT-medium)
to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
Dose range finding test: In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 10^6 cells (10^6 cells/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hour treatment) with a number of test item concentrations increasing by approximately half log steps. The cell cultures for the 3 hour treatment were
placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test item was tested in the absence and presence of S9-mix. Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 40 μg/mL exposure medium. For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in
50 mL growth medium (R10-medium). For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by
removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle counter. The surviving cells of the 3 hour treatment were subcultured twice to determine cytotoxicity. After 24 hour of subculturing, the cells were counted and subcultured again for another
24 hours, after that the cells were counted. The surviving cells of the 24 hour treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 10^5 cells/mL were counted no subculture was performed. The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests.
Mutagenicity tests: Seven to eight doses of the test item were tested in the mutation assay. The test item was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods. Since the test item was not toxic and difficult to dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium. The highest test item concentrations showed slight precipitate in the exposure medium.
Treatment of the cells: Per culture 8 x 10^6 cells (10^6 cells/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C
and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate. In the first experiment, cell cultures were exposed for 3 hours to the test item in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were
exposed to the test item in exposure medium for 24 hours in the absence of S9-mix.
For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in
50 mL growth medium (R10-medium).
For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle
counter.
Expression period: For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).
Determination of mutation frequency: For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
(Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope. - Rationale for test conditions:
- Standard as per OECD guidelines and based on the results of the range finders.
- Evaluation criteria:
- Determination of mutant colonies: The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations.
The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense
colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.
Calculation of mutant frequency: The mutation frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows: MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 10^6 Small and large colony mutation frequencies were calculated in an identical manner. - Statistics:
- Not a requirement of the study guideline
- 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
- Positive controls validity:
- valid
- Additional information on results:
- Solubility: The test item precipitated in the exposure medium at concentrations of 15 μg/mL and above. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 20 μg/mL.
Dose range finder: In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 2 to 20 μg/mL with a 3 hour treatment period in the absence and presence of S9-mix and a test item concentration range of 4 to 40 μg/mL with a 24 hour treatment period in the absence of S9-mix. In the 3 hour treatment no toxicity in the suspension growth was observed up to and including the highest test item concentration of 20 μg/mL compared to the suspension growth of the solvent control both in the absence of S9-mix and presence of S9-mix. In the 24 hour treatment no toxicity in the suspension growth was observed up to and including the highest test item
concentration of 40 μg/mL compared to the suspension growth of the solvent control.
First mutagenicity experiment: Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test in the absence and presence of S9-mix: 0.13, 0.25, 0.5, 1, 2, 4, 8 and 15 μg/mL exposure medium. No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the
numbers of small and large colonies of the solvent controls.
Second mutagenicity experiment: To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. Based on the results of the dose-range finding test, the following dose levels were selected for
mutagenicity testing: 0.1, 0.3, 0.5, 1, 2, 4, 8 and 15 μg/mL exposure medium.
Further investigation showed that at a concentration of 8 μg/mL the test item already precipitated in the exposure medium after 24 hour treatment.
No significant toxicity was observed. To minimize the selection of too many dose levels with precipitation in the exposure medium, the dose level of 15 μg/mL was not selected for mutation frequency measurement. No biologically relevant increase in the mutation frequency at the TK locus was observed after
treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.
Discussion:
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced
significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The suspension growth over the two-day expression period for cultures treated with tetrahydrofuran was between 15 and 19 (3 hour treatment) and 94 and 98 (24 hour treatment). In the absence of S9-mix, the test item did not induce a biologically relevant increase in the
mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment. In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency. - Conclusions:
- In conclusion, the test substance is not mutagenic in the TK mutation test system under the experimental conditions described in this report.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Remarks:
- E.Coli WP2uvrA
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535, TA1537, TA98, TA100; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA)]
- Suitability of cells: The Salmonella typhimurium strains are checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants. The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain is checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year. - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight). Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.
Preparation of S9-Mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment. - Test concentrations with justification for top dose:
- Dose range finder/first mutation experiment: The test item was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix.
Second mutation experiment: To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of 5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Tetrahydrofuran
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: TA1537: ICR-191 (without metabolic activation), all strains: 2-aminoanthracene (with metabolic activation)
- Details on test system and experimental conditions:
- Preparation of test item: No correction was made for the purity/composition of the test item. A solubility test was performed based on visual assessment. The test item formed a clear light yellow solution in THF. The stock solution was treated with ultrasonic waves until the test item had completely dissolved in the first experiment. Test item concentrations were used within 3 hours after preparation.
Cell cultures:
Preparation of bacterial strains: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/mL). Freshly grown cultures of each strain were used for testing.
Agar plates: Agar plates (ø 9 cm) containing 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma).
Top agar: Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.
Environmental conditions: All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 35.5 - 39.3°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Experimental design:
DRF: Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assays was 5000 μg/plate.
Mutation assay: At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain. The above mentioned dose-range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains. The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.05 mL of a dilution of the test item in THF and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of nonactivation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
Colony counting: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Acceptability criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the test lab.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented. - Rationale for test conditions:
- Standard as per test guidelines
- Evaluation criteria:
- No formal hypothesis testing was done. In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment. - Statistics:
- No statistical analysis conducted
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- DRF/first mutation experiment:
The test item was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.
