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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non GLP, not carried out according to recognised guideline although results fully documented.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The Salmonella microsomal assay for bacterial mutagenesis tests the ability of a compound to induce point (reverse) mutations in selected histidine requiring mutant strains of Salmonella typhimurium. Organisms are exposed to varying concentrations of the test agent both with and without the activation of liver microsomal enzyme preparations. These enzyme preparations add an important aspect of mammalian metabolism to the test and allow for the conversion of promutagens (compounds which must be metabolized by mammalian enzyme systems to their ultimate reactive form) to mutagenic derivatives. The mutagenic potential of a test chemical is determined by its ability to induce a significant increase in the number of mutants which occur in treated over control cultures .
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Araclor induced rat liver, S9 mix
Test concentrations with justification for top dose:
100, 10, 5, 1 and 0.1 µl 1% v/v solution of the test substance in DMSO.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
Mutagenesis assay without metabolic activation
The test was performed according to the method of Ames et al. For the assay, the following were added, in order, to I6 x 125 mm screw-capped tubes:
1. 2 ml molten agar (0.6% Noble's agar supplemented with 0.6% NaCl, 0.5 mM biotin, and 0.5 mM histidine).
2. 0.1 ml of bacterial culture.
3. 0.1 ml of test agent in the appropriate solvent.
Tubes were mixed quickly but gently and the contents poured over a base plate of Spizzizen's minimal medium. Plates were rotated to distribute the overlay agar, allowed to harden and incubated at 37° C for 48 hrs at which time the number of mutant colonies was determined. Each agent was tested at five concentrations against all five strains of bacteria. Three plates were made for each control and each dilution of test agent. The following controls were included in each experiment:
1. Bacteria only for spontaneous reversion.
2. Solvent alone.
3. Known positive mutagens: TA1535: MNNG, 5 µg/plate; TA100: MNNG, 5 µg/plate; TA1-537 : 9-AA, 100 µg/plate; TA1538 : 2-NF, 5 µg/plate; TA98: 2-NF, 5 µg/plate

The solvent and each dilution of test agent were checked for sterility by adding 0.1 ml of each to 2 ml overlay agar without bacteria and pouring over a minimal medium base plate. Plates were incubated as above. The solvent was determined by the solubility of the test agent. Wherever possible, distilled water was the solvent of choice. DMSO, ethanol, acetone and ethyl acetate may also be used in descending order of preference. The compound tested in this study was dissolved in DMSO.
Mutagenesis assay with metabolic activation

1. Non-specific induction of liver enzymes:
Aroclor 1254, a polychlorinated biphenyl, was used for the non-specific induction of live r enzymes in rats. Adult male Sprague-Dawley rats, weighing 200-225 g, were given a single IP injection of 500 mg/kg Aroclor 1254 in corn oil. On day 5 post injection, animals were sacrificed by C02 asphyxiation and the livers were removed and the S-9 fraction prepared. Animals had free access to food until 12 to 15 hrs before sacrifice; water was available at all times.

2. Preparation of the S-9 fraction of the liver homogenate
All operations were performed aseptically at 0-4 °C using sterile solutions and glassware. After surgical removal and weighing, livers were minced in three volumes 0.15 M KCl and homogenized in a Potter Elvejhem tissue grinder at 120-150 rpm. The homogenate was centrifuged at 9000 x g for 20 min and the supernatant decanted and saved and designated the S-9 fraction. The S-9 was divided into 1 ml amounts and stored frozen at -80°C until the day of use.

3. Preparation of the S-9 mix
On the day of the assay, sufficient S-9 for that day's use was thawed and mixed with cofactors for use in the assay. S-9 mix contains per ml: S-9 (generally 50 to 100 µl), 0.8 mM MgCl2, 0.33 ml KCl, 0.5 mM glucose-6-phosphate, 4 mM NADP, and 100 mM NaH2PO4.H2O, pH 7.4. Reagents for the S-9 mix were weighed and prepared fresh daily with the exception of sodium phosphate which was made as a stock solution of 0.2 M. The entire S-9 mix was filter sterilized before use through a 0.45 µm Nalgene disposable filter unit. S- 9 mix was kept at 0-4°C until use; unused portions were discarded at the end of the day.

