2,3-Epoxypropyl neodecanoate, oligomeric reaction products with phosphorous acid

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:

2016-03-04 to 2016-03-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

march 2005. OPPTS changed its name to OCSPP,

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,3-Epoxypropyl neodecanoate, oligomeric reaction products with phosphorous acid

EC Number:

500-336-2

EC Name:

2,3-Epoxypropyl neodecanoate, oligomeric reaction products with phosphorous acid

Cas Number:

157348-58-8

Molecular formula:

Exact identification is not feasible

IUPAC Name:

Copolymer of neodecanoic acid oxiranylmethyl ester and phosphorous acid

Test material form:

other: Colourless, viscous liquid

Details on test material:

2,3-Epoxypropyl neodecanoate, oligomeric reaction products with phosphorous acid manufactured by Evonik Degussa GmbH, Batch 1524201 of 2015-12-22.

Method

Target gene:

mutated gene loci resposible for histidine auxotropy

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

- obtained from Trinova Biochem according to Dr. Bruce N. AMES,

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat liver S9; male rats, obtained from Trinova Biochem

Test concentrations with justification for top dose:

Plate incorporation test: 10.0, 31.6, 100, 316, 1000 or 3160 µg per plate;
Preincubation test: 10.0, 31.6, 100, 316, 1000 or 3160 µg per plate;

Vehicle / solvent:

The test item was completely dissolved in dimethylsulfoxide (DMSO) . The vehicle dimethylsulfoxide (DMSO) served as the negative control. Fresh
preparations of the test item were used for the treatment in all experimental parts.

Controls

Untreated negative controls:

no

Remarks:

solvent test will be used as negative reference item

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

for details see below

Positive control substance:

other: without metabolic activation: sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 2-nitroflurene in DMSO for TA 98, 9-amino-acridine in ethanol abs. for TA 1537, Mitomycin C in DMSO for TA 102

Remarks:

with metabolic activation: 2-aminoanthracene in DMSO for TA 100, TA 1535, Benzo(a)pyrene in DMSO for TA 98, TA 102, TA 1537

Details on test system and experimental conditions:

Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment: plate incorporation cytotoxicity test (+/- metabolic activation) with strain TA 100,
- The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100.- Ten concentrations ranging from 0.316 to 5000 µg/plate were tested.
- Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted at concentrations of
3160 and 5000 µg/plate. Hence, 3160 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction.
ADMINISTRATION
- Dosing:
* Plate incorporation test: 10.0, 31.6, 100, 316, 1000 or 3160 µg per plate;
* Preincubation test: 10.0, 31.6, 100, 316, 1000 or 3160 µg per plate;
- Data : 2 independent experiments with and without metabolic activation
- Number of replicates: 3 per concentration and experiment
- Positive and negative control groups and treatment:
- without metabolic activation:
* sodium azide in highly purified water for TA 1535 and TA 100, 10 µg/plate
* 2-nitroflurene in DMSO for TA 98, 10 µg/plate
* 9-amino-acridine in ethanol abs. for TA 1537, 100 µg/plate
* Mitomycin C in in highly purified water for TA 102, 10 µg/plate
- with metabolic acivation
* 2-aminoanthracene in DMSO for TA 100 and TA 1535, 2 µg/plate
* Benzo(a)pyrene in DMSO for TA 98, TA 102 and 1537, 10 µg/plate
- negative control: the vehicle DMSO was used as negative reference item (all test strains).
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

NUMBER OF REPLICATIONS: 3 per concentration and experiment

NUMBER OF CELLS EVALUATED: approximately 10E8 viable cells in the late exponential or early stationary phase

DETERMINATION OF CYTOTOXICITY
- Method: In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce
background lawn and reduction of the number of revertants) was noted at the top concentration of 3160 µg test item/plate in all test strains.

Evaluation criteria:

A test item is considered to show a positive response if
- the number of revertants is significantly increased (p least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent
experiments.
Or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation
coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on
histidine-free agar plates.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Acceptance Criteria
The results of the negative and positive control cultures have to be within the range of the historical data generated by LPT.

Statistics:

According to the OECD Guideline 471, a statistical analysis of the data is not mandatory

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Ccytotoxicity was noted at the top concentration of 3160 µg test item/plate in all strains

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

see Vehicle

Positive controls validity:

valid

Additional information on results:

GENTOXIC EFFECTS:
- With metabolic activation: negativ
- Without metabolic activation: negativ

CYTOTOXICITY EFFECTS:
In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce background
lawn and reduction of the number of revertants) was noted at the top concentration of 3160 µg test item/plate in all test strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

see attchached document

Applicant's summary and conclusion

Conclusions:

In conclusion, under the present test conditions, the test item tested up to a cytotoxic concentration of 3160 µg/plate, caused no mutagenic effect
in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Executive summary:

The purpose of this study was to evaluate the test item for mutagenic activity (gene mutation) in bacteria without and with the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and (1983).

The potential of the test item to induce gene mutations was examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

The test item was completely dissolved indimethylsulfoxide (DMSO).The vehicle DMSOserved as the negative control.

Preliminary test

Test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted at concentrations of 3160 and 5000 µg/plate. Hence, 3160 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 10.0 to 3160 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation

Cytotoxicity

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 3160 µg test item/plate in all test strains.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to acytotoxic

concentration of 3160 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system

In conclusion, under the present test conditions, the test item tested up to a cytotoxic concentration of 3160 µg/plate, caused no

mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.