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EC number: 273-920-5 | CAS number: 69226-44-4
- Life Cycle description
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- Endpoint summary
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- Aquatic toxicity
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- Short-term toxicity to fish
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene Mutation study in Bacteria
Read-Across Studies performed on structurally similar substances:
- DOTE (CAS No 15571-58-1)
- DOTI (CAS No 26401-97-8), and
- DOT bis-(ethylmaleate) (CAS 68109-88-6)
Findings from the 8 studies that are available on the read across substances are considered together in a weight of evidence approach to conclude that the registered substance is unlikely to be mutagenic when tested under the Ames test either with or without metabolic activation.
Mouse Lymphoma Assay (Read-Across Study performed on structurally similar substance (DOTE)(CAS No 15571 -58 -1))
It is concluded that under the conditions used in this study, the test material DOTE is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01 December 2009 to 02 March 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)
- Target gene:
- TK gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- The chromosome number of these cells is 40 (stable aneuploid karyotype, 2n = 40).
The cells were stored as frozen stock cultures in liquid nitrogen.
Each new stock culture is checked for mycoplasma contamination, which was absent.
Cell culturing : The L5178Y cells were grown in culture medium consisting of RPMI 1640 medium (with HEPES and Glutamax-I) supplemented with heat-inactivated horse serum (10 % v/v for growing in flasks, and 20 % for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin.
On the day of exposure, the growth rate (doubling time of 9-14 h) and viability (>90 %; by trypan blue exclusion) of the cells were checked. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver homogenate
- Test concentrations with justification for top dose:
- FIRST test: Without S9 (24hr of treatment): 0.006-10 µg/ml ; With S9 (4 hr of treatment) : 0.01-20 µg/ml
SECOND test: Without S9 (4hr of treatment): 0.86-20 µg/ml; Without S9 (24hr) : 0.02-0.40µg/ml; With S9 : 1.6-60 µg/ml
THIRD test: Without S9 (24hr of treatment): 0.007-0.5 µg/ml ; With S9 (4hr of treatment): 1.4-100 µg/ml
With S9 (Third test, 4 hr of treatment): 1.4-100 µg/ml (11 concentrations) - Vehicle / solvent:
- Ethanol.
The test material was diluted in ethanol to concentrations ranging from 0.04 to 10 mg/ml DOTE. From these stock solutions serial dilutions in ethanol were made and from each of these 0.1 ml were added to a final volume of 10 ml culture medium. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (ethanol)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 4 hours or 24 hours (37°C, 5% CO2)
- Exposure duration: 20-24, and 44-48 hours (37°C, 5% CO2)
- Expression time (cells in growth medium): doubling time of 9-14 hours
- Selection time (if incubation with a selection agent): no data
NUMBER OF REPLICATIONS: single culture for each dose, 2 for negative control group, 1 for positive control group
NUMBER OF CELLS EVALUATED: 2000/well
DETERMINATION OF CYTOTOXICITY
- Method: by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG)
OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no - Evaluation criteria:
- The following criteria were used to validate the data obtained in the gene mutation assay (Cole et al., 1990; Aaron et al. 1994; Clive et al., 1995):
a) the average cloning efficiency of the negative controls should not be less than 60 % or more than 140 %.
b) the average suspension growth of the negative controls should be between 8 and 32.
c) the average mutant frequency of the negative controls should fall within the range of 40-300 TFT-resistant mutants per 1,000,000 clonable cells.
d) the mutant frequency of the positive controls should be higher than 400 TFTresistant mutants per 1,000,000 clonable cells, and should be at least twice that of the corresponding negative control.
e) unless the test substance shows no cytotoxicity at the highest possible concentration (determined by its solubility, pH and osmolar effects), the highest concentration should result in a clear cytotoxic response. The RTG value of one of the data points should be between 10 and 20 %, or one data point between 1 and 10% and another between 20 and 30 %.
The test material was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.
The test material was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test material concentrations. - Statistics:
- no statistical analysis was performed.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- But increase at 72 µg/ml but this increase is not indicative for mutagenicity.
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- Results without S9 (see tables 1 and 2) :
In the absence of S9-mix three assays were performed in which mutagenicity was evaluated; two with 24 hours exposure and one with 4 hours exposure. In the first assay with 24 hours exposure the RTG at the highest concentration was 22 % (not between 10 and 20 %) and only six concentrations could be evaluated due to severe cytotoxicity. In the second assay with 24 hours exposure (results obtained in third test), sufficient cytotoxicity was observed at the highest concentration (RTG of 13 %) and nine concentrations could be evaluated resulting in clear to hardly any cytotoxic response. In the assay with 4 hours exposure (second test) at and above 5 μg/mL no clear dose response in cytotoxicity was observed, however, sufficient toxicity (RTG between 10 and 20 %) was observed for a valid test.
In none of the three assays performed in the absence of S9-mix any increase in mutant frequency (MF) by more than 88 or 126 mutants per 1,000,000 clonable cells, i. e. no equivocal or positive response, compared to the negative control was observed at any dose level.
Results with S9 (see table 3) :
In the presence of S9-mix two assays were performed in which mutagenicity was evaluated. In the first assay not sufficient cytotoxicity was observed at the highest concentration of 20 μg/mL (RTG was 38 %; not between 10 and 20 %). In the second assay (results obtained in third test), although relatively small intervals between the concentrations were used, none of the concentrations resulted in an RTG between 10 and 20 %. Above 44 μg/mL no dose response in cytotoxicity was observed. The RTG at 85 μg/ml was 30% whereas at 72 μg/ml the RTG was 5%. In addition, the relative suspension growth (RSG) at 61 and 85 μg/mL were 24 and 21%, respectively. This means that already in this phase almost 80 % cytotoxicity was observed, however, since the initial cell yield and cloning efficiencies at these concentrations were above 100 % (which is very unusual) this resulted not in RTG values below 20 %. In the presence of S9-mix at 72 μg/mL the mutant frequency was significantly increased by 238 mutants per 1,000,000 clonable cells compared to the negative control. Since relatively small intervals (0.85) were used and the increase was observed at a single concentration causing more than 90% cytotoxicity, it is concluded that this increase is not indicative for mutagenicity.
Colony sizing :
Colony sizing was performed in the third test at 2 concentrations in the presence of S9-mix, since a positive response in mutant frequency was observed. Both at 72 and 85 μg/mL relatively more small then large colonies were formed; 68 and 65 % compared to 32 and 35 %, respectively.
Since the increase in mutant frequency at 72 μg/mL was considered to be not relevant, no conclusions were drawn from these findings.
Positive and negative controls :
Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively; Ethanol served as negative control. The negative controls were within historical background ranges and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls.
RANGE-FINDING/SCREENING STUDIES:
Prior to the main study two dose range finding studies were performed; single cultures were exposed for 24 hours in the absence of S9-mix to 5 concentrations of DOTE ranging from 312 to 5000 μg/mL and 9.4 to 150 μg/mL, in the first and second study, respectively.
COMPARISON WITH HISTORICAL CONTROL DATA:
The negative controls were within historical background ranges and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls. - Conclusions:
- It is concluded that under the conditions used in this study, the test material DOTE is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.
The test material DOTE was tested and evaluated for mutagenicity in both the absence and presence of metabolic activation (S9-mix). In the absence of S9-mix beside treatment for 4 hours, an extended treatment for 24 hours was used. In the three assays performed sufficient cytotoxicity was observed to validate the concentrations used and in none of the assays any indication for a mutagenic potential was observed. - Executive summary:
The test material DOTE was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). Three tests were conducted; in the first and third test single cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively; in the second test cultures were treated for 24 hours and 4 hours in the absence of S9-mix and 4 hours in the presence of S9-mix. The test material was dissolved in ethanol prior to testing.
The highest concentrations of DOTE evaluated for mutagenicity in the absence of S9-mix, were 20 μg/mL and 0.36 μg/mL at 4 and 24 hours exposure, respectively. In the presence of S9-mix and 4 hours exposure, the highest concentration evaluated was 85 μg/mL. The maximum concentrations were limited by cytotoxicity.
DOTE was cytotoxic in both the absence and presence of S9-mix. In the absence of S9-mix and 24 hours exposure cytotoxicity, resulting in a reduction in initial cell yield and/or suspension growth, was observed at and above 0.1 μg/mL. The relative total growth (RTG) at the highest concentration evaluated in the first and third test were 22 and 13 %, respectively. In the absence of S9-mix and 4 hours exposure cytotoxicity was observed at all concentrations included (as from 0.86 μg/mL). The relative total growth (RTG) at the highest concentration evaluated in the second test was 20 %.
In the presence of S9-mix cytotoxicity was observed at and above 1.4 μg/mL. The relative total growth (RTG) at the highest concentration evaluated in the first and third test were 38 and 30 %, respectively. In the absence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. In the presence of S9-mix at 72 μg/mL the mutant frequency was significantly increased by 238 mutants per 1,000,000 clonable cells compared to the negative control. Since relatively small intervals (0.85) were used and the increase was observed at a single concentration causing more than 90 % cytotoxicity compared to six concentrations causing 50-70 % cytotoxicity which showed no increase in mutant frequency, it is concluded that this increase is not indicative for mutagenicity. It is very likely a secondary effect to cytotoxicity and thus not indicative for mutagenicity.
Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and presence of the S9-mix, respectively; ethanol (vehicle) served as negative control. The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls.
