reaction mass of 1-chloro-3-(dodecyloxy)propan-2-ol andalpha-dodecyl-omega-hydroxy-di[oxy[(chloromethyl)-1,2-ethanediyl]] andalpha-dodecyl-omega-hydroxy-tri[oxy[(chloromethyl)-1,2-ethanediyl]]

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:

12 December 2006 - 18 December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

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-chloro-3-(dodecyloxy)propan-2-ol and alpha-dodecyl-omega-hydroxy-di[oxy[(chloromethyl)-1,2-ethanediyl]] and alpha-dodecyl-omega-hydroxy-tri[oxy[(chloromethyl)-1,2-ethanediyl]]

Cas Number:

63727-39-9

Molecular formula:

C15H31ClO2 C18H36Cl2O3 C21H41Cl3O4

IUPAC Name:

reaction mass of 1-chloro-3-(dodecyloxy)propan-2-ol and alpha-dodecyl-omega-hydroxy-di[oxy[(chloromethyl)-1,2-ethanediyl]] and alpha-dodecyl-omega-hydroxy-tri[oxy[(chloromethyl)-1,2-ethanediyl]]

Test material form:

other: liquid

Details on test material:

- Physical state: Colourless to pale yellow thick liquid
- Expiration date of the lot/batch: 04 October 2007
- Storage condition of test material: Room temperature

Method

Target gene:

His+ for S. typhimurium

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):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10 % (v/v) S9 fraction; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254 (500 mg/kg bw) by the intraperitoneal route.

Test concentrations with justification for top dose:

PRELIMINARY TOXICITY TEST
Direct plate incorporation method: 10, 100, 500, 1000, 2500 and 5000 μg/plate for the TA 98, TA 100 and TA 102 strains, with and without S9 mix

MUTAGENICITY EXPERIMENTS
Experiments without S9 mix (Direct plate incorporation method):
0.61, 1.22, 2.44, 4.88, 9.77 and 19.53 μg/plate for the TA 100 strain in both experiments and for the TA 1537 in the second experiment.
4.88, 9.77, 19.53, 39.06, 78.13 and 156.3 μg/plate for the TA 1535 and TA 102 strains in both experiments and for the TA 1537 strain in the first experiment.
156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate for the TA 98 strain in both experiments.

Experiments with S9 mix:
19.53, 39.06, 78.13, 156.3, 312.5 and 625 μg/plate for the TA 102 strain in the second experiment (preincubation method).
39.06, 78.13, 156.3, 312.5, 625 and 1250 μg/plate for TA 100 strain in the second experiment (preincubation method).
156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate for the TA 102 and TA 100 strains in the first experiment (direct plate incorporation method) as well as for the TA 1535, TA 1537 and TA 98 strains in both experiments (direct plate incorporation method for experiment 1 and preincubation method for experiment 2).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Dimethyl sulfoxide - Batch No K35781950 609, VWR, Fontenay-sous-Bois, France)
Formulation procedure: The test item was dissolved in the vehicle at concentrations of:
- 100 mg/mL for the preliminary toxicity test, 100 mg/mL for the first experiment and 100, 25 and 12.5 mg/mL for the second experiment. The preparations were made immediately before use.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine: 2 μg/plate for TA98, TA1537, TA100 and TA1535; 10 μg/plate for TA102

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Strains of S. typhimurium were obtained from B.N. Ames' Laboratory (University of California, Berkeley or Oakland Research Institute, USA).

METHOD OF APPLICATION: In agar (direct plate incorporation and preincubation method)

DURATION
- Preincubation period: 60 minutes at 37 °C
- Incubation period: 48-72 h at 37 °C for both direct plate incorporation and preincubation methods

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single plate/dose for vehicle and test item
- Mutagenicity experiments: 3 plates/dose for test item, vehicle and positive controls

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

OTHER: After 48-72 h of incubation at 37 °C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk, UK). Manual counting was used as needed.

Evaluation criteria:

- A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result.
- Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

- None

Results and discussion

Test results

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
- A moderate emulsion was observed in the Petri plates when scoring the revertants at the dose-levels ≥ 500 μg/plate.
- A moderate to strong toxicity was noted without S9 mix at the dose-levels ≥ 10 μg/plate in the TA 100 strain and at the dose-levels ≥ 500 μg/plate in the TA 102 strain.

MUTAGENICITY EXPERIMENTS
Experiments without S9 mix:
- A moderate emulsion was observed in the Petri plates when scoring the revertants at the dose-levels ≥ 625 μg/plate.
- A moderate to strong toxicity was induced at the dose-levels ≥ 4.88 μg/plate in the TA 1537 strain, at the dose-levels ≥ 9.77 μg/plate in the TA 100 strain, at 156.3 μg/plate in the TA 1535 and TA 102 strains. In the TA 98 strain, a moderate to strong thinning of the bacterial lawn was noted at dose-levels ≥ 1250 μg/plate in the second experiment only.
- In the TA 98 strain, 2-fold increase in the number of revertants was noted in the first experiment at the lowest dose-level tested of 156.3 μg/plate. This slight increase which was due to one out of three individual values was not considered as biologically relevant since it was neither dose-related nor reproducible. The test item did not induce any noteworthy increase in the number of revertants, in either experiment and in any of the remaining tester strains.