4. Standardization of S-9 for use in the assay
Each bulk preparation of S-9 was assayed for aryl hydrocarbon hydroxylase (AHH) activity; protein was determined according to the method of Lowry et al. Prior to use in the assay, each S-9 preparation was titrated over a range of concentrations (10-300 µl S-9 per ml of S-9 mix) with 3-methylcholanthrene ( 3MC), benzo (a) pyrene (13P) and 6- aminochrysene (6-AC) to determine the optimum amount of S-9 for use in a general screening procedure. This was usually in the range of 50 to 100 µl per ml of S-9 mix.

5. Mutagenesis assay with metabolic activation:
The assay was the same as that described above except that 0.5 ml of S-9 mix was added to the overlay agar immediately before it was mixed and poured. Plates were incubated and scored as noted above. Controls for the assay with metabolic activation were:
a. bacteria with S-9 mix to determine the effect of S-9 on spontaneous reversion
b. Positive control mutagens:
TA1535: 5µg 2-AA
TA100: 1 µg AflatoxinB1.
TA1537: 1 µg 6-AC
TA1530: 2 µg 2-AF
TA98: 1 µg Aflatoxin B1
c. Sterility controls on each dilution of test agent were done as noted above. One-half ml of the S-9 mix with neither agent nor bacteria was added to 2 ml overlay agar and poured on to a minimal medium base plate.

Evaluation criteria:
Before an agent is reported to be either active or inactive in the Salmonella/Microsomal Assay, the following criteria must be met: All sterility controls must be negative for bacterial growth. This includes both dilutions and S-9 preparations. The spontaneous revertants for each strain must be within acceptable limits. All three control plates must have approximately equal numbers of revertant colonies. The solvent controls must have approximately the same number of colonies as spontaneous reversion controls. Positive mutagens must produce at least 4x the number of colonies as the controls for spontaneous reversions. Any test with a strain which does not meet these criteria must be repeated on a separate day.
Statistics:
Yes, see results below.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test met each of the evaluation criteria listed above and the activity of the test agent was evaluated accordingly. The test material did not induce a significant increase in the number of revertant colonies over that seen in the untreated control plates in any of the strains tested or at any dilution level.
The exogenous source of liver enzymes did not affect the activity of the test agent in this system. The activity of the S-9 preparation is confirmed by its ability ot activate the control mutagens, 2-AA, 6-AC and aflatoxin B1, which require metabolism to exert their effect to reactive derivatives.
The ability of the selected histidine auxotrophs to be back mutated to histidine independance is shown by their reversion after treatment with both direct acting and metabolically activated control mutagens.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the conditions of this test, the test substance does not induce a significant increase in the number of point mutations in Salmonella typhimurium strains TA1535, TA1538, TA1537, TA98 and TA100 either with or without the addition of an exogenous source of liver enzymes for metabolic activation of the test agent.
Executive summary:

Kronitex 200 was tested for mutagenic potential by the bacterial assay of Ames. Five standard bacterial strains were tested each at 5 dose levels with and without activation by liver enzyme. No mutagenicity was observed. No classification is required.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 14th 2004 to December 6th 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, carried out according to recognised guidelines
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
chromosome aberration.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Peripheral blood lymphocytes were obtained from a healthy non-smoking 28 year old adult female on 12 October 2004 for the preliminary toxicity assay and from the same donor on 26 October 2004 for the definitive assay. The donor had no recent history of radiotherapy, viral infection or the administration of drugs. This system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals (Preston et al., 198 1).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced S9 rat liver
Test concentrations with justification for top dose:
8.75, 17.5,35,65, 85, 115, 150, 200 ug/ml for all treatment / recovery times.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO) was determined to be the solvent of choice based on the solubility of the test article, and compatibility with the target cells.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
MMC was used as the positive control in the non-activated study at final concentrations of 0.3 and 0.6 µg/mL. CP was used as the positive control in the S9 activated study at final concentrations of 20 and 40 µg/mL.
Positive control substance:
other: see above
Details on test system and experimental conditions:
Test system
Peripheral blood lymphocytes were obtained from a healthy non-smoking 28 year old adult female on 12 October 2004 for the preliminary toxicity assay and from the same donor on 26 October 2004 for the definitive assay. The donor had no recent history of radiotherapy, viral infection or the administration of drugs. This system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals (Preston et al., 1981).

Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 was batch prepared and stored at ≤-70°C until used. Each bulk preparation of S9 was assayed for its ability to metabolize 2-aminoanthracene and 7,12-dimethyl-benz(α)anthracene to forms mutagenic to Salmonella typhimurium TA100. Immediately prior to use, the S9 was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 µL S9 per milliliter medium (RPMI 1640 serum-free medium supplemented with 100 units penicillin/mL and 100 µg streptomycin/mL, and 2 mM L-glutamine.

Solubility Test
A solubility test was conducted to select the solvent. The test was conducted using dimethyl sulfoxide (DMSO). The test article was tested to determine the solvent that permitted preparation of the highest soluble stock concentration, up to 500 mg/mL.

Preliminary Toxicity Assay
The toxicity test was performed for the purpose of selecting concentrations for the chromosome aberration assay and consisted of an evaluation of test article effect on mitotic index. Approximately 0.6 mL heparinized blood was inoculated into centrifuge tubes containing 9.4 mL RPMI-1640 complete medium supplemented with 1 % phytohemagglutinin (PHA). The tubes were incubated at 37 +/-1°C in a humidified atmosphere of 5 +/- 1% C02 in air for 44-48 hours. At the time of test article treatment the culture tubes were centrifuged, the supernatant was aspirated, and the cells were resuspended in either 10 mL of fresh RPMI-1640 complete medium containing 1 % PHA for the non-activated study or 10 mL S9 reaction mixture (8 mL seruin free medium containing 1% PHA + 2 mL of S9 cofactor pool), to which was added 0.1 mL test article dosing solution in solvent or solvent alone. The osmolality in treatment medium of the solvent and of the lowest precipitating test article concentration and the highest soluble testarticle concentration were measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape.
The cells were exposed to solvent alone and to nine concentrations of the test article for 4 hours in both the presence and absence of S9 activation, and for 20 hours continuously in the absence of S9 activation. The cells were incubated at 37 +/-1°C in a humidified atmosphere of 5 +/- 1% C02 in air. At the completion of the 4 hour exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline (CMF-PBS), refed with RPMI- 1640 complete medium and retumed to the incubator for an additional 16 hours. Two hours prior to the scheduled cell harvest, Colcemid was added to the cultures at a final concentration of 0.1 µg/mL and the cultures were retumed to the incubator until cell collection.
Cells were collected by centrifugation, treated with hypotonic potassium chloride (0.075M KCl), fixed, stained and the number of cells in mitosis per 500 cells scored was determined in order to evaluate test article effect on mitotic index.

Controls
MMC was used as the positive control in the non-activated study at final concentrations of 0.3 and 0.6 µg/mL. CP was used as the positive control in the S9 activated study at final concentrations of 20 and 40 µg/mL. For both positive controls one dose level exhibiting a sufficient number of scorable metaphase cells was selected for analysis. The solvent vehicle for the test article was used as the solvent control at the same concentration as that found in the test article-treated groups.
Evaluation criteria:
Evaluation of Test Results
The toxic effects of treatment are based upon mitotic inhibition relative to the solvent-treated control and are presented for the preliminary toxicity test and the chromosome aberration assay. The number and types of aberrations per cell, the percentage of structurally and numerically damaged cells (percent aberrant cells), and the frequency of structural aberrations per cell (mean aberrations per cell) in the total population of cells examined was calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.
All conclusions were based on sound scientific judgement; however, as a guide to interpretation of the data, the test article was considered to induce a positive response when the percentages of cells with aberrations were increased in a dose-responsive manner with one or more concentrations being statistically elevated relative to the solvent control group (p≤0.05). A reproducible significant increase at the high dose only with no dose response or a reproducible significant increase at one dose level other than the high dose with no dose response will be considered positive. The test article was concluded to be negative if no statistically significant increase was observed relative to the solvent control.

Criteria for Determination of a Valid Test
The frequency of cells with structural chromosome aberrations in the solvent controls must be within the historical range for solvent controls. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p≤0.05, Fisher's exact test) relative to the solvent control.
Statistics:
Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's exact test at any test article dose level, the Cochran-Amitage test was used to measure dose-responsiveness.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility Test
Dimethyl sulfoxide (DMSO) was determined to be the solvent of choice based on the solubility of the test article, and compatibility with the target cells. The test article was soluble in DMSO at a concentration of 500 mg/mL, the maximum concentration tested for solubility.