It is concluded that under the conditions used in this study, the test material DOTE is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Not specified
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Principles of method if other than guideline:
- Three hundred chemicals were tested for mutagenicity, under code, in Salmonella typhimurium, using a preincubation protocol. All tests were performed in the absence of exogenous metabolic activation, and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His
- Species / strain / cell type:
- other: Salmonella typhimunum strains TA97, TA98, TA100, TA1535, and TA1537
- Details on mammalian cell type (if applicable):
- - Source: Dr. Bruce Ames (University of California, Berkeley)
- Cultures were grown overnight with shaking at 37 °C in Oxoid No. 2 broth, and their phenotypes were analysed prior to their use for mutagenicity assays. - Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 fractions
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 4-nitro-o-phenylenediamine for TA98 in the absence of metabolic activation.With metabolic activation for all strains: 2-aminoanthracene
- Remarks:
- sodium azide (TA1535 and TA 100), 9-aminoacridine (TA97 and TA 1537), and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.
- Details on test system and experimental conditions:
- TEST PROCEDURE
- The pre-incubation assay was performed, with some differences, as described below. The test chemical (0.05 mL), Salmonella culture (0.10 mL), and S-9 mix or buffer (0.50 mL) were incubated at 37 °C, without shaking, for 20 min. Chemicals known or suspected to be volatile were incubated in capped tubes. The top agar was added and the contents of the tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium.
- The histidine-independent (his') colonies arising on these plates were counted following two days incubation at 37 °C. Plates were machine counted unless precipitate was present which interfered with the count, or the colour of the test chemical on the plate reduced the contrast between the colonies and the background agar. At the discretion of the investigators, plates with low numbers of colonies were counted by hand.
- Variations in the protocol among the tested chemicals reflect the evolution of the protocol originally described by Haworth et al. Four protocol variations are evident from the data.
1) Testing in strains TA97, TA98, TA100, and TA1535, with some additional testing in strain TA1537; 10 % S-9 was used.
2)The first test of a chemical was without activation and with 10 % S-9 in the S-9 mix. If a positive result was obtained the test was repeated. If the tests were negative they were repeated without S-9 and with 30 % S-9.
3) The order of use of 10 % and 30 % S-9 was reversed.
4) Initial testing was in strains TA98 and TA100 without activation and with 30 % rat and hamster S-9s. If a positive result was obtained in one of these two strains it was repeated and the other strains were not used. If the tests were negative, the other strains were used with 30 % and 10 % S-9. A chemical was not designated non-mutagenic unless it had been tested in strains TA98, TA100, TA1535, and TA97 and/or TA1537, without activation and with 10 % and 30 % rat and hamster S9. Occasionally, 5 % S-9 was also used in all protocol variations.
- All chemicals were tested initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100 or the system developed by Waleh et al. [1982]. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.
- Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in the preliminary toxicity test. Subsequent trials occasionally used narrower dose increments and may not have included doses in the toxic range. Chemicals that were not toxic were tested, with few exceptions, to a maximum dose of 10 mg/plate. Chemicals that were poorly soluble were tested up to doses defined by their solubility’s. At least five doses of each chemical were tested in triplicate. Experiments were repeated at least one week following the initial trial. A maximum of 0.05 mL solvent was added to each plate.
- Concurrent solvent and positive controls were run with each trial. The positive controls in the absence of metabolic activation were sodium azide (TA1535 and TA 100), 9-aminoacridine (TA97 and TA 1537), and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene. Occasionally, a laboratory used a single solvent and/or positive control for more than one chemical tested on the same day. Each laboratory made its own determination regarding positive control dose levels. - Evaluation criteria:
- DATA EVALUATION
- Evaluations were made at both the individual trial and overall chemical levels. Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+W,” if only a single dose was elevated over the control, or if the increase seen was not dose related. The distinctions between a questionable mutagenic response and a non-mutagenic or weak mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach twofold over background for a chemical to be judged mutagenic.
- A chemical was judged mutagenic (+) or weakly mutagenic (+ W) if it produced a reproducible dose-related response over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials.
- Chemicals were judged non-mutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or non-mutagenicity. - Key result
- Species / strain:
- S. typhimurium, other: TA97, TA98, TA100, TA1535, and TA1537
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- The test material was reported to be mutagenic to the Salmonella typhimunum strains TA97, TA98, TA100, TA1535, and TA1537.
- Executive summary:
The genetic toxicity of the test material was investigated in a study that followed similar principles to the guideline OECD 471.
The test material was examined using the pre-incubation assay, both in the presence and absence of metabolic activation in the form of S9 fractions. At least five doses of the test material were tested in triplicate.
The test material was reported to be mutagenic to the Salmonella typhimunum strains TA97, TA98, TA100, TA1535, and TA1537.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Not reported
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- S. typhimurium: Histidine locus
- Species / strain / cell type:
- S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
- Species / strain / cell type:
- Saccharomyces cerevisiae
- Remarks:
- Strain D4
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 5.000, 1.000, 0.100, 0.010, 0.001 µL/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: deionised water or DMSO
- Methylnitrosoguanidine: Water
- 2-Nitrofluorene: DMO
- Quinacrine mustard: Water
- 2-Anthramine: DMSO
- 2-Acetylaminofluorene: DMSO
- 8-Aminoquinoline: DMSO - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water/ DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- other: Methylnitrosoguanidine, Quinacrine mustard, 2-Anthramine, 2-Acetylaminofluorene, 8-Aminoquinoline
- Details on test system and experimental conditions:
- - Approximately 10^8 cells from an overnight culture of each indicator strain were added to separate test tubes containing 2.0 mL of molten agar supplemented with biotin and a trace of histidine. For non-activation tests, at least four dose levels of the test material were added to the contents of the appropriate tubes and poured over the surfaces of selective agar plates. In activation tests, a minimum of four different concentrations of the test material were added to the appropriate tubes with cells. Just prior to pouring, an aliquot of reaction mixture (0.5 mL containing the 9,000 x g liver homogenate) was added to each of the activation overlay tubes, which were then mixed, and the contents poured over the surface of a minimal agar plate and allowed to solidify.
- The plates were incubated for 48 hours at 37 °C, and scored for the number of colonies growing on each plate.
- Positive and solvent controls using both directly active positive chemicals and those that require metabolic activation were run with each assay.
- The numbers of colonies on each plate were counted and recorded on printed forms. These raw data were analysed in a computer program and reported on a printout. - Evaluation criteria:
- Evaluation Criteria for Ames Assay:
Because the procedures used to evaluate the mutagenicity of the test chemical are semi-quantitative, the criteria used to determine positive effects are inherently subjective and are based primarily on a historical data base. Most data sets are evaluated using the following criteria:
- Strains TA1535, TA1537, and TA1538
If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic.
- Strains TA98, TA100, and D4
If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the highest increase equal to twice the solvent control value for TA100 and two to three times the solvent control value for strains TA98 and D4 is considered to be mutagenic. For these strains, the dose response increase should start at approximately the solvent control value.
- Pattern
Because TA1535 and TA100 were both derived from the same parental strain (G-46) and because TA1538 and TA98 were both derived from the same parental strain (D3052), there is a built-in redundancy in the microbial assay. In general the two strains of a set respond to the same mutagen and such a pattern is sought. It is also anticipated that if a given strain, e.g. TA1537, responds to a mutagen in non-activation tests it will generally do so in activation tests. (The converse of this relationship is not expected.) While similar response patterns are not required for all mutagens, they can be used to enhance the reliability of an evaluation decision.
- Reproducibility
If a chemical produces a response in a single test that cannot be reproduced in one or more additional runs, the initial positive test data loses significance. - Key result
- Species / strain:
- S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Saccharomyces cerevisiae
- Remarks:
- D
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TOXICITY TEST RESULTS
The test material was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The dose range employed for the evaluation of this compound was from 0.001 to 5 µL/ plate.
WITHOUT METABOLIC ACTIVATION
The results of the tests conducted on the test material in the absence of a metabolic system were all negative.
WITH METABOLIC ACTIVATION
The results of the tests conducted on the test material in the presence of the rat liver activation system were all negative.
CONCLUSIONS
The test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic under these test conditions. - Conclusions:
- Under the conditions of the study, the test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic with or without metabolic activation.
- Executive summary:
The genetic toxicity of the test material was investigated in a bacterial reverse mutation assay, broadly similar to OECD 471.
The test material was tested at 5.000, 1.000, 0.100, 0.010, 0.001 µL/plate both with and without metabolic activation, in the form of S9-mix. The following bacterial strains were used: Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA1538, and Saccharomyces cerevisiae strain D4. The overlay method of application was used.
The plates were incubated for 48 hours at 37°C, and scored for the number of colonies growing on each plate. Positive and solvent controls using both directly active positive chemicals and those that require metabolic activation were run with each assay. The numbers of colonies on each plate were counted and recorded.
The test material was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The results of the tests conducted on the test material in the absence of a metabolic system were all negative. The results of the tests conducted on the test material in the presence of the rat liver activation system were all negative.
Under the conditions of the study, the test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic with or without metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- November 1977 to June 1978
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Principles of method if other than guideline:
- The 1978 study was repeated with TA100 at higher test material concentrations due to the slightly increased (and dose-dependent) number of mutants of Salmonella typhimurium TA 100 in the highest tested concentration in the absence of metabolic activation
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- - Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Species / strain / cell type:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- - 0.5, 1, 2, 4, 8, 16, 32, 64 µL/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: Anthracen-2-amine
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- precipitate in agar at highest tested concentration
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- - Nonactivation Test Results: The results of the tests conducted on the compound in the absence of a metabolic system were all negative.
- Activation Test Results: The results of the tests conducted on the compound in the presence of the rat liver activation system were all negative. - Conclusions:
- Under the conditions of this study the test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic under these test conditions.