The test item precipitated on the plates at the dose level of 5000 μg/plate, except in tester strain TA100 in the absence of S9-mix, where no precipitation was observed. To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In strain TA1537 (presence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed. However, since no dose relationship was observed, this reduction is not considered to be caused by toxicity of the test item. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies. No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
Second mutation experiment:
To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of 5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Precipitation of the test item on the plates was observed at concentrations of 1568 μg/plate and upwards. In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix. In the second mutation assay, no increase in the number of revertants was observed upon treatment with Oligomerisation products of beta-pinene under all conditions tested. - Conclusions:
- All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two experiments. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Referenceopen allclose all
Table 1 Preliminary test revertant colonies
| Dose level (ug/plate) | Liver S9 Mix | Strain | ||||
| TA1535 | TA1537 | TA1538 | TA98 | TA100 | ||
| 5000 | - | 16 | 9 | 19 | IL | IL |
| 500 | - | 18 | 9 | 13 | 18 | 111 |
| 50 | - | 13 | 12 | 18 | 26 | 95 |
| 5 | - | 16 | 14 | 16 | 18 | 81 |
| Solvent | - | 10 | 16 | 16 | 19 | 113 |
| 5000 | + | 18 | 12 | 14 | IL | 110 |
| 500 | + | 17 | 11 | 11 | 20 | 129 |
| 50 | + | 16 | 11 | 20 | 22 | 126 |
| 5 | + | 12 | 15 | 12 | 28 | 103 |
| Solvent | + | 15 | 14 | 12 | 28 | 90 |
+ = with S9 mix
- = without S9 mix
IL = incomplete bacterial lawn
Table 2. Revertant colony count from main test 1
| Strain | Dose level (ug/plate) | Liver S9 mix | Mean revertant colony count | Standard deviation | Individual revertant colony count | ||
| Plate 1 | Plate 2 | Plate 3 | |||||
| TA1535 | 5000 | - | 12 | 3.2 | 14 | 13 | 8 |
| 1500 | - | 16 | 3.5 | 16 | 20 | 13 | |
| 500 | - | 16 | 5.5 | 21 | 16 | 10 | |
| 150 | - | 16 | 4.6 | 11 | 17 | 20 | |
| 50 | - | 18 | 4.6 | 21 | 21 | 13 | |
| 0 | - | 16 | 2.6 | 17 | 13 | 18 | |
| Solvent | - | 12 | 5.8 | 19 | 9 | 9 | |
| 5000 | + | 14 | 2.1 | 16 | 12 | 13 | |
| 1500 | + | 13 | 2.1 | 14 | 15 | 11 | |
| 500 | + | 14 | 5.5 | 17 | 8 | 18 | |
| 150 | + | 17 | 0.6 | 17 | 17 | 16 | |
| 50 | + | 12 | 5.6 | 13 | 6 | 17 | |
| 0 | + | 14 | 2 | 16 | 14 | 12 | |
| Solvent | + | 17 | 1 | 17 | 16 | 18 | |
| TA1537 | 5000 | - | 11 | 3.1 | 8 | 14 | 10 |
| 1500 | - | 12 | 6.6 | 13 | 18 | 5 | |
| 500 | - | 10 | 1.7 | 11 | 8 | 11 | |
| 150 | - | 12 | 1.5 | 12 | 10 | 13 | |
| 50 | - | 14 | 4.9 | 20 | 12 | 11 | |
| 0 | - | 12 | 3.8 | 8 | 14 | 15 | |
| Solvent | - | 13 | 3.5 | 10 | 17 | 13 | |
| 5000 | + | 11 | 2.5 | 14 | 9 | 11 | |
| 1500 | + | 13 | 5.6 | 14 | 7 | 18 | |
| 500 | + | 16 | 3 | 19 | 16 | 13 | |
| 150 | + | 11 | 3.5 | 15 | 9 | 9 | |
| 50 | + | 15 | 1.2 | 14 | 16 | 14 | |
| 0 | + | 11 | 2.9 | 13 | 13 | 8 | |
| Solvent | + | 16 | 2.1 | 14 | 18 | 17 | |
| TA1538 | 5000 | - | 12 | 1 | 13 | 11 | 12 |
| 1500 | - | 12 | 3.8 | 14 | 8 | 15 | |
| 500 | - | 10 | 3.1 | 13 | 11 | 7 | |
| 150 | - | 10 | 3 | 10 | 7 | 13 | |
| 50 | - | 8 | 2.5 | 6 | 11 | 8 | |
| 0 | - | 11 | 2.5 | 8 | 11 | 13 | |
| Solvent | - | 11 | 1.2 | 10 | 12 | 10 | |
| 5000 | + | 14 | 2.3 | 15 | 11 | 15 | |
| 1500 | + | 9 | 3.2 | 8 | 7 | 13 | |
| 500 | + | 15 | 4.9 | 18 | 17 | 9 | |
| 150 | + | 13 | 4 | 15 | 15 | 8 | |
| 50 | + | 15 | 2.6 | 16 | 12 | 17 | |
| 0 | + | 12 | 4.6 | 15 | 15 | 7 | |
| Solvent | + | 12 | 4.2 | 7 | 15 | 13 | |
| TA98 | 5000 | - | - | - | IL | IL | IL |
| 1500 | - | 25 | 4.2 | 28 | 26 | 20 | |
| 500 | - | 22 | 5 | 17 | 27 | 23 | |
| 150 | - | 24 | 4.7 | 28 | 26 | 19 | |
| 50 | - | 23 | 3.5 | 19 | 26 | 23 | |
| 0 | - | 22 | 3.8 | 20 | 19 | 26 | |
| Solvent | - | 24 | 3.1 | 27 | 21 | 23 | |
| 5000 | + | - | - | IL | IL | IL | |
| 1500 | + | 28 | 5.7 | 30 | 22 | 33 | |
| 500 | + | 29 | 3.2 | 33 | 28 | 27 | |
| 150 | + | 27 | 4 | 29 | 22 | 29 | |
| 50 | + | 27 | 3.2 | 29 | 23 | 28 | |
| 0 | + | 25 | 4.5 | 29 | 25 | 20 | |
| Solvent | + | 30 | 3.6 | 33 | 26 | 31 | |
| TA100 | 5000 | - | - | - | IL | IL | IL |
| 1500 | - | 108 | 13.7 | 93 | 120 | 110 | |