Preliminary Toxicity Assay
Dose levels for the chromosome aberration assay were selected following a preliminary toxicity test and were based upon a reduction in mitotic index relative to the solvent control. HPBL cells were first exposed to nine concentrations of Reofos 65 ranging from 0.5 µg/mL to S9 activation system for 4 hours, or continuously for 20 hours in the absence of S9 activation. The test article was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at 5000 µg/mL. Concentrations ≤ 1500 µg/mL were soluble in treatment medium. The osmolality in treatment medium of the highest concentration tested, 5000 µg/mL, was 411 mmol/kg. The osmolality in treatment medium of the highest soluble concentration, 1500 µg/mL, was 409 mmol/kg. The osmolality of the solvent (DMSO) in the treatment medium was 407 mmol/kg. The pH of the highest concentration of test article in treatment medium was approximately 7.0. Substantial toxicity (i.e., at least 50% reduction in mitotic index, relative to the solvent control) was observed at dose levels ≥150 µg/mL in all three exposure groups. Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as shown in Table 1 below.

Chromosome Aberration Assay
In the chromosome aberration assay, the test article was soluble in DMSO and in the treatment medium at all concentrations tested. The osmolality in treatment medium of the highest concentration tested, 200 µg/mL, was 417 mmol/kg. The osmolality of the solvent (DMSO) in the treatment medium was 411 mmol/kg. The pH of the highest concentration of test article in treatment medium was approximately 7.0.
The findings of the cytogenetic analysis of the non-activated 4 hour exposure group are documented. At the highest test concentration evaluated microscopically for chromosome aberrations, 115 µg/mL, mitotic inhibition was 54%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 35, 65 and 115 µg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated group was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test). The percentage of structurally damaged cells in the MMC (positive control) group was statistically significant (13.0%).
The findings of the cytogenetic analysis of the S9 activated group are summarized by group are documented. At the highest test concentration evaluated microscopically for chromosome aberrations, 115 µg/mL, mitotic inhibition was 56%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 35, 65 and 115 µg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated group was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test). The percentage of structurally damaged cells in the CP (positive control) group was statistically significant (14.0%).
The findings of the cytogenetic analysis of the non-activated 20 hour exposure group are documented. At the highest test concentration evaluated microscopically for chromosome aberrations, 115 µg/mL, mitotic inhibition was 55%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 35, 65 and 115 µg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated group was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test). The percentage of structurally damaged cells in the MMC (positive control) group was statistically significant (16.0%).
Remarks on result:
other: strain/cell type: lymphocytes
Remarks:
Migrated from field 'Test system'.

Table 1

Treatment condition

Treatment time

Recovery time

Dose levels (µg/ml)

Non activated

4 hr

16 hr

8.75, 17.5, 35, 65, 85, 115, 150,200

20 hr

0 hr

8.75, 17.5, 35, 65, 85, 115, 150,200

S9 activated

4 hr

16 hr

8.75, 17.5, 35, 65, 85, 115, 150,200

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The positive and solvent controls fulfilled the requirements for a valid test. Under the conditions of the assay described in this report, Reofos 65 was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9 activated test systems in the in vitro mammalian chromosome aberration test using human peripheral lymphocytes.
Executive summary:

The test article, ReofosB 65, was tested in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for testing in the cytogenetic test. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article. The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test).

Based on the findings of this study, Reofos 65 was concluded to be negative for the induction of structural and numerical chromosome aberrations in the in vitro mammalian chromosome aberration test using human peripheral lymphocytes.

No classification is required.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August - 8 December 1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, not conducted according to recognised guideline, but fully detailed method and results.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
The mutagenesis assay without metabolic activation test was performed according to Ames, B.N., J. McCann, and E. Yamasaki. Mutation R e s.

The Salmonella/micuosomal assay for bacterial mutagenesis tests the ability of a compound to induce point (reverse) mutations in selected histidinerequiring mutant strairs of Salmonella typhimurium. Organisms are exposed to varying concentrations of the test agent both with and without the activation of Liver microsomal enzyme preparations. These enzyme preparations add an important aspect of mammalian metabolism to the test and allow for the conversion of promutagens (compounds which must be metabolized by mammalian enzyme systems to their ultimate reactive form) to mutagenic derivatives. The mutgenic potential of a test chemical is determined by its ability to induce a significant increase in the number of mutants
which occur in treated over control cultures.