- Executive summary:
The 1978 study was repeated with TA100 at higher test material concentrations due to the slightly increased (and dose-dependent) number of mutants of Salmonella typhimurium TA 100 in the highest tested concentration in the absence of metabolic activation.
The results of the tests conducted on the test material in the absence of a metabolic system were all negative. The results of the tests conducted on the test material in the presence of the rat liver activation system were all negative.
Under the conditions of this study the test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic under these test conditions.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- Restrictions : The number of replicates per test substance and control groups was not reported.
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Missing TA 102 and E.coli strains, The number of replicates per test substance and control groups was not reported.
- Principles of method if other than guideline:
- Study conducts prior to implementation of corresponding guideline. Study performed in accordance to the publications of Ames. The later implemented guideline based to a great extent on these publications.
Method: Ames, B.N., J. McCann, and E. Yamasaki. 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Res. 31:347-364. - GLP compliance:
- no
- Remarks:
- Study predates GLP.
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his
- Species / strain / cell type:
- S. typhimurium, other: TA98, TA1535, TA1537, and TA1538
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- Saccharomyces cerevisiae
- Remarks:
- D4
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 from Sprague-Dawley adult male rat liver induced by Aroclor 1254 five days before sacrifice. The reaction mixture contained 4 mM sodium salt (TPN), 5 mM glucose-6-phosphate, 100 mM sodium phosphate, 8 mM MgCl2, 33 mM KCl, and 0.1 +/- 0.05 ml S9
- Test concentrations with justification for top dose:
- 0.005, 0.01, 0.1, 1.0, 5.0, and 10.0 ul/plate (20.0 ul/plate was used for strain TA1537 without activation)
- Vehicle / solvent:
- acetone
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: without metabolic activation: n-Methyl,n-nitro,n-nitrosoguanidine (10 ug/plate) for strains TA1535 and S. cerevisiae D4. 9-Aminoacridine (50 ug/plate) for strain TA1537. 2-Nitrofluorene (10 ug/plate) for strains TA1538 and TA98. with metabolic activation
- Details on test system and experimental conditions:
- Approximately 10E8 cells from each bacterial strain were added to separate test tubes containing 2.0 ml molten agar supplemented with biotin and a trace of histidine.
For tests with metabolic activation, 0.5 ml containing 9,000 x g liver homogenate was added to the appropriate test tubes. The contents of the tubes were mixed and poured over the surface of minimal agar plates and allowed to solidify. The plates were incubated at 37 °C for 48 hours (D4 yeast plates were incubated at 30 °C for 3-5 days). The number of colonies on each plate were scored and counted.
Positive and negative control assays were conducted concurrently. - Evaluation criteria:
- not reported
- Statistics:
- not reported
- Species / strain:
- S. typhimurium, other: TA98, TA1535, TA1537, and TA1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Toxic to the strain TA1537 at 10 and 20 µL/plate and to the strain TA1538 at 10 µL/plate.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- Saccharomyces cerevisiae
- Remarks:
- D4
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 10 µL/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Solvent and positive controls produced the expected mutant colony counts. The test substance was found to be toxic to the strain TA1537 at 10 and 20 ul/plate and to the strains TA1538 and D4 at 10 ul/plate.
- Conclusions:
- In this study, the test material gave negative results.
- Executive summary:
An 'Ames' test was carried out with a mixture of DOT(2 -EHMA) and MOT(2 -EHMA) (70:30% w/w), with and without metabolic activation. No mutagenic effects were seen for all strains tested in the presence of a metabolic activation system. The test substance was found to be toxic to the strain TA1537 at 10 and 20 µL/plate and to the strains TA1538 and D4 at 10 µL/plate.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- Restrictions : The number of replicates were not reported.
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Study conducts prior to implementation of corresponding guideline. Study performed in accordance to the publications of Ames. The later implemented guideline based to a great extent on these publications.
The test was performed in accordance with the method of Ames et al. (1975): [Ames, B.N., J. McCann, and E. Yamasaki. 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Res. 31:347-364.] - GLP compliance:
- no
- Remarks:
- Study predates GLP.
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- without
- Test concentrations with justification for top dose:
- 0.005, 0.01, 0.1, 1.0, 5.0, and 10 µL/plate
- Vehicle / solvent:
- acetone
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- not specified
- Positive control substance:
- not specified
- Details on test system and experimental conditions:
- The test material was dissolved in acetone (negative control) and was evaluated for mutagenicity at 0.005, 0.01, 0.1, 1.0, 5.0, and 10.0 µL/plate. Plates were incubated at 37 ± 1.5 °C in the absence of light for 48 hours.
- Evaluation criteria:
- The test material was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled in any concentration.
- Statistics:
- The colonies were counted and the arithmetic mean was calculated.
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- not examined
- Additional information on results:
- In the absence of a metabolic activation system, the test substance tested positive for mutagenic activity with the strain TA100. The results were confirmed in a repeat test conducted at 1, 5, 10, and 20 µL/plate.
- Conclusions:
- In this study, DOT(EHMA) was positive in strain TA100 without S9.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Remarks:
- Restrictions : Test was not conducted using E. coli strain WP2 uvrA. Positive controls for the strains TA98, TA1537, and TA1538 were not included in the test with metabolic activation.
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Test was not conducted using E. coli strain WP2 uvrA. Positive controls for the strains TA98, TA1537, and TA1538 were not included in the test with metabolic activation.
- Principles of method if other than guideline:
- Study conducts prior to implementation of corresponding guideline. Study performed in accordance to the publications of Ames. The later implemented guideline (1981) based to a great extent on these publications.
Ames, B.N., W.E. Durston, E. Yamasaki, and F.D. Lee. 1973. Carcinogens are mutagens: A simple test system combining liver homogenates for
activation and bacteria for detection. Proc. Natl. Acad. Sci. USA.
70:2281-2285.
Ames, B.N., F.D. Lee, and W.E. Durston. 1973. An improved bacterial test system for the detection and classification of mutagens and
carcinogens. Proc. Natl. Acad. Sci. USA. 70:782-786.
Ames, B.N., J. McCann, and E. Yamasaki. 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome
mutagenicity test. Mutation Res. 31:347-364. - GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his
- Species / strain / cell type:
- S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix from TiF:RAIF (SPF) rat liver induced with Aroclor 1254.
- Test concentrations with justification for top dose:
- 300, 900, 2700, 8100, and 24,300 µg/0.1 mL
- Vehicle / solvent:
- The test material was dissolved in acetone.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: see Details on test system and conditions
- Details on test system and experimental conditions:
- The test was conducted with triplicate replications. Positive and negative controls were tested concurrently.
In order to confirm the test results, a confirmatory test was performed. The S9 microsomal fraction (for tests with metabolic activation) was obtained from Analabs, Inc., North Haven, CT, USA. One mL of the activation mixture contained 0.3 mL S9 fraction and 0.7 mL of a co-factor solution.
Positive controls (without metabolic activation): Daunorubicin-HCl at 5 and 10 µg/0.1 mL phosphate buffer for TA98. 4-Nitroquinoline-N-oxide at 0.125 and 0.25 µg/0.1 mL phosphate buffer for TA100. n-Methyl-n'-nitro-n-nitrosoguanidine at 3 and 5 µg/0.1 mL phosphate buffer for TA1535. 9(5)aminoacridine hydrochloride monohydrate at 50 and 100 µg/0.1 mL DMSO for TA1537. 2-Nitrofluorene at 5 and 10 µg/0.1 mL DMSO for TA1538.
Positive controls (with metabolic activation): Cyclophosphamide at 250 µg/0.1 mL phosphate buffer for TA1535. 2-Aminoanthracene at 5 µg/0.1 mL DMSO for TA100. Each petri dish contained ~20 mL of minimum agar, 0.1 mL of the solution of the test material or vehicle, and 0.1 mL of the bacterial strains in 2.0 mL of soft agar. The soft agar was composed to 100 mL 0.6% agar solution with 0.6% NaCl, and 10 mL of a solution of 1-histidine (0.5 mM) and biotin (0.5 mM). In the test with metabolic activation, 0.5 mL of the S9 activation mixture was added to the plates.
The plates were incubated at 37 ± 1.5 °C in the absence of light for 48 hours. - Evaluation criteria:
- The test material was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled in any concentration.
- Statistics:
- The colonies were counted and the arithmetic mean was calculated.
- Species / strain:
- S. typhimurium, other: TA98, TA1535 and TA1538
- 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
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- at 300 and 2700 µg/1 mL
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- at 2700 µg/1 mL
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The test material precipitated in the soft agar at concentrations of 8100 and 24,300 µg/0.1 mL. In the tests conducted without microsomal activation, there was a slight increase in the number of back-mutant colonies of strain TA100 in the 2700 ug/0.1 mL concentration and above.
Additionally, a growth-inhibiting effect was reported in the tests without activation for strains TA1535, TA1537, and TA1538 at a concentration of 24,300 µg/0.1 mL. In the first assay with microsomal activation, there was a slight increase in the number of back-mutant colonies of strain TA1537 at 300 and 2700 µg/0.1 mL. The study authors attributed this to fluctuation in the rate of spontaneously occurring back-mutants. Negative and positive controls produced the expected mutant colony counts.
Under the experimental conditions, a mixture of 70% dioctyltin bis(2-ethylhexylmercaptoacetate) and 30% mono-octyltin tris(2-ethylhexylmercaptoacetate) displayed weak mutagenic activity. - Conclusions:
- Under the experimental conditions, a mixture of 70% dioctyltin bis(2-ethylhexylmercaptoacetate) and 30% mono-octyltin tris(2-ethylhexylmercaptoacetate) displayed weak mutagenic activity.