| 500 | - | 100 | 5 | 95 | 105 | 99 | |
| 150 | - | 104 | 5.3 | 106 | 108 | 98 | |
| 50 | - | 106 | 5.3 | 100 | 108 | 110 | |
| 0 | - | 98 | 4.9 | 95 | 96 | 104 | |
| Solvent | - | 109 | 3.2 | 108 | 107 | 113 | |
| 5000 | + | 115 | 11.4 | 128 | 112 | 106 | |
| 1500 | + | 119 | 4.6 | 122 | 114 | 122 | |
| 500 | + | 113 | 4.7 | 117 | 108 | 115 | |
| 150 | + | 124 | 1.7 | 123 | 123 | 126 | |
| 50 | + | 111 | 19.3 | 133 | 99 | 100 | |
| 0 | + | 106 | 11.1 | 94 | 108 | 116 | |
| Solvent | + | 112 | 13.9 | 103 | 105 | 128 | |
+ = with S9 mix
- = without S9 mix
IL = incomplete bacterial lawn
Table 3. Revertant colony count for main test 2
| Strain | Dose level (ug/plate) | Liver S9 mix | Mean revertant colony count | Standard deviation | Individual revertant colony count | ||
| Plate 1 | Plate 2 | Plate 3 | |||||
| TA1535 | 5000 | - | 15 | 3.1 | 12 | 18 | 16 |
| 1500 | - | 12 | 2.1 | 11 | 14 | 10 | |
| 500 | - | 15 | 0 | 15 | 15 | 15 | |
| 150 | - | 15 | 1.5 | 17 | 15 | 14 | |
| 50 | - | 18 | 2.6 | 21 | 16 | 17 | |
| 0 | - | 13 | 0.6 | 13 | 12 | 13 | |
| Solvent | - | 12 | 2.5 | 15 | 10 | 12 | |
| 5000 | + | 16 | 3.8 | 20 | 13 | 14 | |
| 1500 | + | 17 | 3.2 | 15 | 21 | 16 | |
| 500 | + | 15 | 5.2 | 18 | 9 | 18 | |
| 150 | + | 19 | 2.6 | 22 | 18 | 17 | |
| 50 | + | 17 | 6.6 | 24 | 16 | 11 | |
| 0 | + | 17 | 3.8 | 14 | 15 | 21 | |
| Solvent | + | 19 | 3.5 | 16 | 19 | 23 | |
| TA1537 | 5000 | - | 11 | 2.1 | 9 | 13 | 12 |
| 1500 | - | 12 | 1.7 | 14 | 11 | 11 | |
| 500 | - | 9 | 2.1 | 11 | 8 | 7 | |
| 150 | - | 9 | 2.5 | 9 | 12 | 7 | |
| 50 | - | 15 | 3 | 15 | 18 | 12 | |
| 0 | - | 11 | 2 | 11 | 9 | 13 | |
| Solvent | - | 15 | 4.5 | 10 | 15 | 19 | |
| 5000 | + | 11 | 3.2 | 7 | 13 | 12 | |
| 1500 | + | 11 | 1 | 11 | 10 | 12 | |
| 500 | + | 12 | 4.2 | 11 | 17 | 9 | |
| 150 | + | 12 | 5.7 | 17 | 6 | 14 | |
| 50 | + | 13 | 2.5 | 13 | 10 | 15 | |
| 0 | + | 11 | 1.5 | 9 | 12 | 11 | |
| Solvent | + | 19 | 3.1 | 22 | 16 | 18 | |
| TA1538 | 5000 | - | 11 | 4.6 | 7 | 16 | 10 |
| 1500 | - | 13 | 6 | 7 | 19 | 13 | |
| 500 | - | 15 | 4.5 | 10 | 15 | 19 | |
| 150 | - | 15 | 1 | 15 | 14 | 16 | |
| 50 | - | 14 | 2.1 | 12 | 13 | 16 | |
| 0 | - | 10 | 2.5 | 10 | 13 | 8 | |
| Solvent | - | 17 | 0.6 | 16 | 17 | 17 | |
| 5000 | + | 14 | 1.5 | 16 | 13 | 14 | |
| 1500 | + | 15 | 3.1 | 18 | 14 | 12 | |
| 500 | + | 15 | 3.2 | 17 | 16 | 11 | |
| 150 | + | 8 | 0 | 8 | 8 | 8 | |
| 50 | + | 10 | 2.9 | 12 | 7 | 12 | |
| 0 | + | 11 | 4 | 7 | 10 | 15 | |
| Solvent | + | 15 | 2.6 | 13 | 18 | 14 | |
| TA98 | 5000 | - | - | - | IL | IL | IL |
| 1500 | - | 23 | 3.6 | 22 | 20 | 27 | |
| 500 | - | 24 | 4.2 | 25 | 27 | 19 | |
| 150 | - | 26 | 3.6 | 25 | 30 | 23 | |
| 50 | - | 20 | 2.1 | 22 | 21 | 18 | |
| 0 | - | 23 | 4 | 24 | 19 | 27 | |
| Solvent | - | 23 | 2.6 | 21 | 26 | 22 | |
| 5000 | + | - | - | IL | IL | IL | |
| 1500 | + | 29 | 5 | 34 | 24 | 28 | |
| 500 | + | 23 | 1 | 24 | 22 | 23 | |
| 150 | + | 33 | 2.9 | 31 | 36 | 31 | |
| 50 | + | 28 | 5.8 | 31 | 31 | 21 | |
| 0 | + | 25 | 7 | 25 | 18 | 32 | |
| Solvent | + | 27 | 2.9 | 29 | 29 | 24 | |
| TA100 | 5000 | - | - | - | IL | IL | IL |
| 1500 | - | 104 | 5.3 | 108 | 98 | 106 | |
| 500 | - | 117 | 19.3 | 95 | 128 | 129 | |
| 150 | - | 110 | 2.6 | 109 | 113 | 108 | |
| 50 | - | 101 | 14.2 | 96 | 90 | 117 | |
| 0 | - | 118 | 6 | 112 | 119 | 124 | |
| Solvent | - | 115 | 7.8 | 117 | 121 | 106 | |
| 5000 | + | 95 | 5.7 | 90 | 93 | 101 | |
| 1500 | + | 114 | 26.7 | 93 | 105 | 144 | |
| 500 | + | 126 | 13.7 | 114 | 123 | 141 | |
| 150 | + | 107 | 17.7 | 91 | 104 | 126 | |
| 50 | + | 115 | 4.5 | 111 | 115 | 120 | |
| 0 | + | 129 | 6.1 | 134 | 122 | 130 | |
| Solvent | + | 107 | 16.6 | 100 | 95 | 126 | |
+ = with S9 mix
- = without S9 mix
IL = incomplete bacterial lawn
Table 1. Mitotic indices
| Test substance concentration (ug/ml) | S9 mix | Mean mitotic index (%) | Relative mitotic index (%) | |
| First test, 18 hour harvest | Untreated | - | 3.6 | 100 |
| 2 | - | 4.0 | 111 | |
| 3.9 | - | 4.5 | 125 | |
| 7.8 | - | 4.0 | 111 | |
| 15.6 | - | 4.3 | 119 | |
| 31.3 | - | 4.3 | 119 | |
| 62.5 | - | 3.3 | 92 | |
| 125 | - | 4.4 | 122 | |
| 250 | - | 4.3 | 119 | |
| 500 | - | 1.8 | 50 | |
| 1000 | - | 0.3 | 8 | |
| Untreated | + | 3.8 | 100 | |
| 2 | + | 3.6 | 95 | |
| 3.9 | + | 3.0 | 79 | |
| 7.8 | + | 3.0 | 79 | |
| 15.6 | + | 4.5 | 118 | |
| 31.3 | + | 4.0 | 105 | |