Other references:
- Spizzien, J. Proc. Natl. Acad. Sci. USA.
- Lowry, 0.11. , N . J . Koscnbrough, A.L. Fair, and R. J. Randall . J. Bio L Chem.
- Ames, B.N., F.D., Lee , and W.E. Durston. Proc. Natl. Acad. Sci. USA.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Target gene
The strains in this study have a deletion through the uvrB region of the chromosome which has removed excision repair and an rfa mutation which has resulted in removal of the lipopolysaccharide coat down to the ketodeoxyoctanoate core, thus making the organisms more permeable to large agents which ordinarily might not penetrate the organism to interact with the DNA. The lack of excision repair makes the organisms sensitive to agents whose activity might otherwise be masked by the prescence of repair enzymes in the cell.
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine requiring
Metabolic activation:
with and without
Metabolic activation system:
Araclor induced, rat liver S-9
Test concentrations with justification for top dose:
100, 10, 5, 1, and 0.1 μl per plate of a 1% solution (v/v) in DMSO.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Thc solvent w a s determined by the solubility of the test agent.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene, 6-Aminochrysene and Aflatoxin B1.
Details on test system and experimental conditions:
The mutagenesis assay without metabolic activation test was performed according to Ames, B.N., J. McCann, and E. Yamasaki. Mutation R e s.

The Salmonella/micuosomal assay for bacterial mutagenesis tests the ability of a compound to induce point (reverse) mutations in selected histidinerequiring mutant strairs of Salmonella typhimurium. Organisms are exposed to varying concentrations of the test agent both with and without the activation of Liver microsomal enzyme preparations. These enzyme preparations add an important aspect of mammalian metabolism to the test and allow for the conversion of promutagens (compounds which must be metabolized by mammalian enzyme systems to their ultimate reactive form) to mutagenic derivatives. The mutgenic potential of a test chemical is determined by its ability to induce a significant increase in the number of mutants which occur in treated over control cultures.
Evaluation criteria:
Before an agent is reported to be either active or inactive in the Salmonella/Microsomal Assay, the following criteria must be met:
All sterility controls must be negative for bacterial growth. This includes both dilutions and S-9 preparations. The spontaneous revertants for each strain must be within acceptable limits. The range of oberved values along with the mean of these values and the standard deviation around the mean are given below. The figures are from all tests conducted in our laboratory from May 1976 to the present.
1. Without metabolic activation:
- TA1537: Range: 3-8; Mean: 7; SD: 29.
- TA1538: Range: 7-28; Mean: 18; SD: 7.
- TA1535: Range: 3-49; Mean: 20; SD: 7.
- TA100: Range: 80-230; Mean: 146; SD: 29.
- TA98: Range: 6-55; Mean: 25; SD: 10.
2. With metabolic activation:
- TA1537: Range: 1-19; Mean: 9; SD: 4.
- TA1538: Range: 7-55; Mean: 28; SD: 12.
- TA1535: Range: 6-30; Mean: 18; SD: 7.
- TA100: Range: 108-231; Mean: 162; SD: 32.
- TA98: Range: 20-69; Mean: 41; SD: 12.
All three control plates must have approximately equal numbers of revertant colonies. The solventcontrols must have approximately the same number of colonies as spontaneous reversion controls. Positive mutagens must produce at least 4x the number of colonies as the controls for spontaneous reversion. Any test with a strain which does not meet these criteria must be repeated on a separate day. To be considered positive, an unknown agent must exhibit a dose response effect. That is, there must be an increasing number of mutants with increased amounts of test agent. For strains TA1535, TA1537, TA1538 and TA98, the first positive dose must produced 3x the number of colonies as the negative control. For strain TA100, at least one dose tested must produce 3.5x the number of mutants as the spontaneous reversion control.
Statistics:
No data.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Results:
The test substance did not induce a significant increase in the number of revertant colonies over that seen in the untreated control plates in any of the strains tested or at any dilution level. The exogenus source of liver enzymes did not affect the activity of the test agent in this system. The activity of the S-9 preparation is confirmed by its ability to activate the control mutagens, 2 -AA, 6 -AC and Aflatoxin B1, which require metabolism to exert their effect to reactive derivatives. The ability of the selected histidine autotrophs to be back mutated to histidine independence is shown by their reversion after treatment with both direct acting and metabolically activated control mutagens.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Results of the salmonella assay without metabolic activation on compound C-8013 -132 -1