- Executive summary:
An Ames test was caried out with a 70:30 %(w/w) mixture of DOT(2 -EHMA) and MOT(2 -EHMA). The test was considered less reliable due to the fact that the test was not conducted using E. coli strain WP2 uvrA. Positive controls for the strains TA98, TA1537, and TA1538 were not included in the test with metabolic activation. The test material precipitated in the soft agar at concentrations of 8100 and 24,300 µg/0.1 mL.
In the tests conducted without microsomal activation, there was a slight increase in the number of back-mutant colonies of strain TA100 in the 2700 µg/0.1 mL concentration and above. Additionally, a growth-inhibiting effect was reported in the tests without activation for strains TA1535, TA1537, and TA1538 at a concentration of 24,300 µg/0.1 mL. In the first assay with microsomal activation, there was a slight increase in the number of back-mutant colonies of strain TA1537 at 300 and 2700 µg/0.1 mL. The study authors attributed this to fluctuation in the rate of spontaneously occurring back-mutants.
Negative and positive controls produced the expected mutant colony counts.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 17 May 2012 to 03 July 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: guidelines published by the Japanese Regulatory Authorities, including METI, MHLW and MAFF.
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: USA, EPA (TSCA) OPPTS harmonised guidelines.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia coli strain. - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth.
- Properly maintained: yes. Stored at approximately -196 °C in a liquid nitrogen freezer. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). - Additional strain / cell type characteristics:
- other: S. typhimurium: all strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA- mutation.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation Test - Experiments 1 and 2
0, 50, 150, 500, 1500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test material was insoluble in dimethyl sulphoxide at 50 mg/mL; it was soluble in dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. Acetone was selected as the most appropriate vehicle. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- - EXPERIMENT 1
METHOD OF APPLICATION: in agar (plate incorporation)
2 mL molten top agar (0.6 % agar, 0.5 % NaCl with 5 mL of 1.0 mM histidine and 1.0mM biotin for Salmonella typhimurium or 1.0 mM tryptophan solution for E. coli), 0.1 mL of culture of the appropriate strain, 0.1 mL of the appropriate test material solution or the vehicle or positive control substance and 0.5 mL S9-mix (for the plates with metabolic activation) or 0.5 mL phosphate buffer (for the plates without metabolic activation) were thoroughly mixed and the mix was immediately poured onto the surface of Vogel-Bonner Minimal agar plates.
DURATION
- Exposure duration: 48 hours at 37 °C
NUMBER OF REPLICATIONS: The tests were performed in triplicate
- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
0.1 mL of the appropriate bacterial culture was dispensed into a test tube followed by 0.5 mL of S9 mix or phosphate buffer and 0.05 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates.
DURATION
- Exposure duration: 48 hours at 37 °C
NUMBER OF REPLICATIONS: The tests were performed in triplicate
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was assessed as a reduction in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth. - Evaluation criteria:
- The mutagenicity study was considered valid if the mean colony counts of the control plates were within acceptable ranges and if the results of the positive controls met the criteria for a positive response. Furthermore, all tester strains must have demonstrated the required characteristics as determined by their respective strain checks. There should be a minimum of four non-toxic test material dose levels and no evidence of excessive contamination.
A test material was considered to be positive if the increase in the mean number of revertant colonies on the test plates was concentration-related or if a reproducible two-fold or more increase was observed compared to that of negative control plates.
A test substance was considered to be negative in the bacterial gene mutation test if it produced neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points. - Key result
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- PRE-STUDY CHECKS
The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.
PRELIMINARY TOXICITY TEST
The test material was non-toxic to the strains of bacteria used (TA 100 and WP2uvrA). The test material formulation and S9-mix used in the experiment were both shown to be sterile.
MUTATION TEST
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was therefore tested up to the maximum recommended dose level of 5000 µg/plate. A precipitate (creamy and particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. Small statistically significant increases in WP2uvrA revertant colony frequency were observed in the presence of S9-mix at 50 and 5000 µg/plate in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 and 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.
Results for the negative controls were considered to be acceptable. Furthermore, all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. - Conclusions:
- Under the conditions of this study, the test material was not mutagenic to S. typhimurium strains TA1535, TA1537, TA98 and TA100 and in the E. coli strain WP2uvrA in both the absence and presence of metabolic activation.
- Executive summary:
The mutagenicity of the test material was assessed in a bacterial reverse mutation assay (Ames Test) in accordance with standardised guidelines OECD guideline 471 and EU Method B.13/14. During the study, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material using both the plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without metabolic activation. The dose range for the study was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. Under the conditions of the study, the vehicle control plates gave counts of revertant colonies generally within the normal range. All positive controls used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test material precipitate (creamy and particulate in appearance) was noted at and above 1500 µg/plate, though this observation did not prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the presence of S9-mix at 50 and 5000 µg/plate in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 and 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.
Therefore, the test material was concluded to be non-mutagenic under the conditions of the test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Missing TA102 strain and E.coli strains
- Principles of method if other than guideline:
- Study conducts prior to implementation of corresponding guideline. Study performed in accordance to the publications of Ames. The later implemented guideline based to a great extent on these publications.
- Ames, B.N., W.E. Durston, E. Yamasaki, and F.D. Lee. 1973. Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. USA. 70:2281-2285.
- Ames, B.N., F.D. Lee, and W.E. Durston. 1973b. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. USA. 70:782-786.
- Ames, B.N., J. McCann, and E. Yamasaki. 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Res. 31:347-364. - GLP compliance:
- no
- Remarks:
- Study predates GLP.
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- TiF:RAIF (SPF) rat liver induced with Aroclor 1254. One mL of the activation mixture contained 0.3 mL S9 fraction and 0.7 mL of the co-factor solution.
- Test concentrations with justification for top dose:
- 15, 45, 135, 405, and 1215 µg/0.1 mL
- Vehicle / solvent:
- The test substance was dissolved in acetone.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: see Details on test system and conditions
- Details on test system and experimental conditions:
- The test was conducted with triplicate replications. Positive and negative controls were tested concurrently.
Positive controls (without metabolic activation): Daunorubicin-HCl at 5 and 10 µg/0.1 mL phosphate buffer for TA98. 4-Nitroquinoline-n-oxide at 0.125 and 0.25 µg/0.1 mL phosphate buffer for TA100. n-Methyl-n'-nitro-n-nitrosoguanidine at 3 and 5 µg/0.1 mL phosphate buffer for TA1535. 9(5)aminoacridine hydrochloride monohydrate at 50 and 100 µg/0.1 mL DMSO for TA1537.
Positive control (with metabolic activation): Cyclophosphamide at 250 µg/0.1 ml phosphate buffer for TA1535.
Negative (vehicle) control (with and without activation): Acetone
The plates were incubated at 37 ± 1.5°C in the absence of light for 48 hours. - Evaluation criteria:
- The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled in any concentration.
- Statistics:
- The colonies were counted and the arithmetic mean was calculated.
- Key result
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The test material precipitated in the soft agar at 1215 µg/0.1 mL. Solvent and positive controls produced the expected mutant colony counts. Under the experimental conditions, a mixture of 70% dioctyltin bis(2-ethylhexylmercaptoacetate) and 30% mono-octyltin tris(2-ethylhexylmercaptoacetate) displayed no mutagenic activity.
- Conclusions:
- Under the experimental conditions, a mixture of 70% dioctyltin bis(2-ethylhexylmercaptoacetate) and 30% mono-octyltin tris(2-ethylhexylmercaptoacetate) displayed no mutagenic activity.
- Executive summary:
An Ames test was carried out with a mixture of 70% dioctyltin bis(2-ethylhexylmercaptoacetate) and 30% mono-octyltin tris(2-ethylhexylmercaptoacetate). The relatively old test was considered less reliable, because the test was not conducted using an E. coli strain and no positive controls for strains TA98, TA100, and TA1537 were included in the test with metabolic activation. No mutagenic activity was observed.
Referenceopen allclose all
Table 1 : Summary of the results after 24 hours exposure in the absence of S9-mix.
Dose (µg/mL) |
First assay (first test) |
Dose (µg/mL) |
First assay (first test) |
||
MF |
RTG |
MF |
RTG |
||
0.36 |
115 |
13 |
|||
0.31 |
64 |
29 |
|||
0.26 |
63 |
36 |
|||
0.2 |
89 |
22 |
0.22 |
93 |
60 |
0.1 |
72 |
81 |
0.16 |
115 |
79 |
0.05 |
76 |
95 |
0.11 |
59 |
100 |
0.03 |
57 |
84 |
0.079 |
93 |
99 |
0.01 |
74 |
96 |
0.055 |
80 |
108 |
0.006 |
58 |
81 |
0.027 |
72 |
110 |
0 |
93* |
100* |
0 |
74* |
100* |
*Mean of duplicate cultures.
MF = mutant frequency ; RTG = Relative total growth.
Table 2 : Summary of the results after 4 hours exposure in the absence of S9-mix.
Dose (µg/mL) |
Second test |
|
MF |
RTG |
|
20 |
105 |
20 |
17 |
88 |
9 |
14 |
154 |
11 |
10 |
121 |
20 |
7.3 |
72 |
21 |
5.1 |
81 |
21 |
3.6 |
124 |
30 |
1.8 |
105 |
42 |
0.86 |
76 |
64 |
0 |
66* |
100* |
*Mean of duplicate cultures.
MF = mutant frequency ; RTG = Relative total growth.
Table 3 : Summary of the results after 4 hours exposure in the presence of S9-mix.
