Oxirane, 2-methyl-, polymer with oxirane, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1)

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study, but no guideline available

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The "Ames II Assay" is a liquid version of the classical Ames test. It is performed in microwell plates using a modified fluctuation test protocol. Reversion of mutations leads to the growth of the bacteria and thus to an accumulation of catabolites from the metabolic activity. This further leads to a drop of the pH which turns the purple Ames II Reversion Indicator medium into yellow.
The test is carried out based on the description of Gee, P. et al. (Mut. Res., 412,115-130 (1998)) and on the "Users Manual" prepared by XENOMETRIX, Inc., Colorado, USA.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Oxirane, 2-methyl-, oligomeric reaction products with oxirane, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1)

EC Number:

610-877-7

Cas Number:

52624-57-4

Molecular formula:

C6 H14 O3 . 3 (C3 H6 O . C2 H4 O)x

IUPAC Name:

Oxirane, 2-methyl-, oligomeric reaction products with oxirane, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1)

Details on test material:

- Name of test material (as cited in study report): Triol 9053
- Physical state: colorless to yellowish, viscous liquid
- Lot/batch No.: Kreisprobe Tank 16
- Storage condition of test material: room temperature

Method

Target gene:

his

Species / strain

Species / strain 1

Species / strain / cell type:

other: Salmonella typhimurium TA 7000 series, which consists of the strains TA 7001, 7002, 7003, 7004, 7005 and 7006.

Details on mammalian cell type (if applicable):

Histidine auxotrophy for all tester strains is automatically checked in each experiment via the spontaneous rate.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

The Salmonella strain TA 98 is checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (delta uvrB); ampicillin resistance (R factor plasmids). Histidine auxotrophy for all tester strains is automatically checked in each experiment via the spontaneous rate.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix; Aroclor 1254 induced

Test concentrations with justification for top dose:

0, 4, 20, 100, 500, 2500, 5000 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: liquid fluctuation test (the AMAX test procedure: Ames II Mutagenicity Assay by Xenometrix)

OTHER:
- Optical density (OD600 values): The determination of the OD600 values is carried out by adding 100 µl aliquots from overnight cultures of each strain in 1 ml spectrophotometer cuvettes containing 900 pl growth medium. The OD600 values are at least 2.0 for the TA 7000 series strains or TA 98 and about 0.0 for the negative control.

- Liquid exposure: 5 ml of the overnight cultures are added in culture tubes containing 19 ml Ames II Exposure medium (Xenometrix) and are gently mixed. After thorough pipetting af the content the following components are added in 24-well exposure plates using a 8-channel pipettor to a final volume af 250 pl/weIl:
240 µI exposure medium & tester strain (in tests without S-9 mix)
200 µl exposure medium & tester strain (in tests with S-9 mix)
40 µl S-9 mix (delta final concentration af 4.8% S-9 fraction)
10 µl Test solution, control chemicals or vehicle
The 24-well exposure plates are then incubated at 37 °C for 90 minutes, with shaking at 250 rpm using an environmental shaker.

Prototrophic selection: After the 90-minute incubation, 2.8 ml Ames II Reversion lndicator medium (Xenometrix) (containing bromocresol purple as an essential constituent) is pipetted to each well of the 24-well plate. The histidine-deficient indicator medium which selects for prototrophic
reversion is mixed gently several times. When adequately mixed, the contents of each well of a 24-well microtiter plate are distributed in 50 µl aliquots over 48 wells of a 384-well Revertant Colony Selection Plate (RCSP) by Eppendorf pipettes. After sealing the RCSP in plastic bags to prevent evaporation and after incubation at 37 °C for about 48 hours in the dark each 48-well section of the 384-well plates are scored and the number of positive wells (yellow) are counted.

- triplicate plate / dose, control chemical or vehicle

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of positive wells
- clearing or diminution of the background lawn (= reduced his- background growth)
leading from turbid to non-turbid purple wells is recorded for all test groups both with and without S-9 mix.

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
* A dose-related increase in the number of positive wells by a factor of about 2 (calculated primarily on the basis of baseline data) in at least one tester strain either without S-9 mix or after adding a metabolizing system. A test substance is generally considered non-mutagenic in this test if:
* Ihe number of revertant wells for all tester strains were within the historical negative control range under all experimental conditions.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: Salmonella typhimurium TA 7000 series, which consists of the strains TA 7001, 7002, 7003, 7004, 7005 and 7006.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.