E17-194T, (P,P-diethyl, monoester with 1,2 propanediol)_

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 9 2018 - Feb 29 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-hydroxypropan-2-yl diethylphosphinate and 2-hydroxypropyl diethylphosphinate

Cas Number:

2230512-72-6

Molecular formula:

C7H17O3P

IUPAC Name:

Reaction mass of 1-hydroxypropan-2-yl diethylphosphinate and 2-hydroxypropyl diethylphosphinate

Test material form:

liquid

Details on test material:

R&D level

Specific details on test material used for the study:

composition 100%
Clear transparent liquid
The test substance was expected to be stable for the duration of the test
Expiration date: November 3, 2020

Method

Target gene:

S. typhimurium: histidine
E. coli: tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA1535: rfa, uvrB; 1537: rfa, uvrB; 98: rfa, uvrB, R-factor; 100: rfa, uvrB, R-factor

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (cofactor supplemented post-mitochondrial fraction) sourced from mate Sprague-Dawley rats induced with phenobarbital and benzoflavone.

Test concentrations with justification for top dose:

The test was conducted with E17-194T at levels of 1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 microgram per plate, with the high level being the standard limit for this test.

Vehicle / solvent:

sterile water

Controls

Untreated negative controls:

yes

Remarks:

Test material not incorporated

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: Daunomycin ; 2-Aminoanthracene

Details on test system and experimental conditions:

The initial test followed the plate incorporation method, in which the following materials were mixed and poured over the surface of minimal agar plate:
10 microliter of prepared test substance, negative (vehicle) control, or prepared positive control substance.
500 microliter S9 mix or substitution buffer
100 microliter bacteria suspension (ST or EC)
2000 microliter overlay agar maintained at approx. 45 degrees C

Plates were prepared in triplicate at each experimental point and uniquely identified. after pouring, plates were placed on a level surface until the agar gelled then inverted and incubated at approx. 37 degrees C until growth was adequate for enumeration (approx 65 hours). appropriate sterility control check plates (treated with critical components in the absence of bacteria) were incubated as a standard procedural check. For the bacterial strains, plates were prepared for each treatment as follows (see table 1 below).
Conformatory test: this test employed the pre-incubation modification of the plate incorporation test. The test or control substances, bacteria suspension and S9/substitution buffer were incubated under agitation for approximately 30 minutes at approx. 37 degrees C prior to mixing with the overlay agar and pouring onto the minimal agar plates before proceeding as described for the initial test. The atudy design for the confirmatory test, including strains, dose levels ect. was as described above for the initial test.
A supplemental test wes performed to clarify results obtained with TA1537 in the initial phase of testing. The study utilized the same methodolgies and dose levels described above in the original phase of testing. Appropriate vehicle and positive controls were included.

Rationale for test conditions:

The background lawn for vehicle control plates should appear normal. The mean revertant colony counts for each strain treated with the vehicle should lie close to or within the expected range taking into account the laboratory historical control range / or published values. The positive controls (with S9 where required) should produce substantial increases in revertant colony numbers with the appropriate bacterial strain as specified below.

Evaluation criteria:

Evalation of Toxicity: Toxic effects of the test substance are indicated by the partial or complete absence of a background lawn of the non-revertant bacteria or a substantial dose-related reduction in revertant colony counts compared with lower dose levels and concurrent vehicle control taking into account the laboratory historical control range. Where precipitation obscures observations on the condition of the background lawn, the lawn can be considered normal and intact if the revertant colony counts are within the expected range based on results for lower dose levels and historical counts for that strain.
Evaluation of Mutagenicity: For each experiment point, the mutation factor (MF) was calculated by dividing the mean revertant colony count by the mean revertant colony count for the corresponding concurrent vehicle control group. The mutagenic activity of the test item was assessed by applying the following criteria:

Positive results (mutagenic potential): The results for the test item showed a substantial increase in revertant colony counts, i.e., response MF ≥ 2 for strains TA98, TA 100 and WP2 uvrA or MF ≥ 3 for strains TA1535 and TA 1537, with mean value(s) outside the laboratory historical control range. Otherwise, results were considered negative.
The above increae must be dose related and/or reproducible, i.e., increses must be obtained at more than one experimental point. if this criteria is not met, the results may be classified as equivocal and further testing may be appropriate.
A test substance that produces neither a concentration related increase in the number of revertant colonies nor a reproducible substantial increase in revertant colonies to be non-mutagenic in this test system.

Statistics:

means and standard deviations for all quantitative data were collected by the lab.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

pre incorporation method-main test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

pre-incubation method- confirmatory test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

plate incorporation method-supplemental main test 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

pre-incubation method-supplemental confirmatory test 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

plate incorportaion method - main test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

pre-incubation method - confirmatory test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

plate incorporation method - main test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

pre-incubation method-confirmatory test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

plate incorporation method - main test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

pre-incubation method - confirmatory test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, considering the laboratory historical control range and/or published values. The positive control substances caused the expected substantial increses in revertant colony counts in both absence and presence of S9 mix.
No signs of precipitation or contamination were noticed in any of the strains at all dose levels. No signs of toxicity were seen in any of the strains in any dose levels.
There was no contamination-related or substantial test substance related increases in the number of revertant colonies observed with all strains tested in both in the absence and presence of S9 using either plate incorporation or the pre incubation method

Remarks on result:

other: not mutagenic

Applicant's summary and conclusion

Conclusions:

Based on these findings and on evaluation system used E17-194T did not elicit evidence of bacterial mutagenicity in the Ames assay.

Executive summary:

Ames test was conducted with E17 -194T at levels of 1.58, 5.0, 15.8, 50, 158, 500, 1580 and 5000 microgram/plate. The main test was conducted using the plate incorporation method in both the absence and presence of metabolic activation. The results of the test were confirmed using a similar study design but employing the pre-incubation modification of the Ames test.

The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, considering the laboratory historical control range and/or published values. The positive control substances caused the expected substantial increses in revertant colony counts in both absence and presence of S9 mix.

No signs of precipitation or contamination were noticed in any of the strains at all dose levels. No signs of toxicity were seen in any of the strains in any dose levels.

There was no contamination-related or substantial test substance related increases in the number of revertant colonies observed with all strains tested in both in the absence and presence of S9 using either plate incorporation or the pre incubation method.

Based on these findings and on evaluation system used E17-194T did not elicit evidence of bacterial mutagenicity in the Ames assay.