| 62.5 | + | 4.2 | 111 | |
| 125 | + | 4.7 | 124 | |
| 250 | + | 4.0 | 105 | |
| 500 | + | 3.7 | 97 | |
| 1000 | + | 4.9 | 129 | |
| Second test, 18 hour harvest | Untreated | - | 4.0 | 100 |
| 62.5 | - | 3.3 | 83 | |
| 125 | - | 2.9 | 73 | |
| 250 | - | 2.7 | 68 | |
| 375 | - | 2.0 | 50 | |
| 500 | - | 1.8 | 45 | |
| 750 | - | 0.3 | 8 | |
| 1000 | - | 0.0 | 0 | |
| Untreated | + | 2.6 | 100 | |
| 125 | + | 2.5 | 96 | |
| 250 | + | 2.2 | 85 | |
| 500 | + | 2.3 | 88 | |
| 1000 | + | 2.2 | 85 | |
| Second test, 32 hour harvest | Untreated | - | 4.8 | 100 |
| 15.6 | - | 4.2 | 88 | |
| 31.3 | - | 3.0 | 63 | |
| 62.5 | - | 3.3 | 69 | |
| 125 | - | 2.8 | 58 | |
| 250 | - | 1.6 | 33 | |
| 375 | - | 1.6 | 33 | |
| 500 | - | 0.5 | 10 | |
| 750 | - | 0.1 | 2 | |
| 1000 | - | 0.0 | 0 | |
| Untreated | + | 3.6 | 100 | |
| 125 | + | 3.9 | 108 | |
| 250 | + | 3.1 | 86 | |
| 500 | + | 3.5 | 97 | |
| 1000 | + | 2.2 | 61 |
+ = Presence of S9 mix
- = Absence of S9 mix
Table 2. Metaphase analysis
| Test | Test material | Concentration | S9 mix | Number of aberrant cells | |
| Mean % (Excluding gaps) | Mean % (Including gaps) | ||||
| First test | Untreated | - | - | 1.25 | 1.25 |
| Nu-Film-17 (Pinolene) | 62.5 | - | 1.00 | 1.00 | |
| 250 | - | 1.00 | 1.00 | ||
| 500 | - | 2.50 | 2.50 | ||
| Ethyl methanesulphonate | 500 | - | 19.00* | 19.5* | |
| Untreated | - | + | 1.25 | 1.25 | |
| Nu-Film-17 (Pinolene) | 125 | + | 3.50 | 3.50 | |
| 500 | + | 3.00 | 3.50 | ||
| 1000 | + | 3.00 | 3.50 | ||
| Cyclophosphamide | 15 | + | 16.5* | 16.5* | |
| Second test, 18 hour harvest | Untreated | - | 2.50 | 2.50 | |
| Nu-Film-17 (Pinolene) | 32.5 | - | 1.50 | 1.50 | |
| 250 | - | 1.00 | 1.00 | ||
| 500 | - | 4.50 | 4.50 | ||
| Ethyl methanesulphonate | 500 | - | 18.5* | 18.5* | |
| Untreated | - | + | 0.50 | 0.50 | |
| Nu-Film-17 (Pinolene) | 125 | + | 0.00 | 0.00 | |
| 500 | + | 0.00 | 0.50 | ||
| 1000 | + | 0.50 | 1.00 | ||
| Cyclophosphamide | 10 | + | 13.0* | 13.0* | |
| Second test, 32 hour harvest | Untreated | - | - | 1.50 | 1.50 |
| Nu-Film-17 (Pinolene) | 31.3 | - | 0.50 | 0.50 | |
| 125 | - | 4.50 | 4.50 | ||
| 250 | - | 5.5* | 5.5* | ||
| Ethyl methanesulphonate | 500 | - | 32.5* | 32.5* | |
| Untreated | + | 1.50 | 1.50 | ||
| Nu-Film-17 (Pinolene) | 125 | + | 2.00 | 2.00 | |
| 500 | + | 1.00 | 1.00 | ||
| 1000 | + | 1.50 | 1.50 | ||
| Cyclophosphamide | 5 | + | 24.7* | 25.2* | |
* Statistically significant
+ = Presence of S9 mix
- = Absence of S9 mix
Dose | RSG | CE day 2 | RCE | RTG | Mutation frequency per 10^6 survivors | ||
(µg/mL) | (%) | (%) | (%) | (%) | Total | Small | Large |
3-hour treatment (without metabolic activation) | |||||||
SC1 | 100 | 97 | 100 | 100 | 90 | 40 | 46 |
SC2 | 94 | 104 | 45 | 54 | |||
0.13 | 104 | 93 | 97 | 101 | 79 | 26 | 49 |
0.25 | 89 | 85 | 89 | 79 | 107 | 22 | 81 |
0.5 | 68 | 99 | 104 | 71 | 84 | 39 | 41 |
1 | 83 | 89 | 93 | 77 | 92 | 44 | 44 |
2 | 101 | 99 | 104 | 105 | 79 | 17 | 60 |
4 | 52 | 80 | 84 | 44 | 121 | 47 | 68 |
8 | 46 | 93 | 97 | 45 | 115 | 34 | 76 |
15* | 49 | 86 | 91 | 44 | 106 | 32 | 69 |
MMS | 77 | 59 | 62 | 48 | 788 | 427 | 274 |
3-hour treatment (with metabolic activation) | |||||||
SC1 | 100 | 76 | 100 | 100 | 88 | 28 | 57 |
SC2 | 93 | 66 | 19 | 44 | |||
0.13 | 121 | 76 | 90 | 109 | 70 | 21 | 47 |
0.25 | 110 | 95 | 113 | 125 | 64 | 20 | 42 |
0.5 | 100 | 102 | 122 | 122 | 62 | 22 | 38 |
1 | 123 | 93 | 110 | 135 | 98 | 36 | 58 |
2 | 111 | 88 | 104 | 115 | 87 | 24 | 60 |
4 | 106 | 84 | 100 | 106 | 85 | 32 | 51 |
8 | 83 | 91 | 108 | 90 | 76 | 15 | 59 |
15* | 111 | 69 | 82 | 91 | 110 | 32 | 74 |
CP | 107 | 36 | 43 | 46 | 1406 | 705 | 528 |
RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency;
RTG = Relative Total Growth; SC = Solvent control = THF;
MMS = Methylmethanesulfonate; CP = Cyclophosphamide
(*) = the test item precipitated in the exposure medium
Dose | RSG | CE day 2 | RCE | RTG | Mutation frequency per 10^6 survivors | ||
(µg/mL) | (%) | (%) | (%) | (%) | Total | Small | Large |
24-hour treatment (without metabolic activation) | |||||||
SC1 | 100 | 121 | 100 | 100 | 68 | 24 | 41 |
SC2 | 98 | 99 | 18 | 78 | |||
0.1 | 103 | 107 | 97 | 100 | 75 | 16 | 57 |
0.3 | 86 | 88 | 80 | 69 | 94 | 24 | 67 |
0.5 | 88 | 97 | 88 | 78 | 89 | 23 | 63 |
1 | 98 | 99 | 91 | 89 | 90 | 19 | 68 |
2 | 93 | 99 | 91 | 84 | 87 | 29 | 54 |
