Acetic acid, 2-(diethoxyphosphinyl)-

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:

2009

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(diethoxyphosphoryl)acetic acid

EC Number:

608-560-3

Cas Number:

3095-95-2

Molecular formula:

C6 H13 O5 P

IUPAC Name:

2-(diethoxyphosphoryl)acetic acid

Details on test material:

Test substance: Diethylphosphonoacetic acid (impurity of BIBW 2992)
Batch no.: 1041020
Appearance: white, crystalline substance
Purity: 99.6 %
Certificate of analysis: 11 March 2009
To be retested: February 2010
Storage conditions: At room temperature in the dark (ambient humidity)
Manufacturer: Boehringer Ingelheim Pharma GmbH & Co. KG,
88397 Biberach (Germany)

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 mix: Aroclor 1254-unduced rat liver microsomal fraction and cofactors

Test concentrations with justification for top dose:

100-5000 ug/plate

A maximum concentration of 5000 μg/plate should be investigated according to relevant
guidelines. Diethylphosphonoacetic acid did not precipitate up to the highest concentrations
of 5000 μg/plate in a non-GLP solubility test. Therefore, 5000 μg/plate was investigated as
the highest concentration.

Vehicle / solvent:

DMSO was used as solvent vehicle and negative control.

Controls

Untreated negative controls:

yes

Remarks:

DMSO only

Negative solvent / vehicle controls:

yes

Remarks:

DMSO only

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2.Aminoanthracene

Remarks:

2-Nitrofluorene, Sodium azide, Mitomycin C and 9-Aminoacridine without activation; 2-Aminoanthracene with activation

Details on test system and experimental conditions:

Revertant his+ colonies were counted using the automatic colony counter IPI Analyzer 982 B
after incubation at 37°C for 2 days (TA 102: 3 days) and the data were collected by computer.
For all replicate platings, the mean number of revertants per test concentration was
calculated. The condition of the background bacterial lawn (residual growth on minimum
histidine) was evaluated macroscopically for evidence of bacteriotoxicity induced by the test
substance. If extreme thinning or complete lack of the microcolony lawn compared to the
negative, vehicle control plates was observed, no revertants were counted. Evidence of test
substance precipitates in the agar overlay was recorded.

Evaluation criteria:

A reproducible, concentration-dependent increase in the number of revertants of at least one
tester strain over the vehicle control value and/or outside the historical control range is
indicative of genotoxic activity.

Results and discussion

Test results

Species / strain:

S. typhimurium 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:

not specified

Positive controls validity:

valid

Additional information on results:

Diethylphosphonoacetic acid did not increase the number of revertant colonies in different
tester strains of S. typhimurium compared to the negative control when tested up to
bacteriotoxic concentrations. Furthermore, metabolic activation by S9 mix did not alter the
mutation frequency of these bacterial strains. All values were clearly within the historical
background data range.
The validity of this study is given since the vehicle control plates showed spontaneous
revertants in different tester strains of S. typhimurium at frequencies similar to those
described in the literature and within the historical control range experienced in our
laboratory.
The diagnostic mutagens NaN3, 9-AA, 2-NF, MMC and 2-AA showed the expected strain
specific responses in the absence and presence of mammalian liver enzymes, respectively,
demonstrating the validity of this study.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Diethylphosphonoacetic acid caused neither base-pair substitutions nor frameshift mutations
in different strains of S. typhimurium in the presence and absence of metabolic activation
when tested up to bacteriotoxic concentrations. Based on these results it was concluded, that
the test substance is "Ames negative".

Executive summary:

SUMMARY

Diethylphosphonoacetic acid, a possible impurity in BIBW 2992, triggered a structural alert for mutagenicity in the DEREK in silico system. Therefore, Diethylphosphonoacetic acid was investigated under GLP conditions to see whether it induces mutations in the Salmonella typhimurium strains TA 1535, TA 100, TA 102 (sensitive to base-pair substitution), TA 1537 and TA 98 (sensitive to frameshift mutagens) both in presence and absence of a metabolic activation system (S9 mix: Aroclor 1254-induced rat liver microsomal fraction and cofactors). Diethylphosphonoacetic acid was dissolved in dimethylsulfoxide (DMSO) and added to bacterial cultures in triplicates to give the final concentrations of 100 to 5000 μg/plate in the plate test. No repeat experiment was performed due to a clear negative result.

Solubility and Toxicity

Diethylphosphonoacetic acid did not precipitate up to the highest concentration of 5000 μg/plate. A strain-dependent bacteriotoxicity was observed primarily at concentrations ≥1000 μg/plate.

Mutagenicity

Diethylphosphonoacetic acid did not increase the number of revertant colonies in different tester strains of S. typhimurium compared to the negative control when tested up to bacteriotoxic concentrations. Furthermore, metabolic activation by S9 mix did not alter the mutation frequency of these bacterial strains. All values were clearly within the historical background data range. The validity of this study is given since the vehicle control plates showed spontaneous revertants in different tester strains of S. typhimurium at frequencies similar to those described in the literature and within the historical control range experienced in our laboratory. The diagnostic mutagens NaN3, 9-AA, 2-NF, MMC and 2-AA showed the expected strain specific responses with and without metabolic activation.

Conclusion

Diethylphosphonoacetic acid caused neither base-pair substitutions nor frameshift mutations in different strains of S. typhimurium in the presence and absence of metabolic activation when tested up to bacteriotoxic concentrations. Based on these results it was concluded, that the test substance is "Ames negativ