 Compound

Concentration 

His+ 

Rever 

tants 

per 

plate 

 

(units/plate) 

TA1535 

TA100 

TA1537 

TA1538 

TA98 

Bacteria only 

 100µl

 6.0 +/- 1.0

 91.7 +/- 8.1

 7.0 +/- 0

 6.3 +/- 2.3

 11.0 +/- 5.0

DMSO 

 100µl

 5.3 +/- 1.5

 88.3 +/- 6.8

 6.0 +/- 1.0

 4.3 +/- 2.1

 7.0 +/- 4.2

MNNG 

 5µg

 474.? +/- 225.3

 TNTC

 NT

 NT

 NT

2 -NF 

 5µg

 NT

 NT

 NT

 510.7 +/- 117.7

 277.3 +/- 14.0

9 -AA 

 100µg

 NT

 NT

 772.7 +/- 72.0

 NT

 NT

C-8013 -132 -1 

 100µl

 7.3 +/- 2.1

94.3 +/- 15.3

5.7 +/- 3.5

 6.0 +/- 1.7

 12.7 +/- 5.5

(1% solution 

 10µl

 6.7 +/- 3.2

94.3 +/- 5.9 

5.7 +/- 1.2

 5.7 +/- 1.2

 8.0 +/- 1.7

v/v in DMSO)

 5µl

 7.3 +/- 2.1

94.0 +/- 9.5

13.7 +/- 2.1

 6.3 +/- 1.5

 11.7 +/- 0.6

 1µl

 9.0 +/- 2.0

87.3 +/- 5.5

12.7 +/- 2.1

 5.0 +/- 1.0

 9.0 +/- 2.0

 0.1µl

 9.0 +/- 2.1

107.7 +/- 20.6

 9.3 +/- 3.8

 4.3 +/- 1.5

 13.0 +/- 1.0

NT = Not tested.

TNTC = Too numerous to count.

HIS+ = Histidine Positive.

Note: Each value represents the mean of the counts or three plates and the standard deviation.

Table 2: Results of the Salmonella assay with meatbolic activation on compound C-8013 -132 -1:

 Compound

Concentration 

His+ 

Rever 

tants 

per 

plate 

 

(units/plate) 

TA1535 

TA100 

TA1537 

TA1538 

TA98 

Bacteria Only 

100µl 

6.0 +/- 1.0 

91.7 +/- 8.1 

7.0 +/- 0 

6.3 +/- 2.3 

11.0 +/- 5.0 

S-9 

100µl 

15.0 +/- 4.0 

118.3 +/- 17.8 

17.0 +/- 3.6 

10.7 +/- 2.1 

11.0 +/- 2.0 

DMSO 

100µl 

9.0 +/- 1.0 

114.0 +/- 13.8 

17.0 +/- 1.0 

9.0 +/- 2.0 

12.3 +/- 2.5 

2 -AA 

5µg 

146.0 +/- 20.5 

NT 

NT 

NT 

NT 

2 -AF 

2µg 

NT 

NT 

NT 

615.3 +/- 278.7 

NT 

Aflatoxin B1 

1µg 

NT 

460.3 +/- 144.5 

NT 

NT 

437.3 +/- 186.9 

6-AC 

1µg 

NT 

NT 

91.7 +/- 13.3 

NT 

NT 

C-8013 -132 -1 

100µl 

8.3 +/- 4.9 

124.7 +/- 18.5 

12.3 +/- 5.1 

11.0 +/- 4.4 

18.3 +/- 0.6 

(1% solution 

10µl 

10.3 +/- 4.2 

112.3 +/- 24.1 

11.3 +/- 2.5 

15.3 +/- 2.1 

16.3 +/- 2.5 

v/v in DMSO) 

5µl 

6.0 +/- 1.7 

112.7 +/- 11.7 

12.7 +/- 5.0 

12.0 +/- 1.7 

16.7 +/- 4.9 

 

1µl 

10.3 +/- 3.8 

113.7 +/- 18.3 

18.0 +/- 9.0 

9.7 +/- 4.0 

14.7 +/- 4.7 

 

0.1µl 

10.3 +/- 6.8 

111.0 +/- 14.7 

18.0 +/- 2.6 

8.3 +/- 2.3 

18.0 +/- 4.0 

NT = Not tested

T = Toxic

HIS+ = Histidine Positive

NOTE: Each value represents the mean of the counts on three plates and the standard deviation.