4 | 89 | 108 | 99 | 88 | 90 | 18 | 69 |
8* | 90 | 89 | 81 | 73 | 94 | 22 | 70 |
MMS | 84 | 93 | 84 | 71 | 547 | 175 | 307 |
RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency;
RTG = Relative Total Growth; SC = Solvent control = THF; MMS = Methylmethanesulfonate
(*) = the test item precipitated in the exposure medium
Experiment 1
Dose (µg/plate) | Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium. | ||
TA1535 | TA1537 | TA98 | |
Without S9 Mix | |||
Positive control | 760 ± 39 | 811 ± 72 | 876 ± 89 |
Solvent control | 9 ± 2 | 6 ± 2 | 9 ± 7 |
52 | 8 ± 6 | 4 ± 2 | 13 ± 3 |
164 | 15 ± 4 | 3 ± 3 | 12 ± 2 |
512 | 7 ± 1 | 6 ± 5 | 8 ± 2 |
1600 | 11 ± 3 NP | 5 ± 3 NP | 12 ± 5 NP |
5000 | 14 ± 4 n SP | 4 ± 1 n SP | 13 ± 4 n SP |
With S9 Mix | |||
Positive control | 282 ± 59 | 253 ± 71 | 945 ± 36 |
Solvent control | 6 ± 1 | 6 ± 2 | 19 ± 5 |
52 | 12 ± 5 | 5 ± 2 | 19 ± 6 |
164 | 6 ± 2 | 6 ± 2 | 23 ± 3 |
512 | 13 ± 2 | 2 ± 2 | 18 ± 8 |
1600 | 12 ± 2 NP | 3 ± 1 NP | 20 ± 3 NP |
5000 | 10 ± 2 n SP | 6 ± 3 n SP | 20 ± 11 n SP |
NP – No precipitate
SP – Slight precipitate
N – Normal bacterial background lawn
Experiment 2
Dose (µg/plate) | Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium. | ||||
TA1535 | TA1537 | TA98 | TA100 | WP2uvrA | |
Without S9 Mix | |||||
Positive control | 878 ± 200 | 909 ± 43 | 1152 ±14 | 822 ± 34 | 1329 ± 124 |
Solvent control | 9 ± 4 | 6 ± 2 | 17 ± 1 | 108 ± 7 | 26 ± 8 |
492 | 8 ± 4 | 3 ± 3 | 15 ± 5 | 105 ± 14 | 22 ± 8 |
878 | 8 ± 3 NP | 6 ± 2 NP | 16 ± 5 NP | 111 ± 12 NP | 20 ± 2 NP |
1568 | 8 ± 3 SP | 3 ± 1 SP | 17 ± 3 SP | 106 ± 3 SP | 25 ± 2 SP |
2800 | 11 ± 2 SP | 5 ± 3 SP | 20 ± 4 SP | 103 ± 14 SP | 25 ± 2 SP |
5000 | 11 ± 3 n SP | 5 ± 2 n SP | 21 ± 6 n SP | 109 ± 13 n SP | 27 ± 5 n SP |
With S9 Mix | |||||
Positive control | 251 ± 6 | 369 ± 76 | 637 ± 76 | 1281 ± 69 | 426 ± 50 |
Solvent control | 8 ± 4 | 5 ± 5 | 15 ± 8 | 68 ± 10 | 32 ± 6 |
492 | 11 ± 3 | 4 ± 1 | 26 ± 3 | 87 ± 15 | 28 ± 7 |
878 | 9 ± 3 NP | 7 ± 4 NP | 18 ± 6 NP | 76 ± 4 NP | 34 ± 8 NP |
1568 | 13 ± 4 SP | 7 ± 2 SP | 27 ± 1 SP | 86 ± 10 SP | 33 ± 6 SP |
2800 | 15 ± 4 SP | 8 ± 6 SP | 18 ± 4 SP | 87 ± 14 SP | 38 ± 14 SP |
5000 | 15 ± 4 n SP | 5 ± 2 n SP | 28 ± 3 n SP | 88 ± 21 n SP | 45 ± 4 n SP |
NP – No precipitate
SP – Slight precipitate
N – Normal bacterial background lawn
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Two in vivo studies are available for the substance including a mouse micronucleus assay and a rat UDS assay. Both provided negative results.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Principles of method if other than guideline:
- Mouse bone marrow micronucleus test
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK), Margate, Kent, England.
- Age: 4 - 5 weeks old on arrival
- Weight: 14.9 - 21.6 g on arrival
- Assigned to test groups randomly: yes, using computer generated random numbers
- Fasting period before study:
- Housing: Animals were housed in polypropylene cages with stainless steel tops.
- Diet: Laboratory animal diet RM1 (E)SQC (Special Diet Services Ltd., Witham, Essex, England) ad libitum
- Water: Drinkning water from the public supply, ad libitum
- Acclimation period: At least 4 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 45 - 60
- Air changes (per hr): 15
- Photoperiod: 12 hour light / 12 hour dark - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle: sterile water
- Details on exposure:
- The test material formed a doseable emulsion when mixed with sterile water (purified by reverse osmosis) at a maximum concentration of 200 mg/ml. Dosing formulations were prepared with sterile water on the day of dosing. The test material or dosing vehicle (sterile water) was administered once in a dose volume of 10 ml/kg via intraperitoneal injection to groups of male and female mice.
- Duration of treatment / exposure:
- Preliminary study: 48 hours
Main study: 24 and 48 hours - Frequency of treatment:
- Single dose (both preliminary and main study)
- Post exposure period:
- 24 hours after treatment, 5 test animals of each sex from each dose group were killed and smears were prepared from bone marrow cells of the femur. 48 hours after treatment the remaining 5 animals (per sex) from the 2000 mg/kg dose group and vehicle control group were killed and smears prepared from the bone marrow cells of the femur.
