dodecane-12-lactam, manufacturing of, by-products from, distillation residues

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:

11 August 2011 - 17 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

dodecane-12-lactam, manufacturing of, by-products from, distillation residues

EC Number:

923-400-5

IUPAC Name:

dodecane-12-lactam, manufacturing of, by-products from, distillation residues

Test material form:

other: brown solid

Details on test material:

- Name of test material: Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues
- Physical state: brown solid
- Lot/batch No.: AVRIL 2011
- Expiry date: 30 April 2012
- Composition of test material: UVCB
- Storage condition: at room temperature.

Method

Target gene:

Histidine operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Experiment without S9 :
The selected treatment-levels were:
. 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains in the first experiment,
. 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains in the second experiment.

Experiment with S9:
The selected treatment-levels were:
. 312.5, 625, 1250, 2500 and 5000 µg/plate for the TA 98, TA 100, TA 1535 and
TA 1537 strains in the first experiment,
. 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the strain TA 102 in the first experiment and for the five strains in the second experiment,
. 625, 1250, 1875, 2500, 3750 and 5000 µg/plate for the TA 1537, TA 98 and TA 100 strains in
the third experiment.

Vehicle / solvent:

- Vehicle used: 0.9% NaCl
- Justification for choice: According to the results of the solubility assay, the test item was prepared as a suspension in 0.9% NaCl. Using a concentration of 25 mg/mL and a treatment volume of 200 µL/plate, it was possible to reach the dose-level of 5000 µg/plate which is the highest recommended dose-level.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar
see Executive summary

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn

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.

Statistics:

not applicable

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

negative for TA 1535

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium 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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation

The test item showed mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, after metabolic activation system
with S9 mix.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium.

The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice.

 

Methods

A preliminary toxicity test was performed to define the dose-levels of AMINOCAL to be used for the mutagenicity study. The test item was then tested in three independent experiments, with and/or without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Experiments were performed according to the direct plate incorporation method except for the second and the third tests with S9 mix, which were performed according to the pre-incubation method (60 minutes, 37°C).

 

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at, the revertant colonies were scored.

The 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.

 

The test item AMINOCAL was suspended in 0.9% NaCl.

 

Results

The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered valid.

Since the test item was poorly soluble but also cytotoxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

 

The selected treatment-levels were ranged from 156.3 to 5000 µg/plate.

 

A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at all tested dose-levels.

 

Experiments without S9 mix

A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at 5000 µg/plate in the TA 1535, TA 1537 and TA 98 strains and at dose-levels = 2500 µg/plate in the TA 100 and TA 102 strains.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strain

Experiments with S9 mix

A moderate toxicity was noted at 5000 µg/plate in the TA 102, TA 98 and TA 1535 strains and at dose-levels = 3750 µg/plate in the TA 1537 and TA 100 strains.

No noteworthy increase in the number of revertant colonies was noted in the first experiment (using the direct plate incorporation method).

 

Noteworthy increases in the number of revertants, exceeding the positive threshold, were noted in the TA 1537 and TA 98 strains in the second experiment using the pre-incubation method (up to 3.3- and 2.8-fold the vehicle control value, respectively). These increases were then reproduced in the third experiment (up to 4.3- and 3.4-fold the vehicle control value, respectively). Consequently, they were considered as biologically relevant.

 

A slight increase in the number of revertants, remaining below the positive threshold (up to 1.9-fold the vehicle control value) was noted in the TA 100 strain in the second experiment. Then an increase in the number of revertants, exceeding the positive threshold was noted in the third experiment (up to 3.5-fold the vehicle control value), without any clear evidence of a dose-response relationship. Since the increase did not exceed the positive threshold in the second experiment and was not dose-related in the third experiment, it remained equivocal.

 

Conclusion

The test item showed mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, after metabolic activation system with S9 mix.