Reaction Products of Dimethyl hydrogen phosphite and Alcohols C14-18, Even, Linear

Genetic toxicity: in vitro

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

Study period:

2 July 1993 - 28 July 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study using some noncurrent practices

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The study used the concurrent control, 2-anthramine, as the sole indicator of the efficacy of the S9 mix. The concentration of the post-mitochondrial fraction in the S9 mix was 6% which boarders on the very lower limit of acceptability (5%).

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

All Salmonella strains contain a uvrB deletion mutation (affecting DNA excision repair), as well as an rfa mutation (affecting membrane permeability). In addition, strains TA98 and TA100 contain the plasmid pKM101, which enhances the error-prone DNA repair system normally present in this organism. Strains TA1535 and TA100 detect base pair substitution mutations affecting the hisG locus. Strains TA1538 and TA98 detect frameshift mutations affecting the hisD locus, while TA1537 detects frameshift mutations at the hisC locus. E. coli strain WP2 uvrA contains a uvrA deletion mutation (affecting DNA excision repair) and can detect base pair substitution mutations affecting the trp locus.

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium strains TA1535, TA1537, TA1538, TA 98, TA100 and Escherichia coli strain WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The exogenous metabolic activation (S9) mixture contained 8 mM MgCl2, 33 mM KCl, 4 mM NADP, 5 mM glucose-6-phosphate, 100 mM Na2HP04 (pH 7.4) and 6% (v/v) Aroclor 1254-induced male Sprague-Dawley rat liver homogenate.

Test concentrations with justification for top dose:

1.67, 5.00, 16.7, 50.0, 167, 500, 1670 and 5000 mg/plate for the original assay with and without S9; 16.7, 50.0, 167, 500, 1670 and 5000 mg/plate for the confirmatory assay and the retest with and without S9; and 1.67, 5.00, 16.7, 50.0, 167, 500, 1670 and 5000 mg/plate for thesecond retest without S9

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Concurrent positive controls without S9: sodium azide, 9-aminoacridine, 2-nitrofluorene, N-ethyl-N-nitro-N-nitroguanidine; Concurrent positive controls with S9: 2-anthramine

Remarks:

The report notes that according to SOP, the concentration of liver homogenate in the metabolic activation mixture is optimized for each lot of S9 prepared, using three positive control articles (2-acetylaminofluorene, 2-anthramine, and benzo[a]pyrene.

Details on test system and experimental conditions:

Fresh cultures for mutagenesis testing were prepared by quickly thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 25 mL of Oxoid Nutrient Broth #2. After growth for approximately 6 hours at 37°C in an orbital shaking incubator, samples of each culture were diluted 1:4 in de-ionized water (di-H20) and optical densities were determined at 650 nm. Cultures with optical densities of 0.40 to 0.60, representative of cells in late exponential or early stationary phase (approximately 1-2 x 109 cellsfmL), were utilized for this study. Triplicate cultures of each strain were evaluated with the appropriate solvent and positive controls.

Evaluation criteria:

A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value. If the test article does not induce a statistically significant, dose-dependent increase in revertant frequency, but induces a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine- or tryptophan­ independent revertants. Statistical analyses are performed only when a 50% increase in revertant frequency, relative to the concurrent negative controls, is observed. This 50% "trigger" was selected based upon the normal, spontaneous variation observed among replicate negative control cultures (see next page), as well as spontaneous fluctuation observed in this laboratory among groups of cultures treated with a variety of test articles judged to be negative in this assay.

Statistics:

Statistical analyses were performed using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. [Snee, R.D. and J.D. Irr (1981) Design of a statistical method for the analysis of mutagenesis at the hypoxanthine guanine phosphoribosyl transferase locus of cultured Chinese hamster ovary cells, Mutation Res., 85:77-93.]

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium strains TA1535, TA1537, TA1538, TA 98, TA100 and Escherichia coli strain WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

other: The positive controls overall are considered valid recognizing that the S9 mix was checked with 2-acetylaminofluorene, 2-anthramine, and benzo[a]pyrene prior to testing but that only 2-anthramine was used as the positive control in the tests.

Additional information on results:

Prescreen: the test material was not toxic to any strain at a dose of 50 ug/plate, or to strain WP2 uvrA at doses of 167 and 500 u/g/plate. Inhibited growth was observed in strains TA1538 and TA100 at doses of ≥167 ug/plate and in strain WP2 uvrA at doses ≥1670 ug/plate. The test article was incompletely soluble at doses ≥500 ug/plate. First test: The test material again was found to be incompletely soluble at doses ≥1670 ug/plate, but normal growth was observed in all tester strains at all doses evaluated with and without S9. Second test: In the confirmatory the test article again was found to be incompletely soluble at doses ≥500 ug/plate. Inhibited growth was observed in tester strains TA1537 and TA1538 at doses of 1670 and/or 5000 ug/plate with and/or without S9. Third test: The test article again was found to be incompletely soluble at doses ≥1670 g/plate, and inhibited growth again was observed in tester strains TA1537 and TA100 at a dose of 5000 ug/plate with or without S9. Fourth test: The test material was incompletely soluble at doses ≥1670 ug/plate, and inhibited growth was observed at doses of 167, 500, 1670 and 5000 ug/plate. Analysis: The authors concluded that the apparent discrepancy in toxicity between the prescreen and the mutation assays likely was related to interference resulting from test article insolubility.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