- Remarks:
- Doses / Concentrations:
Main study: 500, 1000 and 2000 mg/kg
Basis:
nominal in water
intraperitoneal injection - Remarks:
- Doses / Concentrations:
Preliminary study: 500, 1000, 1500 and 2000 mg/kg
Basis:
nominal in water
intraperitoneal injection - No. of animals per sex per dose:
- Preliminary test: 2 animals/sex/dose
Main test: For the 500 and 1000 mg/kg test substance dose groups used 5 animals of each sex. For the 2000 mg/kg test substance dose group and vehicle control group, 10 animals of each sex were used. - Control animals:
- yes
- Positive control(s):
- Chlorambucil (reference substance) was administered by intraperitoneal injection at a dosage of 30 mg/kg in aqueous 10% ethanol.
- Tissues and cell types examined:
- Smears were prepared from the bone marrow cells of the femur.
- Details of tissue and slide preparation:
- Following carbon dioxide inhalation, animals were killed by cervical dislocation. The femurs of each animal were dissected out and cleaned of adherent tissue. The epiphyses were cut off to gain access to the marrow canal. Marrow cells were washed out with 2.5 ml foetal calf serum using a syringe and needle. These cells were centrifuged and the majority of the supernantant fluid was discarded, the remaining fluid was used to resuspend the cell pellet. Single drops of the cell suspension were placed onto clean dry slides, three smears (two smears for the preliminary study) were prepared and the slides left to dry. Cells were then fixed with methanol and stained using 5% Giemsa stain. After staining, slide were washed with buffer, air-dried and cleared in xylene, coverslips were applied using a DPX mountant.
- Evaluation criteria:
- The test substance can be considered to have caused clastogenic or other damage leading to micronucleus production if the following criteria are met:
- A statistically significant increase in micronucleated polychromatic erythrocyte are observed at least one dose levels in either or both sex.
- the increases are reproducible in animals of the same sex and same treatment group(s)
- the increases exceed the lab's historical range of the control group
- Evidence of a dose-response relationship increases in both sexes or increases at more than one sacrifice time will be considered to support the conclusion. - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Remarks:
- weight loss seen in all mice at 1000 and 2000 mg/kg was of uncertain significance
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary study: All animals survived to the scheduled termination. Treatment related effect were observed at all test concentrations and included: underactivity (14 mice), piloerection (4 mice) and flat posture (1 mouse). One male of the 2000 mg/kg dose group was unclean at the base of the tail (observed from day 2 until scheduled termination).
Weight loss was apparent in 16 mice 24 hours after treatment and in 14 mice after 48 hours.
The polychromatic:mature erythrocyte ratios are given in table 1 (below). Bone marrow toxicity was suggested by a reduction in the ratio of polychromatic to mature erythrocytes to 0.7 and 0.6 in mice of the 1500 and 2000 mg/kg dose groups respectively. In view of the small sample of animals used in this test and absence of concurrent control, this data should be treated with caution. From this data the highest dose selected for the main test was 2000 mg/kg.
Main test: although changes in bodyweight were observed in the majority of test animals in this study, these were not considered to be significant changes due to the short timespan between weighings. In the positive control group (chlorambucil) weight loss was observed in all mice during the 24 hour period between dosing and sacrifice. Clinical signs were observed at all concentrations tested, these included: underactivity (29 mice), piloerection (8 mice) and hunched postures (5 mice). Mice of the 2000 mg/kg dose group showed signs that included flat posture (2 mice) and unclean fur at the base of the tail (6 mice).
No statistically significant differences in the frequencies of micronucleated polychromatic cells were observed between sexes in any dose group (see table 2). The positive control group (chlorambucil) produced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes and this increase confirms the sensitivity of the test system.
The polychromatic to mature erythrocyte ratio for each dose group was similar to corresponding vehicle control group values at each sacrifice time. - Conclusions:
- Interpretation of results (migrated information): negative
Under the conditions of this study, no evidence of chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes was obtained following intraperitoneal administration at high dosage. - Executive summary:
In this mouse bone marrow micronucleus assay, 5 animals/sex/dose (the highest dose group and negative control group used 10 animal/sex) were treated intraperitoneally at doses of 0, 500, 1000 and 2000 mg/kg bw. Bone marrow cells were harvested at 24 h (for all dose groups) and 48 hours (high dose group only) post-treatment. The vehicle was sterile water.
There were signs of toxicity during the study which included underactivity (29 mice), piloerection (8 mice) and hunched postures (5 mice). Mice of the 2000 mg/kg dose group showed signs that included flat posture (2 mice) and unclean at the base of the tail (6 mice). The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow in test group animals.
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Remarks:
- Type of genotoxicity: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study, performed under GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
- GLP compliance:
- yes
- Type of assay:
- unscheduled DNA synthesis
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Assigned to test groups randomly: yes
- Housing: Animals were housed in groups of 4 in cages
- Diet: Expanded rat and mouse No.1 maintenance diet (SQC grade obtained from Special Diet Services Ltd, Witham, Essex, UK), ad libitum
- Water: tap water ad libitum
- Acclimation period: 6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 21
- Humidity (%): 36 - 54
- Photoperiod: 12 hours light / 12 hours dark - Route of administration:
- oral: gavage
- Vehicle:
- Corn oil
- Details on exposure:
- Based on the results of a preliminary study, doses of 600 and 2000 mg/kg were selected for use in the main DNA repair test. Animals from all dose groups received test material or vehicle by oral gavage at a dose volume of 10 ml/kg. The negative control group received only the vehicle (corn oil) and the positive control group received 4 mg/kg dimethylnitrosamine in the 2-hour expression group and 50 mg/kg 2-acetylaminofluorene in the 14-hour expression group. Two expression times were used to allow for variations in rate of absorption, metabolism and accumulation of DNA damage.
- Duration of treatment / exposure:
- 2 and 14 hours
- Frequency of treatment:
- Single dose
- Remarks:
- Doses / Concentrations:
600 and 2000 mg/kg
Basis:
other: nominal in corn oil vehicle - No. of animals per sex per dose:
- 4 animals/dose (except for positive control group where only 2 animals were used).
- Control animals:
- yes
- Positive control(s):
- For the 2-hour expression group, dimethylnitrosamine was used as the reference substance. For the 14-hour expression group, 2-acetylaminofluorene was used as the reference substance.