Dose (µg/mL) |
First assay (first test) |
Dose (µg/mL) |
Second assay (third test) |
||
MF |
RTG |
MF |
RTG |
||
85 |
143 |
30 |
|||
20 |
117 |
38 |
72 |
318 |
5 |
14 |
105 |
49 |
61 |
89 |
39 |
9.8 |
83 |
60 |
44 |
111 |
35 |
6.9 |
80 |
80 |
32 |
140 |
42 |
3.4 |
73 |
88 |
22 |
71 |
57 |
1.6 |
75 |
93 |
11 |
103 |
85 |
0.8 |
69 |
101 |
5.4 |
83 |
89 |
0.4 |
87 |
84 |
2.7 |
95 |
83 |
0.2 |
78 |
93 |
1.4 |
100 |
71 |
0 |
86* |
100* |
0 |
80* |
100* |
*Mean of duplicate cultures.
MF = mutant frequency ; RTG = Relative total growth.
Table 1: Summary of Experiment
± S9 Mix |
Concentration (µL/plate) |
Revertants/plate |
|||||
TA1535 |
TA1537 |
TA1538 |
TA98 |
TA100 |
D4 |
||
- |
Solvent 0.001 0.010 0.100 1.000 5.000 |
31 50 25 38 31 30 |
6 4 6 9 9 7 |
20 11 21 21 16 17 |
42 21 31 38 52 33 |
201 211 194 204 204 319 |
42 51 32 50 40 40 |
+ |
Solvent 0.001 0.010 0.100 1.000 5.000 |
35 38 31 14 16 23 |
14 18 21 14 12 7 |
25 17 30 25 35 21 |
51 45 42 40 49 43 |
226 224 237 243 254 272 |
44 46 47 38 45 42 |
Positive Controls |
|||||||
- |
Name |
MNNG |
QM |
NF |
NF |
MNNG |
MNNG |
Concentration (µg/plate) |
10 |
10 |
100 |
100 |
10 |
10 |
|
Revertants/plate |
935 |
448 |
834 |
939 |
>1000 |
925 |
|
+ |
Name |
ANTH |
AMQ |
AAF |
AAF |
ANTH |
DMNA |
Concentration (µg/plate) |
100 |
100 |
100 |
100 |
100 |
100(µM) |
|
Revertants/plate |
402 |
208 |
451 |
791 |
>1000 |
68 |
MNNG = Methylnitrosoguanidine
QM = Quinacrine mustard
ANTH = 2-Anthramine
NF = 2-Nitrofluorene
AAF = 2-Acetylaminofluorene
AMQ = 8-Aminoquinoline
Table 1: Results
Salmonella typhimurium TA100 |
|||
Substance |
Conc. (µg/plate) |
with S9 |
without S9 |
DMSO (vehicle control) |
0 |
114 |
112 |
Anthracen-2-amin |
2 |
770; 810 |
114 |
Benzo(a)pyrene |
5 |
787 |
107 |
Test material |
0.5 |
116 |
132 |
1 |
113 |
152 |
|
2 |
122 |
190 |
|
4 |
142 |
244 |
|
8 |
130 |
306 |
|
16 |
136 |
513 |
|
32 |
155 |
687 |
|
64 |
42** |
46** |
** Lawn growth slightly impaired. Precipitates in agar.
Table 1: Experiment 1 - Mean Number of Revertants
Dose (µg/plate) |
|
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
-S9 Mix |
+S9 Mix |
-S9 Mix |
+S9 Mix |
-S9 Mix |
+S9 Mix |
-S9 Mix |
+S9 Mix |
-S9 Mix |
+S9 Mix |
||
0 |
Mean |
115 |
118 |
24 |
12 |
38 |
51 |
35 |
36 |
13 |
12 |
SD |
13.3 |
14.6 |
4.5 |
0.0 |
4.6 |
6.4 |
5.3 |
6.1 |
4.4 |
3.5 |
|
50 |
Mean |
103 |
103 |
20 |
9 |
42 |
47 |
32 |
31 |
9 |
12 |
SD |
6.1 |
8.1 |
1.0 |
2.0 |
1.7 |
6.2 |
7.5 |
2.0 |
5.9 |
1.2 |
|
150 |
Mean |
112 |
113 |
22 |
10 |
43 |
45 |
24 |
27 |
8 |
12 |
SD |
0.6 |
2.1 |
1.7 |
4.2 |
4.6 |
5.5 |
1.2 |
6.7 |
1.0 |
0.6 |
|
500 |
Mean |
99 |
99 |
22 |
12 |
31 |
36 |
35 |
28 |
13 |
8 |
SD |
4.5 |
4.5 |
2.6 |
1.0 |
5.2 |
8.5 |
3.8 |
10.3 |
6.0 |
5.0 |
|
1500 |
Mean |
108 P |
107 P |
23 P |
11 P |
42 P |
41 P |
30 P |
30 P |
12 P |
8 P |
SD |
1.7 |
4.0 |
3.5 |
1.7 |
5.6 |
10.5 |
10.5 |
4.0 |
8.0 |
6.4 |
|
5000 |
Mean |
96 P |
75 P |
22 P |
11 P |
35 P |
41 P |
30 P |
34 P |
10 P |
11 P |
SD |
8.1 |
6.2 |
1.5 |
2.0 |
3.6 |
6.1 |
3.2 |
2.5 |
1.2 |
4.5 |
|
Positive Control |
Substance (µg/plate) |
ENNG 3 |
2AA 1 |
ENNG 5 |
2AA 2 |
ENNG 2 |
2AA 10 |
4NQO 0.2 |
BP 5 |
9AA 80 |
2AA 2 |
Mean |
409 |
1513 |
141 |
288 |
550 |
427 |
137 |
195 |
561 |
278 |
|
SD |
82.4 |
77.9 |
9.9 |
24.1 |
28.1 |
19.4 |
12.7 |
3.1 |
202.6 |
3.1 |
Table 2: Experiment 2 - Mean Number of Revertants
Dose (µg/plate) |
|
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
-S9 Mix |
+S9 Mix |
-S9 Mix |
+S9 Mix |
-S9 Mix |
+S9 Mix |
-S9 Mix |
+S9 Mix |
-S9 Mix |
+S9 Mix |
||
0 |
Mean |
110 |
99 |
22 |
11 |
40 |
39 |
19 |
31 |
12 |
10 |
SD |
10.6 |
20.1 |
4.9 |
3.8 |
9.0 |
6.7 |
4.0 |
10.2 |
4.0 |
5.2 |
|
50 |
Mean |
97 |
106 |
23 |
12 |
45 |
50* |
19 |
32 |
8 |
12 |
SD |
2.9 |
10.1 |
0.6 |
3.1 |
0.0 |
4.6 |
4.0 |
7.0 |
4.6 |
7.2 |
|
150 |
Mean |
100 |
112 |
20 |
10 |
35 |
46 |
30 |
31 |
13 |
8 |
SD |
12.9 |
11.6 |
4.6 |
1.2 |
4.0 |
4.2 |
7.6 |
6.8 |
5.5 |
1.0 |
|
500 |
Mean |
108 |
103 |
21 |
15 |
43 |
48 |
16 |
28 |
10 |
11 |
SD |
20.3 |
5.0 |
2.0 |
4.6 |
11.6 |
7.0 |
4.6 |
4.5 |
4.0 |
3.2 |
|
1500 |
Mean |
104 P |
105 P |
14 P |
12 P |
33 P |
46 P |
20 P |
20 P |
12 P |
14 P |
SD |
3.8 |
11.7 |
6.6 |
0.0 |
2.1 |
2.1 |
2.3 |
1.2 |
0.0 |
2.1 |
|
5000 |
Mean |
103 P |
91 P |
20 P |
9 P |
32 P |
52 P* |
15 P |
24 P |
17 P |
8 P |
SD |
14.2 |
0.6 |
3.6 |
2.6 |
3.0 |
4.0 |
8.5 |
7.8 |
1.7 |
3.1 |
|
Positive Control |
Substance (µg/plate) |
ENNG 3 |
2AA 1 |
ENNG 5 |
2AA 2 |
ENNG 2 |
2AA 10 |
4NQO 0.2 |
BP 5 |
9AA 80 |
2AA 2 |
Mean |
466 |
1474 |
180 |
280 |
596 |
341 |
129 |
669 |
463 |
236 |
|
SD |
24.9 |
187.6 |
48.6 |
29.3 |
47.4 |
31.9 |
17.1 |
41.4 |
87.2 |
19.7 |
ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO = 4 -nitroquinoline-1 -oxide
9AA = 9 -aminoacridine
2AA = 2 -aminoanthracene
BP = benzo(a)pyrene
P = precipitate
* = p ≤ 0.05
SD = standard deviation
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Micronucleus Tests SHG 182/79184 and SHG 191 -79738 (Read-Across Study performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))
In both mouse micronuclues tests, it was concluded that the test material failed to show any evidence of mutagenic potential, when administered orally. However, at the higher dose levels evidence of toxicity, as shown by bone marrow depression, was observed.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- Not specified
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- not specified
- Type of assay:
- other: mammalian erythrocyte micronucleus test (migrated information)
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: 23 - 25 g
- Assigned to test groups randomly: yes
- Fasting period before study: animals were starved overnight prior to dosing
- Housing: groups of 5 mice in plastic disposable cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 5 %
- Air changes: 20 per hour
- Photoperiod: illuminated by artificial light for 12 hours per day - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: 1 % Methyl cellulose
- Amount of vehicle: 0.1 mL/ 10 g bw per dose - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- The test material was prepared in the vehicle using a Silverson high speed mixer - Duration of treatment / exposure:
- - The total doses were given as two equal administrations separated by an interval of 24 hours.
- Frequency of treatment:
- - Two equal administrations separated by an interval of 24 hours.