Conclusions:
Interpretation of results (migrated information):
negative with or without metabolic activation

It was determined that the registered substance does not induce a significant increase in the number of point mutations in Salmonella typhimurium strains TA1535, TA1538 , TA1537 , TA98 and TA100 either with or without metabolic activation.
Executive summary:

It was determined that the registered substance does not induce a significant increase in the number of point mutations in Salmonella typhimurium strains TA1535, TA1538 , TA1537 , TA98 and TA100 either with or without metabolic activation.

No classification is applicable.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1987, not specified.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP. Reported under EC 793/93 for Existing substances regulations. Full methodology etc not available. Deemed reliable as evaluated by the European Commission. This data is considered the property of the data submitter. However it has not been possible to locate the study report due to re-organisation associated with the merger.
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Version / remarks:
Described as OECD Guideline 475 "Genetic toxicology: In vivo mammalian bone marrow cytogenic test - Chromosomal analysis".
Deviations:
not specified
GLP compliance:
yes
Type of assay:
other: somatic mutation assay (Chinese hamster)
Species:
hamster, Chinese
Strain:
other: Cricetulus grinseus
Sex:
male/female
Details on test animals and environmental conditions:
Not specified.
Route of administration:
oral: gavage
Vehicle:
Not specified.
Details on exposure:
24 male and 24 female hamsters were administered the test material once at a dosage of 5000 mg/kg. Eight hamsters were sacrificed 16 hours, 24 hours and 48 hours later and bone marrow from the femurs were evaluated for aberrations. Both positive and negative control groups were also run.
Duration of treatment / exposure:
16, 24 or 48 hours
Frequency of treatment:
single dose, 5000 mg/kg
Post exposure period:
None.
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes
Positive control(s):
yes, not specified.
Tissues and cell types examined:
bone marrow from the femurs
Details of tissue and slide preparation:
Not specified.
Evaluation criteria:
Not specified.
Statistics:
Not specified.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
There was no evidence of mutagenic effects in this study
Conclusions:
Interpretation of results (migrated information): negative
There was no evidence of mutagenic effects in this study
Executive summary:

There was no evidence of mutagenic effects in this study.

This data is provided as supporting information only.

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

Additional information

In summary, an extensive battery of genetoxicity studies have been conducted on a variety of batches of the test substance over the years, evaluating the end points of mutagenicity, primary DNA damage and chromosome aberrations.   Negative results were found in all categories.   The weight of the evidence indicates that isopropylated triaryl phosphates are not genotoxic.   The equivocal result in the mouse lymphoma assay, and the positive result in the somatic mutation assay in Chinese hamster bone marrow cells in one test must be seriously questioned in light of the negative results in the chromosome aberration test and the sister chromatid exchange test.  In addition, all materials were generally negative in the cell transformation test.

This test correlates well with potential carcinogenicity and evaluates a chemicals potential to change normal cells to malignant ones.   The negative result in the cell transformation test indicates that the test substance does not demonstrate the potential to transform cells in vitro. The weight of the evidence of numerous genetoxicity studies with isopropylated triaryl phosphates and other aryl phosphates indicates a lack of genotoxicity.

Short description of key information:

Various different types of studies for this endpoint have been conducted; all are negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The above studies have all been ranked reliability 2 or 3 according to the Klimish et al system. This ranking was deemed appropriate because the studies were not conducted to GLP or in compliance with agreed protocols. Some of the reports do not detail a specific method; however it documents dose levels and responses in detail, so is deemed appropriate for use in the support of a formal registration. Sufficient dose ranges, experimental types and numbers are detailed; hence it is appropriate for use based on reliability as a weight of evidence type approach.

Justification for classification or non classification

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) or the CLP Regulation (EC No 1272/2008).