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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1977
Report Date:
1977

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Kronitex 200 (triaryl phosphate ester)
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): see Fig. no
- Substance type: not specified
- Physical state: liquid
- Analytical purity: not specified
- Impurities (identity and concentrations): not specified
- Composition of test material, percentage of components: Triphenyl phosphate 9%, 2-isopropylphenyl phosphates 10%, 3-isopropylphenyl phosphates 2%, 4-isopropylphenyl phosphates 11%, higher substituted triphenyl phosphate 90%, average molecular weight 420
- Isomers composition: see above
- Purity test date: not specified
- Lot/batch No.: not specified
- Expiration date of the lot/batch: not specified
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Locations of the label (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): not applicable
- Stability under test conditions: not specified
- Storage condition of test material: room temperature
- Other:

Method

Species / strain
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
Controls
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.

Results and discussion

Test results
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'.

Applicant's summary and conclusion

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.