- Post exposure period:
- - Five male and five female mice from each dosage group were killed by cervical dislocation, at intervals of 12, 24, 36 and 48 hours after the last dose.
- Dose / conc.:
- 4 500 mg/kg bw (total dose)
- No. of animals per sex per dose:
- 20 animals per sex per dose
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- - Methotrexate
- Route of administration: intraperitoneal injection
- Doses / concentrations: 40 mg/kg (total dose)
- Vehicle: 0.9 % saline - Tissues and cell types examined:
- - The incidence of micronucleated cells per 2000 normochromatic and 2000 polychromatic erythrocytes per animalwas determined.
- In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal. - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: The dosage of test material used in this study was the intermediate dose level used in a previous experiment.
TREATMENT AND SAMPLING TIMES: The chosen total dosage of 4500 mg/kg bodyweight was given as two equal administrations separated by an interval of 24 hours. The mice were starved overnight prior to dosing with the standard volume of 0.1 mL/10 g bodyweight per dose.
DETAILS OF SLIDE PREPARATION:
- The femurs were cleared of tissue and one epiphysis removed. Direct bone marrow smears were made onto slides containing a drop of calf serum.
- The slides were cleaned by immersion in methanol for 24 hours and air-dried immediately before use. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol overnight. After fixation, the smears were air-dried and placed in buffered distilled water (pH 6.8) for 10 minutes prior to staining in Giemsa (dilution factor 1 part Giemsa: 9 parts buffered distilled water pH 6.8) for 10 minutes. After rinsing in buffered distilled water (pH 6.8), the slides were air-dried.
- Prior to mounting in DPX, the slides were defatted by immersion in xylene for 10 minutes.
METHOD OF ANALYSIS: The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 normochromatic and 2000 polychromatic erythrocytes per animal. In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal. - Statistics:
- Statistical analysis of the micronucleated normochromatic and polychromatic erythrocyte counts:
- Due to heterogeneity of variance (Bartlett's test; P < 0.001) non-parametric methods based on Kruskal-Wallis mean ranks were used to analyse all the data from this experiment. These methods have been found to be more robust against inequality of variance than classical analysis of variance methods.
Statistical analysis of the ratios of normochromatic to polychromatic erythrocytes:
- The non-parametric methods of statistical analysis detailed above were used to analyse the ratio of normochromatic to polychromatic erythrocytes. - Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
Clinical signs and mortalities:
- After administration of 1 % Methylcellulose, the vehicle control, no toxic reactions or mortalities were observed.
- After administration of the test material no toxic reactions were observed until 36 hours after the second dose when a reaction consisting of a period of lethargy and ptosis was observed. At 48 hours after the second dose an additional toxic reaction consisting of hypopnoea and tremors was observed. There were four mortalities in this group, animals died overnight between 36 and 48 hours.
- After administration of the positive control compound, no toxic reactions or mortalities were observed.
Induction of micronuclei:
- In this experiment the negative control group gave mean counts of 2.4, 2.6, 2.6 and 2.3 micronucleated polychromatic erythrocytes at the 12, 24, 36 and 48 hour kills respectively. This group also gave mean counts of 2.0, 2.3, 2.0 and 2.0 micronucleated normochromatic erythrocytes at the 12, 24, 36 and 48 hour kills respectively.
- After administration of the test material the group mean micronucleated polychromatic cell counts were comparable with the concurrent control value at the four periods tested. The group mean micronucleated normochromatic erythrocyte counts at the 12, 24 and 36 hour kills were also comparable with the concurrent control value. After 48 hours however the group mean micronucleated normochromatic cell count was slightly increased over the concurrent control value.
- The positive control, administered intraperitoneally, gave group mean counts of 14.2, 19.6 and 42.3 micronucleated polychromatic cells at the 12, 24 and 36 hour kills respectively. There were too few cells to count at the 48 hour kill. This group also gave mean counts of 11.3, 13.8 and 19.3 micronucleated normochromatic cells at the 12, 24 and 36 hour kills respectively. At the 48 hour kill the group mean count was 2.7 micronucleated normochromatic cells which was comparable with the concurrent control value.
Ratio of PCE/NCE (for Micronucleus assay):
- In this experiment, the negative control group, gave group mean ratios of 0.97, 1.01, 1.01 and 1.06 normochromatic to polychromatic cells, at the 12, 24, 36 and 48 hour kills respectively.
- After administration of the test material the group mean ratio at each period was higher than that obtained with the concurrent control. The ratio increased with time after administration of the second dose.
- The positive control, administered intraperitoneally, gave group mean ratios of 2.07, 10.76 and 19.18 normochromatic to polychromatic cells at the 12, 24 and 36 hour kills respectively. After 48 hours there were too few polychromatic cells to count and therefore no cell ratio value was recorded.
Statistical analysis of the micronucleated normochromatic and polychromatic erythrocyte counts:
- The positive control group and the groups dosed with the test material were compared with the vehicle control group using the non-parametric equivalent of the method of L.S.D.'s (least, significant differences).
- The positive control group and the vehicle control group were the same as those used in a previous report.
- After administration of the test material, the micronucleated normochromatic erthrocyte counts at the 12, 24 and 36 hour kills were not significantly different from the respective concurrent control values. At the 48 hour kill the micronucleated normochromatic cell count was significantly different from the concurrent control value P < 0.05. The micronucleated polychromatic erythrocyte counts at each kill were not significantly different from the respective concurrent control values.
- The positive control group gave micronucleated polychromatic and normochromatic cell counts at the 12, 24 and 36 hour kills which were significantly different from the respective concurrent control values P < 0.001. At the 48 hour kill the micronucleated normochromatic cell count was not significantly different from the concurrent control value.
Statistical analysis of the ratios of normochromatic to polychromatic erythrocytes:
- After administration of the test material the ratio of normochromatic to polychromatic erythrocytes were significantly different from the respective concurrent control values at each kill. At the 12 and 24 hour kills P < 0.05 and at the 36 and 48 hour kills P < 0.01.
- The positive control group, gave ratios at the 12, 24 and 36 hour kills which were significantly different from the respective concurrent control values, P < 0.001. - Conclusions:
- Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential when administered orally. However, evidence of toxicity as shown by bone marrow depression was observed.
- Executive summary:
A study was performed to investigate the genetic toxicity in vivo of the test material. The study was conducted in a style similar to OECD 474 using the micronucleus test on male and female CD-1 mice.
The chosen total dosage of 4500 mg/kg bodyweight (the intermediate dose level from a previous study) was given as two equal administrations separated by an interval of 24 hours. The mice were starved overnight prior to dosing with the standard volume of 0.1 mL/10 g bodyweight per dose.
Following the last dose, the animals were observed and all mortalities and signs of malreaction during the experiment were recorded. Five male and five female mice from each dosage group were killed by cervical dislocation, at intervals of 12, 24, 36 and 48 hours after the last dose and both femurs dissected from each animal. Direct bone marrow smears were made onto slides containing a drop of calf serum. The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 normochromatic and 2000 polychromatic erythrocytes per animal. In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal.
The small, but statistically significant, rise in the micronucleated normochromatic erythrocytes at the 48 hour kill cannot be taken as evidence of mutagenic potential due to anti-metabolite action. In the event of cell cycle delay a corresponding rise in micronucleated polychromatic cells would have been expected prior to an increase in micronucleated normochromatic cells. In this experiment the test material did not cause this rise in the micronucleated polychromatic cells and therefore the slight increase in the micronucleated normochromatic cells must remain an unexplained phenomenon. It is possible that it may be related to the high toxicity of the test material as shown by the degree of bone marrow depression.
The positive and negative controls used in the study were judged to be valid.
Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential when administered orally. However, evidence of toxicity as shown by bone marrow depression was observed.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study conducted on read-across material
- Justification for type of information:
- Read-across to DOTI (Diisooctyl 2,2'-[(dioctylstannylene)bis(thio)]diacetate) (CAS 26401-97-8), see attached justification.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Not specified
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- no
- Type of assay:
- other: mammalian erythrocyte micronucleus test (migrated information)
- Species:
- mouse
- Strain:
- other: CFLP
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: 19 to 23 g
- Assigned to test groups randomly: yes
- Fasting period before study: animals were starved overnight before dosing
- Housing: each group of 5 mice were kept in a plastic disposable cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week
ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 50 ± 5 %
- Air changes (per hr): 20
- Photoperiod: The room was illuminated by artificial light for 12 hours per day - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Dosing volume: 0.1 mL/10 g bw per dose - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Preparation: The formulations were prepared using a Silverson high speed mixer. - Duration of treatment / exposure:
- The total doses were given as two equal administrations separated by an interval of 24 hours.
- Frequency of treatment:
- Two equal administrations separated by an interval of 24 hours.
- Post exposure period:
- 6 hours following the second dose.
- Dose / conc.:
- 2 250 mg/kg bw (total dose)
- Dose / conc.:
- 4 500 mg/kg bw (total dose)
- Dose / conc.:
- 9 000 mg/kg bw (total dose)
- No. of animals per sex per dose:
- Phase I (preliminary toxicity study): 2 animals per sex per dose
Phase II (micronucleus study): 5 animals per sex per dose - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- - Mitomycin C
- Doses / concentrations: Prepared at 0.7 mg/mL for two doses of 7 mg/kg, in 0.9 % saline
- Route of administration: Intra-peritoneally - Tissues and cell types examined:
- - The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic and 2000 normochromatic erythrocytes per animal.
- In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal. - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
- Doses for the micronucleus assay were selected from the results of the preliminary toxicity study.
DETAILS OF SLIDE PREPARATION:
- Following the last dose, the animals were observed for a further six hours before killing and all mortalities and signs of malreaction during the experiment were recorded. The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and one epiphysis removed from each bone.
