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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity of the test substance in Ames, Mouse Lymhoma and Sister Chromatid Exchange Assays in vitro.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Chemical Company.
- Purity: 97.7%
Target gene:
- S. typhimurium: His-locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced Sprague-Dawley rats or Syrian hamsters liver S9 (due to the nature of the study as an inter-laboratory trial, tests were run using both types).
Test concentrations with justification for top dose:
For the standard plate method: 0, 100, 3.333, 1000, 6,666, 10,000 µg/mL

For the desiccator method designed to take account of the volatility of the test substance: 0.000, 0.001, 0.005, 0.007, 0.010, 0.020, 0.025, 0.035, 0.050, 0.100, 0.500, 1.000 mL/chamber.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine
Evaluation criteria:
Evaluations of the results were made at both the individual trial and overall chemical levels. Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or non-mutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose-response. A trial was 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 a weak increase 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 trial to be judged "+" or "+ W". A chemical was judged mutagenic (+) or weakly mutagenic (+ W) if it gave a reproducible dose-related response over the solvent control in replicate trials, and judged questionable (?) if the results of individual trials were not reproducible 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.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not applicable
Remarks:
No vehicle used
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not applicable
Remarks:
No vehicle used
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not applicable
Remarks:
No vehicle used
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA97, TA1535, TA1537
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
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: desiccator method
Conclusions:
The test substance was considered positive for mutagenicity in salmonella typhimurium in the Ames test.
Executive summary:

Introduction

The study was conducted to determine the mutagenic potential of the test substance to Salmonella typhimurium using a test method comparable to the OECD 471 Bacterial Reverse Mutation Test

Method

S. typhimurium strains TA97, TA98, TA100, TA1535, and TA1537 were used.  Because the testing was done as part of an inter-laboratory trial testing was conducted in more than one laboratory on coded samples.   The test used a preincubation procedure with and without exogenous metabolic activation.  The metabolising system was liver S-9 derived from Aroclor 1254-induced Sprague-Dawley rats and Syrian hamsters. Experiments were conducted with both types of S9.

The test substance is a volatile chemical and following testing in the preincubation procedure it produced either negative or equivocal responses in some laboratories.  It was therefore retested at one laboratory using exposure in a desiccator. Test strains were TA98, TA100, TA1535 and TA1537. In this procedure, S-9 or buffer was incorporated into the top agar and poured onto the plate. The lids of the plates were removed and the plates were stacked on a perforated porcelain plate in a 9 litre glass desiccator jug containing a magnetic stirring bar. A measured volume of test chemical in liquid form was introduced into a watch glass suspended below the porcelain plate, and the desiccator was sealed and placed on a magnetic stirrer in a 37°C incubator.  After 24 hr, the plates were removed from the desiccator and incubated at 37°C in air for an additional 24 hr.

 

Results

In the desiccator protocol which is considered the most reliable method the test substance was positive for mutagenicity in strain TA 98 in the absence of S9 and positive in strain TA100 in the presence of S9.

Conclusion

The test substance was considered positive for mutagenicity in salmonella typhimurium the Ames test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Version / remarks:
The study was part of an inter-laboratory trial using two laboratories. Only the results from one laboratory are reported in this study summary as the test in the second laboratory was negative. However, this may have been due to the lower top dose used and hence the relability of the result from the second laboratory is uncertain.
GLP compliance:
no
Type of assay:
sister chromatid exchange assay in mammalian cells
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Chemical Company.
- Purity: 97.7%

Further specific information on the test material is in Zeiger E. (1990): Mutagenicity of 42 chemicals in Salmonella. Environ Molec Mutagen 16(Suppl 18):32-54.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO cells were cloned at Litton Bionetics Inc. and supplied to the other laboratories. Cells were not used beyond 15 passages after cloning. Cells for experiments were thawed and grown in McCoys 5A medium supplemented with antibiotics and 10% foetal calf serum at 37°C using 5% CO2. Cells were routinely checked for mycoplasma contamination; the results of these analyses disclosed no evidence of contamination.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from male Sprague-Dawley rats induced with Aroclor 1254.  
Test concentrations with justification for top dose:
The study was part of an inter-laboratory trial. Only the results from one laboratory are reported in this study summary (see additional information on results section).

Laboratory One (laboratory to which the results apply)
0, 29, 96, 290 µg/mL with S9
0, 96, 290 and 968 µg/mL without S9

Doses were selected based on trial experiments designed optimse dose levels.


Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
triethylenemelamine
cyclophosphamide
Evaluation criteria:
Data were evaluated for both trend and dose point increase over the solvent control. A trend of P < 0.005 or an individual dose with a 20% increase over the solvent control was considered significant.

If a trial had a positive trend and no significant doses, or if there was no trend and only one significant dose, the trial was judged equivocal (?); if a trial had significant trend and one significant dose it was judged weak positive (+ W); and if the trial had two significant doses it was judged positive (+), whether or not a positive trend was obtained. In the chromsome aberrations trials, if only one dose was significant and the increase over the control was P < 0.0005 the trial was denoted (+ W*). The term “weak positive” (+ W) refers to the strength of the evidence for the positive call, not to the potency of the response.