First test: The test material was evaluated in triplicate cultures in all five Salmonella tester strains at doses of 1.67, 5.00, 16.7, 50.0, 167 and 500 ug/plate, and in E. coli strain WP2 uvrA at doses of 16.7, 50.0, 167, 500, 1670 and 5000 ug/plate with and without S9. Revertant frequencies for all doses in all tester strains with and without S9 approximated or were less than those observed in the concurrent negative solvent controls.

Second test: A confirmatory assay was conducted in all Salmonella and E. coli strains at doses of 16.7, 50.0, 167, 500, 1670 and 5000 ug/plate with and without S9. Revertant frequencies for all doses of the test material in all tester strains with and without S9 again approximated or were less than in the negative solvent controls.

Third test: The test material again was re-evaluated under identical conditions in all five Salmonella tester strains (16.7, 50.0, 167, 500, 1670 and 5000 ug/plate with and without S9) to confirm the results observed at the higher dose levels evaluated in the second assay where cytotoxicity was observed (5000 ug/plate with and without S9 for TA 1537 and 1670 and 5000 ug/plate without S9 for TA1538). Revertant frequencies for all doses in all tester strains with S9, and in tester strains TA1535, TA1537, TA98 and TA100 without S9, approximated or were less than negative solvent control values. Statistically significant increases in revertant frequencies, approximately 2.4- to 2.7-fold negative solvent control values, were observed in strain TA1538 at doses of 16.7, 50, and 500 g/plate without S9. However, these increases were not dose related.

Fourth test: The test material was re-evaluated in strain TA1538 doses of 1.67, 5.00, 16.7, 50.0, 167, 500, 1670 and 5000 ug/plate without S9. Revertant frequencies for all doses of the test material approximated or were less than control values, thus not confirming the response observed in test three and indicating that the slight increases observed in strain TA1538 in previous trial were statistical aberrations due to random fluctuation of the spontaneous revertant frequency.

Controls: All positive and negative control values in all assays were within acceptable limits.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was not mutagenic in the Ames/Salmonella-E. coli Liquid Pre-incubation Assay under
the conditions and the criteria of the test protocol.

Executive summary:

The in vitro genetic toxicity potential of this substance was evaluated in the Ames/Salmonella-E. coli Liquid Pre-incubation Assay using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA 98, TA100 and Escherichia coli strain WP2 uvrA. An apparent difference in toxicity was observed between the prescreen and the mutation assays, which likely was related to interference resulting from test article insolubility. In the first, test the test material was evaluated in triplicate cultures in all five Salmonella tester strains at doses of 1.67, 5.00, 16.7, 50.0, 167 and 500 ug/plate, and in E. coli strain WP2 uvrA at doses of 16.7, 50.0, 167, 500, 1670 and 5000 ug/plate with and without S9. Revertant frequencies for all doses in all tester strains with and without S9 approximated or were less than those observed in the concurrent negative solvent controls. In the second, confirmatory assay that was conducted in all Salmonella and E. coli strains at doses of 16.7, 50.0, 167, 500, 1670 and 5000 ug/plate with and without S9, revertant frequencies for all doses of the test material in all tester strains with and without S9 again approximated or were less than in the negative solvent controls. In the third, test the test material again was evaluated under identical conditions in all five Salmonella tester strains (16.7, 50.0, 167, 500, 1670 and 5000 ug/plate with and without S9) to confirm the results observed at the higher dose levels evaluated in the second assay where cytotoxicity was observed (5000 ug/plate with and without S9 for TA 1537 and 1670 and 5000 ug/plate without S9 for TA1538). Revertant frequencies for all doses in all tester strains with S9, and in tester strains TA1535, TA1537, TA98 and TA100 without S9, approximated or were less than negative solvent control values. Statistically significant increases in revertant frequencies, approximately 2.4- to 2.7-fold negative solvent control values, were observed in strain TA1538 at doses of 16.7, 50, and 500 g/plate without S9. However, these increases were not dose related. In the fourth test, the test material was re-evaluated in strain TA1538 doses of 1.67, 5.00, 16.7, 50.0, 167, 500, 1670 and 5000 ug/plate without S9. Revertant frequencies for all doses of the test material approximated or were less than control values, thus not confirming the response observed in test three and indicating that the slight increases observed in strain TA1538 in previous trial were statistical aberrations due to random fluctuation of the spontaneous revertant frequency. All positive and negative control values in all assays were within acceptable limits. Based on these results, the test material was not mutagenic in the Ames/Salmonella-E. coli Liquid Pre-incubation Assay under the conditions and the criteria of the test protocol.