- A direct bone marrow smear was made onto a slide containing a drop of calf serum. The slide was cleaned by immersion in methanol for 24 hours and air-dried immediately before use. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol overnight.
- After fixation, the smears were air-dried and placed in buffered distilled water (pH 6.8) for 10 minutes prior to staining in Giemsa (dilution factor 1 part Giemsa: 9 parts buffered distilled water pH 6.8) for 10 minutes. After rinsing in buffered distilled water (pH 6.8), the slides were air-dried. Prior to mounting in DPX, the slides were defatted by immersion in xylene for 10 minutes.
METHOD OF ANALYSIS:
- The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic and 2000 normochromatic erythrocytes per animal. In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal. - Statistics:
- STATISTICAL ANALYSIS OF THE RATIOS OF NORMOCHROMATIC TO POLYCHROMATIC ERYTHROCYTES
- Due to heterogeneity of variance (Barlett's test; P < 0.001) non-parametric methods based on Kruskal-Wallis mean ranks were used to analyse the ratios of normochromatic to polychromatic erythrocytes. These methods have been found to be more robust against inequality of variance than classical analysis of variance methods.
- The positive control group dosed with Mitomycin C and the groups dosed with the test material were compared with the vehicle control using the non-parametric equivalent of the method of L.S.D.'s (least significant differences).
- After administration of the test material at a total dosage of 2250 mg/kg bodyweight, the ratio of normochromatic to polychromatic erythrocytes was not significantly different from the concurrent control value. At total dosages of 4500 and 9000 mg/kg body weight the ratios were significantly different from the concurrent control value, P < 0.01 and P < 0.001 respectively
- The positive control group dosed with Mitomycin C, gave a ratio which was significantly different from the concurrent control value, P < 0.001.
STATISTICAL ANALYSIS OF THE MICRONUCLEATED NORMOCHROMATIC AND POLYCHROMATIC ERYTHROCYTE COUNTS
- The non-parametric methods of statistical analysis detailed above were used to analyse the micronucleated normochromatic and polychromatic erythrocyte counts.
- After administration of the test material at all dosages, the micronucleated normochromatic and polychromatic erythrocyte counts were not significantly different from the respective concurrent control values.
- The positive control group dosed with Mitomycin C gave micronucleated cell counts which were significantly different from the respective concurrent control values, P < 0.001 - Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- Bone marrow depression
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 4000, 8000, 9000, 10000, 11000, 12000, 14000 and 20000 mg/kg bw
- Clinical signs of toxicity in test animals: No toxic reactions were observed at 2000, 4000, 8000 and 9000 mg/kg bw. At 10000 and 11000 mg/kg bw, a toxic reaction consisting of hypopnoea was observed. At total dosages of 12000, 14000 and 20000 mg/kg bw, a toxic reaction consisting of hypopnoea and a period of lethargy was observed.
- From the above results, a top dosage of 9000 mg/kg bodyweight was chosen for the micronucleus test which, it was indicated, would cause one or two deaths.
RESULTS OF DEFINITIVE STUDY
SIGNS AND MORTALITIES
- After administration of 1 % methylcellulose, the vehicle control, no toxic reactions or mortalities were observed.
- After administration of the test material at all dosages, no toxic reactions were observed. At a total dosage of 9000 mg/kg bodyweight, there were three mortalities. Two animals died overnight after the administration of the first dose, between 8 and 24 hours. Advanced visceral autolysis prevented any post-mortem examination. The third died after administration of the second dose between 24 and 30 hours. Macroscopic examination at post-mortem failed to reveal any abnormalities.
- After administration of Mitomycin C, the positive control compound, no toxic reactions or mortalities were observed.
MICRONUCLEUS COUNTS
- The group mean counts and ranges obtained with the test material are shown in Table 1.
- In this experiment the negative control group gave a mean count of 2.8 micronucleated polychromatic cells which was just outside the range for previous unrelated experiments. However, the individual counts were within the laboratory standard range of individual counts for negative controls obtained in previous unrelated experiments.
- After administration of the test material at all dosages, the group mean polychromatic cell counts were comparable with the concurrent control value and within the laboratory standard range for negative controls obtained in previous unrelated experiments. In addition, the group mean micronucleated normochromatic cell counts of the treatment groups were comparable with the concurrent control value.
- The positive control, Mitomycin C, administered intraperitoneally gave a mean count of 89.5 micronucleated cells per 2000 polychromatic erythrocytes. This group also gave a mean count of 11.4 micronucleated cells per 2000 normochromatic erythrocytes. - Conclusions:
- Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential, when administered orally. However, at the higher dose levels evidence of toxicity, as shown by bone marrow depression, was observed.
- Executive summary:
A study was performed to investigate the genetic toxicity in vivo of the test material. The study was conducted in a style similar to OECD 474.
In this assessment of the effect of the test material on the incidence of micronucleated polychromatic erythrocytes in mice, total dosages of 2250, 4500 and 9000 mg/kg bodyweight were administered by oral gavage, in two equal dosages, separated by an interval of 24 hours.
A negative control group was dosed in an identical manner with the vehicle, 1 % methylcellulose. A positive control group was dosed by intraperitoneal injection with Mitomycin C, at a total dosage of 14 mg/kg bodyweight. The mice were sacrificed six hours after the second dose and bone marrow smears examined for the presence of micronuclei in 2000 polychromatic and 2000 normochromatic erythrocytes per mouse. The ratio of normochromatic to polychromatic cells was also examined in each mouse.
A preliminary toxicity study indicated that a top dosage of 9000 mg/kg bodyweight would cause one or two deaths and led to the selection of the dose concentrations for the main test.
At all dosages of the test material both of the group mean micronucleated cell counts were comparable with the concurrent control values.
After administration of the test material at a total dosage of 2250 mg/kg bodyweight, the ratio of normochromatic to polychromatic cells was comparable with the concurrent control value. At total dosages of 4500 and 9000 mg/kg bodyweight, the ratios were higher than the concurrent control value. Statistical analysis, using Kruskal-Wallis methods, showed these increases to be significantly different. The positive control compound, Mitomycin C, produced the expected large increase in the group mean polychromatic micronucleated cell count and in the normochromatic to polychromatic cell ratio. This group also produced a small increase in the group mean micronucleated normochromatic cell count.
Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential, when administered orally. However, at the higher dose levels evidence of toxicity, as shown by bone marrow depression, was observed.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study conducted on read-across material
- Justification for type of information:
- Read-across to DOTI (Diisooctyl 2,2'-[(dioctylstannylene)bis(thio)]diacetate) (CAS 26401-97-8), see attached justification.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- Bone marrow depression
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
Referenceopen allclose all
CONCLUSIONS
- From these results, it is concluded that the test material does not show any evidence of mutagenic potential when administered orally, in this test procedure. However evidence of bone marrow depression was observed in the smears from each kill.
- The small, but statistically significant, rise in the micronucleated normochromatic erythrocytes at the 48 hour kill cannot be taken as evidence of mutagenic potential due to anti-metabolite action. In the event of cell cycle delay a corresponding rise in micronucleated polychromatic cells would have been expected prior to an increase in micronucleated normochromatic cells. In this experiment the test material did not cause this rise in the micronucleated polychromatic cells and therefore the slight increase in the micronucleated normochromatic cells must remain an unexplained phenomenon. It is possible that it may be related to the high toxicity of the test material as shown by the degree of bone marrow depression.
- The positive control compound, gave the expected rises in the micronucleated normochromatic and polychromatic erythrocyte counts in mice killed 12, 24 and 36 hours after the second dose. In mice killed 48 hours after the second dose there were too few polychromatic erythrocytes to count. In addition, at the 48 hour kill, the value of the mean micronucleated normochromatic erythrocyte count dropped so as to become comparable with the concurrent control value. These two findings may be explained by the high level of toxicity to the bone marrow cells resulting in massive numbers of deaths in the cell population, so causing a reduction in the number of polychromatic erythrocytes and the consequent shunting of peripheral blood into the bone marrow to compensate for this. Hence the frequency of micronucleated normochromatic erythrocytes from the bone marrow exposed to Methotrexate would be reduced to control levels due to the influx of cells from the circulating blood.
Table 1: Group mean number of micronucleated cells per 2000 normochromatic erythrocytes and 2000 polychromatic erythrocytes per animal and the group mean ratio of normochromatic to polychromatic erythrocytes
Treatment |
Total dosage over 24 h (mg/kg) |
Number of micronucleated cells per 2000 erythrocytes per animal |
Ratio of normochromatic to polychromatic erythrocytes |
||||
Normochromatic |
Polychromatic |
|
|||||
Mean |
Range |
Mean |
Range |
Mean |
Range |
||
Vehicle |
- |
2.5 |
0 to 6 |
2.8 |
1 to 6 |
1.09 |
0.93 to 1.22 |
Test material |
2250 |
2.3 |
0 to 6 |
2.5 |
0 to 5 |
1.38 |
1.19 to 1.69 |
4500 |
2.4 |
0 to 5 |
2.4 |
0 to 6 |
1.72** |
1.47 to 2.09 |
|
9000 |
2.3 |
0 to 6 |
2.6 |
1 to 5 |
3.93*** |
2.56 to 5.39 |
|
Positive control |
14 |
11.4*** |
8 to 15 |
89.5*** |
74 to 115 |
10.46*** |
7.75 to 15.38 |
** P < 0.01
*** P < 0.001
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
IN VITRO
Gene Mutation study in Bacteria
Thompson 2012 (Read-Across Study performed on structurally similar substance DOT bis-(ethylmatelate) (CAS 68109-88-6)
The mutagenicity of the test material was assessed in a bacterial reverse mutation assay (Ames Test) in accordance with standardised guidelines OECD guideline 471 and EU Method B.13/14. During the study, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material using both the plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without metabolic activation. The dose range for the study was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. Under the conditions of the study, the vehicle control plates gave counts of revertant colonies generally within the normal range. All positive controls used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test material precipitate (creamy and particulate in appearance) was noted at and above 1500 µg/plate, though this observation did not prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the presence of S9-mix at 50 and 5000 µg/plate in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 and 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.