- Tissues and cell types examined:
- Liver (hepatocytes)
- Details of tissue and slide preparation:
- 2 or 14 hours after dosing, each animals was killed using carbon dioxide. Livers were exposed and cannulated (via hepatic portal vein) and EGTA medium perfused for 3 minutes to deplete the liver of calcium ions thus reducing cellular adhesion. The liver was then perfused with a collagenase soltuion, excised and placed into a petri dish containing collagenase solution. Liver suspensions were centrifuged, supernatant discarded and the pellet resuspended in Williams' medium E (complete).
A viable cell count was carried out after diluting an aliquot of cell with an equal volume of trypan blue solution. Using these cell count results, cells from 4 animals in each of the vehicle and test substance dose groups and two animals from the positive control group were selected for plating out.
Isolated cells were suspended in Williams medium E (complete) at a density of 0.2E06 cells/ml. 2ml aliquots of this cell suspension were added to a multi-well tissue culture plate (each well wil a coverslip). 12 replicate cultures were initiated per animal. Cultures were incubated at 37 degrees C in a humid atmosphere with 5% CO2 for at least 90 minutes, allowing hepatocytes to attach to coverslips. After this attachment period, the supernatant was discarded and cell were rinsed (using Williams medium E, incomplete).
The medium was replaced with Williams medium E, incomplete containing high specific activity (methyl-3H)thymidine (specific activity 90 Ci/mmol) for both the 2-hour and 14-hour expression times. Cultures were incubated with this medium for 4 hours. After this period, the supernatant was removed and replaced with Williams medium E (incomplete) containing cold unlabelled thymidine (TdR) and incubated for 20 hours (chase period).
After the chase period, coverslips with attached hepatocytes were removed and washed in Hanks balanced salt solution and fixed in 2.5% v/v acetic acid in ethanol and left to air-dry. They were subsequently mounted onto glass microscope slide using DPX mountant and processed for autoradiography. DNA repair was evaluated by comparing the labelling levels in hepatocytes of treated animals with the control group. - Evaluation criteria:
- A positive response is observed when a statistically significant increase in the net nuclear grain count is accompanied by a substantial increase in the gross nuclear grain count over concurrent control values. A negative response is observed when the mean net nuclear grain count is not significantly greater than the concurrent control. An equivocal response is seen when the results do not meet the criteria specified for a positive or negative response.
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- No mortalities or clinical signs were observed during this test in animals of the treated, vehicle control and positive control groups. Upon examination of slides, animals receiving test substance did not show a statistically significant increase in the gross or net nuclear grain count at any dose level or expression time (see tables 1 and 2). Animals of the positive control groups (dimethylnitrosamine and 2-acetylaminofluorene) demonstrated a significant increase i net nuclear grain count along with an increase in gross nuclear grain count.
- Conclusions:
- Interpretation of results (migrated information): negative
Under the conditions of this study, no significant increase in nuclear grain count was seen at any dose level or timepoint. Thus there was no indication of DNA damage in the livers of treated rats.
Referenceopen allclose all
Table 1. Preliminary study: polychromatic: mature erythrocyte ratios
| Group | Treatment (mg/kg) | Animal number | Polychromatic cells (P) | P:M ratio | |||
| Mature cells (M) | By animal | By sex | By group | ||||
| 1 | 500 | 1M | 1184 | 100 | 1.2 | 1 | 0.9 |
| 2M | 1001 | 1110 | 0.9 | ||||
| 9F | 1013 | 1184 | 0.9 | 0.8 | |||
| 10F | 1042 | 1249 | 0.8 | ||||
| 2 | 1000 | 3M | 1023 | 1092 | 0.9 | 1 | 0.9 |
| 4M | 1017 | 1050 | 1 | ||||
| 11F | 1006 | 1066 | 0.9 | 0.8 | |||
| 12F | 1030 | 1365 | 0.8 | ||||
| 3 | 1500 | 5M | 1000 | 1838 | 0.5 | 0.5 | 0.7 |
| 6M | 954 | 2054 | 0.5 | ||||
| 13F | 1013 | 1090 | 0.9 | 0.8 | |||
| 14F | 1026 | 1419 | 0.7 | ||||
| 4 | 2000 | 7M | 1005 | 1702 | 0.6 | 0.6 | 0.6 |
| 8M | 1004 | 1827 | 0.5 | ||||
| 15F | 1001 | 2022 | 0.5 | 0.6 | |||
| 16F | 1019 | 1593 | 0.6 | ||||
P:M ratio = Ratio of polychromatic to mature erythrocytes
Table 2. Main study. Polychromatic: mature erythrocyte ratio