Experiments with S9 mix
- A moderate emulsion was observed in the Petri plates when scoring the revertants at the dose-levels ≥ 625 μg/plate.
- No toxicity was noted in any of the five tester strains using the direct plate incorporation method (first experiment) up to the highest dose-level of 5000 μg/plate.
- Using the preincubation method (second experiment) no toxicity was noted in the TA 1535 and TA 98 strains up to the highest dose-level of 5000 μg/plate. A moderate thinning of the bacterial lawn was noted in the TA 1537 strain at dose-levels ≥ 2500 μg/plate.
- For the TA 100 and TA 102 strains, the second experiment was in first instance performed up to 5000 μg/plate. At least four dose-levels showed toxicity following this treatment, therefore the experiment was not validated and repeated at lower dose-levels.
- In this repeat experiment, a moderate to strong thinning of the bacterial lawn was noted in the TA 100 strain at dose-levels ≥ 312.5 μg/plate.
- In the TA 102 strain, no toxicity was observed in the repeat experiment up to 156.3 μg/plate , therefore the treatment was repeated up to 625 μg/plate which showed an emulsion but no noteworthy toxicity.
- Test item did not induce any noteworthy increase in the number of revertants, in either experiment and in any of the five strains.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical data from the period 4 November 2004 to 23 August 2005.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

See attached Document for Tables of Results

Applicant's summary and conclusion

Conclusions:

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

Under the test conditions, the test item is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) strains.

Executive summary:

In a GLP-compliant reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471, 5 different strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to the test item at the following concentrations:

PRELIMINARY CYTOTOXICITY ASSAY:

Direct plate incorporation method: 10, 100, 500, 1000, 2500 and 5000 μg/plate for the TA 98, TA 100 and TA 102 strains, with and without S9 mix

MUTAGENICITY EXPERIMENTS

Experiments without S9 mix:

- Experiment 1 and 2 (direct plate incorporation method): 0.61, 1.22, 2.44, 4.88, 9.77 and 19.53 μg/plate (TA 100); 4.88, 9.77, 19.53, 39.06, 78.13 and 156.3 μg/plate (TA 1535 and TA 102); 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate (TA 98)

- Experiment 1 (direct plate incorporation method): 4.88, 9.77, 19.53, 39.06, 78.13 and 156.3 μg/plate (TA 1537)

- Experiment 2 (direct plate incorporation method): 0.61, 1.22, 2.44, 4.88, 9.77 and 19.53 μg/plate (TA 1537)

Experiments with S9 mix:

- Experiment 1 (direct plate incorporation method): 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate (TA 102 and TA 100)

- Experiment 1 and 2 (direct plate incorporation method for experiment 1 and preincubation method for experiment 2): 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate (TA 1535, TA 1537 and TA 98)

- Experiment 2 (preincubation method): 19.53, 39.06, 78.13, 156.3, 312.5 and 625 μg/plate (TA 102); 39.06, 78.13, 156.3, 312.5, 625 and 1250 μg/plate for (TA 100)

The metabolic activation system used in this test was10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254 (500 mg/kg bw) by the intraperitoneal route. Vehicle and positive control groups were also included in mutagenicity tests.

In a preliminary toxicity test, a moderate emulsion was observed in the Petri plates when scoring the revertants at the dose-levels ≥ 500 μg/plate. A moderate to strong toxicity was noted without S9 mix at dose-levels ≥ 10 μg/plate in the TA 100 strain and at dose-levels ≥ 500 μg/plate in the TA 102 strain. In mutagenicity experiments, a moderate emulsion was observed in the Petri plates when scoring the revertants at the dose-levels ≥ 625 μg/plate, with and without S9 mix. Without S9 mix, a moderate to strong toxicity was induced, depending on the dose-level and the tester strain. With S9 mix, no toxicity was noted in any of the five tester strains using the direct plate incorporation method. In the preincubation method, no or a moderate to marked toxicity was noted, depending on the tester strain and the dose-levels. In the TA 98 strain, 2-fold increase in the number of revertants was noted in the first experiment at the lowest dose-level tested of 156.3 μg/plate, without S9 mix. This slight increase which was due to one out of three individual values was not considered as biologically relevant since it was neither dose-related nor reproducible. The test item did not induce any noteworthy increase in the number of revertants, in either experiment and in any of the remaining tester strains, without S9 mix. Test item did not induce any noteworthy increase in the number of revertants, in either experiment and in any of the five strains, with S9 mix. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

 

Under the test conditions, the test item is considered as not mutagenic in this bacterial system.