Therefore, the test material was concluded to be non-mutagenic under the conditions of the test.
Jagannath 1978 (Read-Across Study performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))
The genetic toxicity of the test material was investigated in a bacterial reverse mutation assay, broadly similar to OECD 471. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
The test material was tested at 5.000, 1.000, 0.100, 0.010, 0.001 µL/plate both with and without metabolic activation, in the form of S9-mix. The following bacterial strains were used: Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA1538, and Saccharomyces cerevisiae strain D4. The overlay method of application was used.
The plates were incubated for 48 hours at 37 °C, and scored for the number of colonies growing on each plate. Positive and solvent controls using both directly active positive chemicals and those that require metabolic activation were run with each assay. The numbers of colonies on each plate were counted and recorded.
The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The results of the tests conducted on the test material in the absence of a metabolic system were all negative. The results of the tests conducted on the test material in the presence of the rat liver activation system were all negative.
Under the conditions of the study, the test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic with or without metabolic activation.
Gene Mutation study in Bacteria
Gunzel 1979 (Read-Across Study performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))
The 1978 (Jagannath) study was repeated with TA100 at higher test material concentrations due to the slightly increased (and dose-dependent) number of mutants of Salmonella typhimurium TA 100 in the highest tested concentration in the absence of metabolic activation.
The results of the tests conducted on the test material in the absence of a metabolic system were all negative. The results of the tests conducted on the test material in the presence of the rat liver activation system were all negative.
Under the conditions of this study the test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered as not mutagenic under these test conditions.
Gene Mutation study in Bacteria
Zieger 1988 (Read-Across Study performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))
The genetic toxicity of the test material was investigated in a study that followed similar principles to the guideline OECD 471.
The test material was examined using the pre-incubation assay, both in the presence and absence of metabolic activation in the form of S9 fractions. At least five doses of the test material were tested in triplicate.
The test material was reported to be mutagenic to the Salmonella typhimunum strains TA97, TA98, TA100, TA1535, and TA1537.
Gene Mutation study in Bacteria
Read-across data (mixture DOTE: MOTE. 70:30) (CAS No 15571-58-1 and CAS 27107-89-7)
- Anonymous (1979):An Ames test was carried out with a mixture of 70% dioctyltin bis(2-ethylhexylmercaptoacetate) and 30% mono-octyltin tris(2-ethylhexylmercaptoacetate). The relatively old test was considered less reliable, because the test was not conducted using an E. coli strain and no positive controls for strains TA98, TA100, and TA1537 were included in the test with metabolic activation. No mutagenic activity was observed.
-Anonymous (1978) Two studies: In these studies the test material was positive in strain TA100 without S9. The other strains were negative.
- Anonmymous (1983):This Ames test was considered less reliable due to the fact that the test was not conducted using E. coli strain WP2 uvrA. Positive controls for the strains TA98, TA1537, and TA1538 were not included in the test with metabolic activation. The test material precipitated in the soft agar at concentrations of 8100 and 24,300 µg/0.1 mL. In the tests conducted without microsomal activation, there was a slight increase in the number of back-mutant colonies of strain TA100 in the 2700 µg/0.1 mL concentration and above. Additionally, a growth-inhibiting effect was reported in the tests without activation for strains TA1535, TA1537, and TA1538 at a concentration of 24,300 µg/0.1 mL. In the first assay with microsomal activation, there was a slight increase in the number of back-mutant colonies of strain TA1537 at 300 and 2700 µg/0.1 mL. The study authors attributed this to fluctuation in the rate of spontaneously occurring back-mutants. Negative and positive controls produced the expected mutant colony counts.
Mouse Lymphoma Assay Read-across to (DOTE) (CAS No 15571 -58 -1).
The test material DOTE was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). Three tests were conducted; in the first and third test single cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively; in the second test cultures were treated for 24 hours and 4 hours in the absence of S9-mix and 4 hours in the presence of S9-mix. The test material was dissolved in ethanol prior to testing.
The highest concentrations of DOTE evaluated for mutagenicity in the absence of S9-mix, were 20 μg/mL and 0.36 μg/mL at 4 and 24 hours exposure, respectively. In the presence of S9-mix and 4 hours exposure, the highest concentration evaluated was 85 μg/mL. The maximum concentrations were limited by cytotoxicity.
DOTE was cytotoxic in both the absence and presence of S9-mix. In the absence of S9-mix and 24 hours exposure cytotoxicity, resulting in a reduction in initial cell yield and/or suspension growth, was observed at and above 0.1 μg/mL. The relative total growth (RTG) at the highest concentration evaluated in the first and third test were 22 and 13 %, respectively. In the absence of S9-mix and 4 hours exposure cytotoxicity was observed at all concentrations included (as from 0.86 μg/mL). The relative total growth (RTG) at the highest concentration evaluated in the second test was 20 %.
In the presence of S9-mix cytotoxicity was observed at and above 1.4 μg/mL. The relative total growth (RTG) at the highest concentration evaluated in the first and third test were 38 and 30 %, respectively. In the absence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. In the presence of S9-mix at 72 μg/mL the mutant frequency was significantly increased by 238 mutants per 1,000,000 clonable cells compared to the negative control. Since relatively small intervals (0.85) were used and the increase was observed at a single concentration causing more than 90 % cytotoxicity compared to six concentrations causing 50-70 % cytotoxicity which showed no increase in mutant frequency, it is concluded that this increase is not indicative for mutagenicity. It is very likely a secondary effect to cytotoxicity and thus not indicative for mutagenicity.
Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and presence of the S9-mix, respectively; ethanol (vehicle) served as negative control. The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls.
It is concluded that under the conditions used in this study, the test material DOTE is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.
IN VIVO
Mouse Micronucleus Test 1980, SHG 182/79184 (Read-Across Study performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))
A study was performed to investigate the genetic toxicity in vivo of the test material. The study was conducted in a style similar to OECD 474. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
In this assessment of the effect of the test material on the incidence of micronucleated polychromatic erythrocytes in mice, total dosages of 2250, 4500 and 9000 mg/kg bodyweight were administered by oral gavage, in two equal dosages, separated by an interval of 24 hours.
A negative control group was dosed in an identical manner with the vehicle, 1 % methylcellulose. A positive control group was dosed by intraperitoneal injection with Mitomycin C, at a total dosage of 14 mg/kg bodyweight. The mice were sacrificed six hours after the second dose and bone marrow smears examined for the presence of micronuclei in 2000 polychromatic and 2000 normochromatic erythrocytes per mouse. The ratio of normochromatic to polychromatic cells was also examined in each mouse.
A preliminary toxicity study indicated that a top dosage of 9000 mg/kg bodyweight would cause one or two deaths and led to the selection of the dose concentrations for the main test.
At all dosages of the test material both of the group mean micronucleated cell counts were comparable with the concurrent control values.
After administration of the test material at a total dosage of 2250 mg/kg bodyweight, the ratio of normochromatic to polychromatic cells was comparable with the concurrent control value. At total dosages of 4500 and 9000 mg/kg bodyweight, the ratios were higher than the concurrent control value. Statistical analysis, using Kruskal-Wallis methods, showed these increases to be significantly different. The positive control compound, Mitomycin C, produced the expected large increase in the group mean polychromatic micronucleated cell count and in the normochromatic to polychromatic cell ratio. This group also produced a small increase in the group mean micronucleated normochromatic cell count.
Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential, when administered orally. However, at the higher dose levels evidence of toxicity, as shown by bone marrow depression, was observed.
Mouse Micronucleus Test 1980, SHG 191/79738 (Read-Across Study performed on structurally similar substance (DOTI)(CAS No 26401 -97 -8))
A study was performed to investigate the genetic toxicity in vivo of the test material. The study was conducted in a style similar to OECD 474 using the micronucleus test on male and female CD-1 mice. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).
The chosen total dosage of 4500 mg/kg bodyweight (the intermediate dose level from a previous study) was given as two equal administrations separated by an interval of 24 hours. The mice were starved overnight prior to dosing with the standard volume of 0.1 mL/10 g bodyweight per dose.
Following the last dose, the animals were observed and all mortalities and signs of malreaction during the experiment were recorded. Five male and five female mice from each dosage group were killed by cervical dislocation, at intervals of 12, 24, 36 and 48 hours after the last dose and both femurs dissected from each animal. Direct bone marrow smears were made onto slides containing a drop of calf serum. The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 normochromatic and 2000 polychromatic erythrocytes per animal. In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal.
The small, but statistically significant, rise in the micronucleated normochromatic erythrocytes at the 48 hour kill cannot be taken as evidence of mutagenic potential due to anti-metabolite action. In the event of cell cycle delay a corresponding rise in micronucleated polychromatic cells would have been expected prior to an increase in micronucleated normochromatic cells. In this experiment the test material did not cause this rise in the micronucleated polychromatic cells and therefore the slight increase in the micronucleated normochromatic cells must remain an unexplained phenomenon. It is possible that it may be related to the high toxicity of the test material as shown by the degree of bone marrow depression.
The positive and negative controls used in the study were judged to be valid.
Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential when administered orally. However, evidence of toxicity as shown by bone marrow depression was observed.
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.
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