Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September-December 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP- and OECD 471-compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
- OECD guideline No. 471, adopted on 21st July 1997,
- Commission Directive No. 2000/32/EC, B13/14, 8 June 2000.
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid - liquid: aqueous solution
Details on test material:
Substance tested as different commercial products:
- Mackam 2CY-75, 15% solids (active + impurities) [batch L-48049] in Acute Oral Toxicity, Skin Irritation and Eye Irritation
- Miranol J2M Conc, 49% solids (typical) [batch 2902D77] in Acute Oral Toxicity, or [batch 4349D78] in Skin Irritation and Eye Irritation
- Miranol J2M Anhydrous Acid, 100% solids (assumed) [batch 5958D80] in Acute Oral Toxicity
- Miranol J2M Freeze Dried, 100% solids (measured) [batch R-0469-203-19] in Genetic Toxicity In Vitro
Specific details on test material used for the study:
Trade name: Miranol J2M Freeze Dried
Other name: DV 7581
Purity: 100%
Chemical active ingredient: Disodium caprylo amphoacetate
CAS No.: 68608-64-0
Batch No.: R-0469-203-19
Appearance: granular whitish powder
Storage conditions: at room temperature
Expiry date: August 2005

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Liver post-mitochondrial fraction of Aroclor 1254 intraperitoneally-treated rats (S9 mix)
Test concentrations with justification for top dose:
Experiments without S9 mix:
- 156.3, 312.5, 625, 1250 and 2500 μg/plate, for the TA 98 strain in the first experiment and for the TA 1537 and TA 102 strains in both experiments,
- 312.5, 625, 1250, 2500 and 5000 μg/plate, for the TA 1535 and TA 100 strains in both experiments and for the TA 98 strain in the second experiment.
Experiments with S9 mix:
- 312.5, 625, 1250, 2500 and 5000 μg/plate, for all the strains.
in both mutagenicity experiments.
Based on the levels of toxicity observed in the preliminary assay.
Vehicle / solvent:
Water for injections, batch No. FVC01A (Fresenius Kabi, Sèvres, France)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9 mix: sodium azide, 9-Aminoacridine, 2-Nitrofluorene, Mitomycin C / With S9 mix: 2-Anthramine
Details on test system and experimental conditions:
The test item was tested in a preliminary test and two mutagenicity experiments.
In the preliminary assay, to assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix. 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 preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.
The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
The preincubation method was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate. After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK).
Evaluation criteria:
Treatment of results:
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle), are presented in tabular form.

Acceptance criteria:
This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with the historical data of the testing facility,
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.

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.

Results and discussion

Test results
Key result
Species / strain:
other: 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

Any other information on results incl. tables

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

Without S9 mix:

A moderate to marked toxicity was noted at dose-levels ≥ 2500 μg/plate in the TA 1535, TA 1537 and TA 102 strains and at 5000 μg/plate in the TA 98 and TA 100 strains.

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

With S9 mix:

A moderate toxicity was noted in the TA 1535 strain in the first experiment (using the direct plate incorporation method).

A moderate to marked toxicity was noted at 5000 μg/plate in all the strains in the second experiment (using the preincubation method).

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

Applicant's summary and conclusion

Conclusions:
Under such experimental conditions, Miranol J2M Freeze Dried did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

The mutagenic potential of disodium caprylo amphodiacetate, as Miranol J2M Freeze Dried, was assessed in the Salmonella typhimurium microsomal assay according to OECD 471 test guideline and in compliance with Good Laboratory Practice.

The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used in the presence and the absence of metabolic activation system from the liver fraction of Aroclor 1254-induced rats (S9-mix). Each strain was exposed to 5 dose levels. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

A preliminary toxicity assay was performed to define the 5 dose levels to be used. The test substance, a slight pale yellow solid, was then tested in 2 independent experiments performed according to the direct plate incorporation method, except for the second test with S9 mix, which was performed according to the preincubation method (60 min, 37°C).

The evaluation of 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 article was dissolved in water. Because the test substance was toxic in the preliminary assay, the choice of the highest dose level for the main assay was based on the level of toxicity:

-         156.3, 312.5, 625, 1250 and 2500 µg/plate, for TA 98 strain in the first experiment and for the TA 1537 and TA 102 strains in both experiments without S9 mix

-         312.5, 625, 1250, 2500 and 5000 µg/plate, for the TA 1535 and TA 100 strains in both experiments and for the TA 98 strain in the second experiment without S9 mix

-         312.5, 625, 1250, 2500 and 5000 µg/plate, for all the strains in both experiments with S9 mix

 

All determinations were made in triplicate. Simultaneous negative (solvent) and positive controls were used in all experiments.

 

A moderate to marked toxicity was noted without S9 mix at dose-levels ≥ 2500 µg/plate in the TA 1535, TA 1537 and TA 102 strains and at 5000 µg/plate in the TA 98 and TA 100 strains. With S9 mix, a moderate toxicity was noted in the TA 1535 strain in the first experiment (using the direct plate incorporation method) and a moderate to marked toxicity was noted at 5000 µg/plate in all the strains in the second experiment (using the preincubation method).

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. Positive controls gave the expected increases in the number of revertants, with and without S-9 mix.

 

Under such experimental conditions, Miranol J2M Freeze Dried did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.