In general, positive responses were repeated, whereas repeats were not required for the trials concluded to be negative. If positive responses were obtained both with and without S9, the laboratories were required to repeat only one activation condition. When combining the conclusions of individual trials, more emphasis was given to trials in which experimental conditions were optimised. For example, a trial that included an extended harvest time was generally given more weight, whereas a trial that had a positive response only at doses judged to be toxic was given less weight. However, when trials were considered to be equivalent the following scheme was used in combining trial calls (+) and (-) = (?); (+) and (+W) or (?) = (+); (+W) and (?) = (+ W); (-) and (+ W) or (?) = (-); (W*) was considered to be equivalent to a (+) when combining calls.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
other: weak positive
Cytotoxicity / choice of top concentrations:
other: For toxic compounds, cells from the highest dose likely to yield analysable metaphases were fixed, together with cells from five successively lower dose levels.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: For toxic compounds, cells from the highest dose likely to yield analysable metaphases were fixed, together with cells from five successively lower dose levels.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Testing was undertaken as an inter-laboratory trial with testing being undertaken at two laboratories. The maximum dose tested was higher at one laboratory which observed a weak positive response in the test. The other laboratory tested at lower doses and obtained negative results in both the presence and absence of metabolic activation. The results from this laboratory have not been included as they are not considered relevant given the results from the laboratory which tested at a higher concentration.
Remarks on result:
other: The view of the literature paper authors was that this weak positive result needed confirmation.
Conclusions:
The test substance produced a weak positive result in the sister chromatid exchange assay in the avsence of metabolic activation.
Executive summary:

Introduction

The study was conducted to determine the potential for the test substance to induce chromosome aberrations in Chinese Hamster Ovary Cells (CHO) using a test method comparable to OECD method No. 479: Genetic Toxicology: In vitro Sister Chromatid Exchange Assay in Mammalian Cells.


Method

Chinese hamster lung cells were used for the study. Cultures were initiated 24 hours prior to treatment. Treatment was with or without S9 metabolising system. Concurrent solvent and positive controls were included. Mitomycin C (MMC) was the positive control in experiments without S9 and cyclophosphamide (CPA) was used in experiments with S9.

 

For the tests without S9 the positive controls and test chemicals were added and the flasks were incubated at 37°C for 2 hr prior to the addition of bromodeoxyuridine (BrdUrd) (M), after which the incubation was continued for an additional 24 hr. The cells were then washed and medium containing BrdUrd and Colcemid was added to the flasks for an additional 2-2.5 hours incubation period. Twenty-four hours later Colcemid was added and the cells were incubated for an additional 2-2.5 hours. At the end of the treatment regimen a visual estimate of the confluency of each flask was made in order to evaluate toxicity.

 

At the end of treatment cells were harvested and resuspended in fixative fixed before slides were prepared and stained with Hoechst 33258 and scored for sister chromatid exchanges.

Results and Conclusion

The test substance produced a weak positive result in the sister chromatid exchange assay in the absence of metabolic activation.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
not applicable
GLP compliance:
no
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Test chemicals used for the study were supplied coded by the National Toxicology Program's (NTP) chemical repository (Radian Corporation, Austin, Tex). The original source and purity are in Zeiger E, Haseman J. Shelby M. Margolin B, Tennant R (1990): Evaluation of Four In Vitro Genetic Toxicity Tests for Predicting Rodent Carcinogenicity: Confirmation of Earlier Results with 41 Additional Chemicals. Environ Mol Mutagen 16(Suppl 18): 1-14.
Target gene:
tk locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Hepatic S9 mix prepared from Aroclor 1254-induced male Fischer 344 rats.
Evaluation criteria:

Solvent control cultures
1. The cloning efficiency must be in the 50-120% range.
2. At least two acceptable cultures must be available.
3. The average mutant frequency for all acceptable cultures must be between 15 x 10^-6 and 110 x 10^-6 for negative evaluations. For mutagenic test chemicals the range is extended to 10 x 10^-6 to 150 x 10^-6.
4. A Chi-square test for consistency among the mutant frequencies of the acceptable cultures must be significant at P ≤ 0.05.

Positive control cultures
1. The cloning efficiency must be in the 10-120% range.
2. The relative total growth must not be less than 1%.
3. At least one acceptable culture must be available.
4. The average mutant frequency for all acceptable cultures must be within the historical range


Test chemical cultures
1. The cloning efficiency must be in the 10-120% range.
2. The relative total growth must not be less than 1%.
3. The relative suspension growth for the second day of expression must be 40% or greater
4. The test chemical must remain soluble during treatment.
5. The maximum dose is 5 mg/ml for solids and 5 µg/ml for liquids.
6. Each dose level must have two or more acceptable cultures.
7. A Chi-square test for consistency among the mutant frequencies of the acceptable cultures must be significant at P ≤ 0.05.
8. At least three acceptable dose sets must be available, except when no response is obtained and sets are rejected due to precipitation.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: Publication states that the high dose was determined by solubility or cytotoxcity, but did not exceed 5 mg/mL.
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Publication states that the high dose was determined by solubility or cytotoxcity, but did not exceed 5 mg/mL.
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
The test substance was considered positive for mutagenicity in mouse lymphoma L5178Y cells in the absence of S9 and negative in the presence of S9.
Executive summary:

Introduction

The mutagenic potential of the test substance to L5178Y Mouse Lymphoma Cells was Investigated using a test method similar to the OECD 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene).

 

Method

The highest dose of the compound tested was determined by solubility or toxicity but did not exceed 5 mg/ml. All doses were tested at least in duplicate. Cells (6 x 106) were treated for 4 hr at 37°C, washed, suspended in medium, and incubated for 48 hr at 37°C. After expression, 3 x 106 cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT resistant (TFTr) cells, and 600 cells were plated in nonselective medium and soft agar to determine the cloning efficiency. After plating, the cells were incubated for 9-12 days at 37°C before colonies were counted on an Artek 880 colony counter fitted with a 10-turn size discriminator.

 

Results and Conclusion

The test substance was considered positive for mutagenicity in mouse lymphoma L5178Y cells in the absence of S9 and negative in the presence of S9.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Justification for classification or non-classification