Reaction mass of Phenol, dodecyl-, sulfurized, calcium salts and benzene and butene and calcium carbonate and calcium dihydroxide and sulphur trioxide

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:

November 2011- July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A GLP study conducted in accordance with OECD test guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phenol, paraalkylation products with C12-rich branched olefins derived from propene oligomerisation, reaction products with sulphur monochloride and decene, reaction products with polybutenyl benzenesulphonic acid, carbon dioxide and calcium hydroxide

EC Number:

903-161-3

IUPAC Name:

Phenol, paraalkylation products with C12-rich branched olefins derived from propene oligomerisation, reaction products with sulphur monochloride and decene, reaction products with polybutenyl benzenesulphonic acid, carbon dioxide and calcium hydroxide

Test material form:

liquid: viscous

Details on test material:

-Test Article I.D.: EC 903-161-3
- Physical state: dark brown/blcak viscous liquid
- Lot/batch No.:LN09010643
-Expiration date: 07 November 2013
- Storage condition of test material: Room temperature, stored protected from light
*EC 903-161-3 is exclusively synthesised and handled in solvent oil. Therefore testing was conducted on sample that contained 40.2% base oil and 59.8 % EC 903-161-3.

Method

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:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Acrol-induce rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity assay- Dose level tested : 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, 5000 ug per plate.
Mutagenicity assay- Dose level tested: 50, 150, 500, 1500, 5000 ug per plate

Vehicle / solvent:

- Solvent used: tetrahydrofuran (THF) (99.92%, CAS No. 109-99-9, Lot No. 48347, Exp. Date April 2012)
- Justification for choice of solvent: THF was selected based on the solubility of the test article and compatibility with the target cells.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Remarks:

All positive controls were diluted in dimethyl sulfoxide (DMSO) except for sodium azide, which was diluted in water.

Details on test system and experimental conditions:

Solubility Test
A solubility test was conducted to determine the vehicle. The test was conducted using water, dimethyl sulfoxide (DMSO), ethanol (EtOH), acetone, tetrahydrofuran (THF) and dimethylformamide (DMF) to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents.

Preliminary Toxicity Assay
The preliminary toxicity assay was used to establish the dose-range over which the test article would be assayed. Vehicle control and a minimum of eight dose levels of the test article were plated, one plate per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.

Mutagenicity Assay
The mutagenicity assay was used to evaluate the mutagenic potential of the test article. A minimum of five dose levels of test article along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

Plating and Scoring Procedures
The test system was exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 µM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).

Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain and activation, as described in detail in BioReliance's Standard Operating Procedures.

One-half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain (cells seeded) and 25 µL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the attached table (see Attachment 1).
Revertant colonies for a given tester strain and activation condition, except for positive controls,were counted either entirely by automated colony counter or entirely by hand unless the assay was the preliminary toxicity assay or the plate exhibited toxicity.

Evaluation criteria:

The following criteria must be met for the mutagenicity assay to be considered valid. All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the characteristic mean number of
spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60. To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x109 cells/mL. The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).

Statistics:

no data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Solubility Test

THF was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article formed workable suspensions in THF from approximately 100 to 500 mg/mL and a clear solution at 75 mg/mL in the solubility test.

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions and the S9 and Sham mixes.

Tester Strain Titer Results

Experiment	TA98	TA100	TA1535	TA1537	WP2 uvrA
B1	0.9	1.1	2.3	1.4	1.2

Titer Value (x 10 9 cells per mL)

Preliminary Toxicity Assay

The results of the preliminary toxicity assay are presented in Tables 1 and 2 (see attachment 2). In the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 200 mg/mL and a 25 µL plating aliquot. The dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg per plate. In the larger volume needed to dose this trial, the test article formed clear solutions in THF from 0.27 to 200 mg/mL. Because only a minimal amount of test article is used in the solubility test, it is not unusual to see differences in the solubility profile when a larger volume is prepared. Precipitate was observed beginning at 667 or 1000 µg per plate. No toxicity was observed. Based on the findings of the toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 µg per plate.

Mutagenicity Assay

The results of the mutagenicity assay are presented in Tables 3 and 4 (see attachment 3) . These data were generated in Experiment B1. In Experiment B1 (Mutagenicity Assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 µg per plate. Precipitate was observed beginning at 500 µg per plate. No toxicity was observed.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The study was concluded to be negative.

Executive summary:

The study was conducted in accordance with OECD guidelines 471. All critieria for a valid study were met. Under the condition of this study EC 903-161-3 did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9. The study was concluded to be negative. The interpretation of the study data was based on the nominal dose levels due to lack of dose formulation analysis for concentration and stability. Nevertheless, precipitate in the assay demonstrated the test system was dosed up to the regulatory-required level.