| Group | Treatment (mg/kg) | Frequency of micronucleated polychromatic erythrocytes | P:M ratio | |
| Mean +/- SD | Range | |||
| 24 hour sacrifice time | ||||
| 1 | Sterile water | 0.6 +/-0.8 | 0.0 - 1.8 | 1 |
| 2 | Nu-Film-17 (500 mg/kg) | 0.9 +/- 0.7 | 0.0 - 2.0 | 0.9 |
| 3 | Nu-Film-17 (1000 mg/kg) | 0.6 +/- 0.6 | 0.0 - 1.5 | 0.9 |
| 4 | Nu-Film-17 (2000 mg/kg) | 0.9 +/- 1.0 | 0.0 - 2.8 | 0.8 |
| 5 | Chlorambucil (30 mg/kg) | 45.6 +/- 18.9* | 20.7 - 87.1 | 1 |
| 48 hour sacrifice time | ||||
| 1 | Sterile water | 1.0 +/- 0.8 | 0.0 - 2.0 | 1 |
| 4 | Nu-Film-17 (2000 mg/kg) | 0.4 +/- 0.6 | 0.0 - 1.8 | 0.9 |
* Highly significant (P<0.01)
P:M ratio: ratio of polychromatic to mature erythrocytes
Table 1. Grain counts for the 2 -hour expression time
| Treatment | Dosage (mg/kg) | Animal | Gross nuclear grain count | Cytoplasmic grain count | Net nuclear grain count | ||||||||||||
| R1 | R2 | R3 | Mean | Group mean | R1 | R2 | R3 | Mean | Group mean | R1 | R2 | R3 | Mean | Group mean | |||
| Vehicle control | - | 1 | 8 | 10.2 | 8 | 8.7 | 8.5 | 8.8 | 13 | 10.1 | 10.6 | 11 | -0.8 | -2.8 | -2.1 | -1.9 | -2.5 |
| 2 | 10.3 | 7.2 | 7.2 | 8.2 | 12.3 | 10.1 | 8.8 | 10.4 | -2 | -2.9 | -1.6 | -2.2 | |||||
| 3 | 9.2 | 8.2 | 7.8 | 8.4 | 12.8 | 11.2 | 10.7 | 11.6 | -3.6 | -3 | -2.9 | -3.2 | |||||
| 4 | 7.5 | 9.4 | 8.5 | 8.5 | 10 | 12.1 | 11.6 | 11.2 | -2.5 | -2.7 | -3.1 | -2.8 | |||||
| Pinolene | 600 | 1 | 6.8 | 5.5 | 7.2 | 6.5 | 7.7 | 10.4 | 7.8 | 9.9 | 9.4 | 10.5 | -3.6 | -2.3 | -2.6 | -2.9 | -2.8 |
| 2 | 7.3 | 7 | 9.9 | 8.1 | 10.7 | 11.4 | 13.2 | 11.8 | -3.4 | -4.3 | -3.2 | -3.6 | |||||
| 3 | 7.6 | 7.3 | 8.2 | 7.7 | 10.3 | 8.3 | 9.8 | 9.5 | -2.7 | -1 | -1.6 | -1.8 | |||||
| 4 | 8.6 | 8.9 | 7.9 | 8.5 | 11 | 12.8 | 10.4 | 11.4 | -2.4 | -3.9 | -2.4 | -2.9 | |||||
| 2000 | 1 | 7 | 10.7 | 8.3 | 8.7 | 7.8 | 9.3 | 13.2 | 10.9 | 11.1 | 10.7 | -2.3 | -2.5 | -2.6 | -2.5 | -2.9 | |
| 2 | 10.4 | 10 | 6.6 | 9 | 13 | 14.3 | 10.6 | 12.6 | -2.6 | -4.3 | -4 | -3.6 | |||||
| 3 | 4.2 | 4.9 | 8.4 | 5.8 | 5.9 | 7 | 9.9 | 7.6 | -1.7 | -2.1 | -1.5 | -1.8 | |||||
| 4 | 7 | 8.3 | 7.4 | 7.6 | 11.3 | 11.9 | 10.9 | 11.4 | -4.2 | -3.5 | -3.5 | -3.7 | |||||
| Dimethylnitrosamine(Positive control) | 4 | 1 | 31.6 | 29.1 | 23.7 | 28.1 | 31.1* | 14.8 | 12.4 | 11.8 | 13 | 12.6 | 16.8 | 16.7 | 11.9 | 15.1 | 18.5* |
| 2 | 36.7 | 27.4 | 38 | 34 | 10.4 | 10.5 | 15.4 | 12.1 | 26.3 | 16.8 | 22.6 | 21.9 | |||||
R1, R2, R3: Replicates
* Significant (P<0.001)
Table 2. Grain counts for the 14 -hour expression time
| Treatment | Dosage (mg/kg) | Animal | Gross nuclear grain count | Cytoplasmic grain count | Net nuclear grain count | ||||||||||||
| R1 | R2 | R3 | Mean | Group mean | R1 | R2 | R3 | Mean | Group mean | R1 | R2 | R3 | Mean | Group mean | |||
| Vehicle control | - | 1 | 7.1 | 7.8 | 9.0 | 8.0 | 8.4 | 9.7 | 10.7 | 12.2 | 10.9 | 10.7 | -2.6 | -2.9 | -3.1 | -2.9 | -2.4 |
| 2 | 7.6 | 7.8 | 8.6 | 8.0 | 10.6 | 11.2 | 11.1 | 11.0 | -3.0 | -3.4 | -2.5 | -3.0 | |||||
| 3 | 9.4 | 8.5 | 10.4 | 9.4 | 12.3 | 9.2 | 12.9 | 11.5 | -3.0 | -0.8 | -2.5 | -2.1 | |||||
| 4 | 8.4 | 8.9 | 6.8 | 8.0 | 10.6 | 9.5 | 8.5 | 9.5 | -2.2 | -0.6 | -1.7 | -1.5 | |||||
| Pinolene | 600 | 1 | 7.6 | 8.6 | 7.2 | 7.8 | 8.4 | 9.6 | 10.6 | 8.3 | 9.5 | 10.8 | -2.0 | -2.0 | -1.2 | -1.7 | -2.4 |
| 2 | 8.3 | 9.6 | 7.0 | 8.3 | 9.8 | 13.7 | 10.9 | 11.5 | -1.5 | -4.1 | -3.9 | -3.2 | |||||
| 3 | 7.8 | 8.1 | 8.2 | 8.0 | 9.9 | 11.5 | 10.5 | 10.6 | -2.1 | -3.4 | -2.4 | -2.6 | |||||
| 4 | 8.5 | 10.3 | 9.7 | 9.5 | 10.2 | 13.3 | 11.5 | 11.7 | -1.7 | -3.1 | -1.8 | -2.2 | |||||
| 2000 | 1 | 8.1 | 11.0 | 9.8 | 9.6 | 8.9 | 11.1 | 14.8 | 12.9 | 12.9 | 11.6 | -3.0 | -3.7 | -3.1 | -3.3 | -2.7 | |
| 2 | 11.9 | 11.5 | 8.8 | 10.7 | 16.5 | 15.4 | 12.1 | 14.7 | -4.6 | -3.9 | -3.3 | -3.9 | |||||
| 3 | 7.1 | 9.3 | 6.5 | 7.6 | 9.9 | 10.3 | 8.9 | 9.7 | -2.7 | -1.0 | -2.5 | -2.1 | |||||
| 4 | 8.4 | 6.9 | 7.9 | 7.7 | 9.4 | 8.3 | 9.3 | 9.0 | -1.0 | -1.4 | -1.4 | -1.3 | |||||
| 2-acetylaminofluorene (Positive control) |
50 | 1 | 28.4 | 37.9 | 37.0 | 34.4 | 35.0* | 13.0 | 13.7 | 19.2 | 15.3 | 14.3 | 15.4 | 24.2 | 17.8 | 19.1 | 20.7* |
| 2 | 31.7 | 28.2 | 46.8 | 35.6 | 14.6 | 10.0 | 15.3 | 13.3 | 17.2 | 18.3 | 31.5 | 22.3 | |||||
R1, R2, R3: Replicates
* Significant (P<0.001)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
No evidence of mutagenic activity has been found across a range of in vitro and in vivo studies which addressed different genetic endpoints. Based on the data no classification is warranted for mutagenicity under the CLP Regulation (EC No. 1272/2008, as adapted).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
