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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, Fully OECD 417 compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Also OECD guideline No 471
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): CECAJEL 210
- Chemical Name in report: N-Alkyl "oleyl" N,N-bishydroxyethylamine
- Substance type: Organic
- Physical state:light yellow liquid
- Analytical purity:98.5% (CoA)
- Impurities (identity and concentrations): free amine 2.78%, moisture 0.14%
- Purity test date: 8th March 2005
- Lot/batch No.: 9194, Arkema filing number GRL 0004/05
- Expiration date of the lot/batch: Not stated
- Stability under test conditions:stable
- Storage condition of test material:in a well ventilated place at room temperature

Method

Target gene:
The five strains of Salmonella typhimurium (a): TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine.
In addition, to increase their sensitivity to mutagenic items, further mutations have been added:
• the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of
the bacteria and increases permeability to large molecules that do not penetrate the normal
bacteria cell wall,
• the uvrB mutation is a deletion of a gene coding for the DNA excision repair system, which
renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
• the addition of the plasmid pKM 101 to strains TA 98, TA 100 and TA 102 enhances their
sensitivity of detection to some mutagens,
• in case of TA 102 strain, the histidine mutation is located on the multicopy plasmid pAQ1.
Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100 and TA102
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from Aroclor 1254 treated rats
Test concentrations with justification for top dose:
PRELIMINARY TOXICITY TEST
The test item was freely soluble in the vehicle (DMSO) at 100 mg/mL. Consequently, with a treatment volume of 50 μL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate.

MUTAGENICITY EXPERIMENTS
Since the test item was toxic 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.
Experiments without S9 mix:
The selected treatment-levels were:
⋅ 2.34, 4.69, 9.38, 18.75, 37.5 and 75 μg/plate, for all the strains in the first experiment,
⋅ 1.25, 2.5, 5, 10 and 20 μg/plate, for all the strains in the second experiment

Experiments with S9 mix:
The selected treatment-levels were:
⋅ 18.75, 37.5, 75, 150 and 300 μg/plate, for the TA 98, TA 1537 and TA 102 strains in the first experiment,
⋅ 9.38, 18.75, 37.5, 75 and 150 μg/plate, for the TA 100 and TA 1535 strains in the first experiment,
⋅ 6.25, 12.5, 25, 50 and 100 μg/plate, for all the strains in the second experiment


2.1.3 Positive controls
without S9 mix:
• 1 μg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,
• 50 μg/plate of 9-Aminoacridine (9AA): TA 1537 strain,
• 0.5 μg/plate of 2-Nitrofluorene (2NF): TA 98 strain,
• 0.5 μg/plate of Mitomycin C (MMC): TA 102 strain.
with S9 mix:
• 2 μg/plate of 2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,
• 10 μg/plate of 2-Anthramine (2AM): TA 102 strain.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO batch K33208450 435 (Merck Eurolab, Fontenay-Sous-Bois, France or distilled water for the Mitomycin C positive control.
- Justification for choice of solvent/vehicle: To achieve necessary solubility for test substance and the positive control substances.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Anthramine
Remarks:
With S9 Mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium Azide TA 1535 -TA100, 9-Aminoacridine TA 1537, 2-nitrofluorene TA 98, Mitomycin C TA102
Remarks:
Without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) was used for the preliminary test and for the first experiment both without S9 mix and the first experiment with S9 mix.
preincubation; was used for the second experiment with S9.

The direct plate incorporation method was performed as follows: test item solution (0.05 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 (b, c) was performed as follows: test item solution (0.05 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 (see § 2.5), revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK).

Preliminary toxicity test

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.

Mutagenicity experiments

In two independent experiments, using three plates/dose-level, each strain was tested, with and without S9 mix, with:
• at least five dose-levels of the test item,
• the vehicle control,
• the appropriate positive control.
The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.
Evaluation criteria:
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
Species / strain:
other: 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 examined
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

RANGE-FINDING/SCREENING STUDIES:

The test item was freely soluble in the vehicle (DMSO) at 100 mg/mL.

 

Consequently, with a treatment volume of 50 μL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate. A marked precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 2500 μg/plate.

 

A moderate to marked toxicity was noted at dose-levels ≥ 100 μg/plate towards the three strains used without S9 mix and in the TA 100 strain with S9 mix and ≥ 500 μg/plate in the TA 98 and TA 102 strains with S9 mix

 

COMPARISON WITH HISTORICAL CONTROL DATA:

 

All values were within the historical control means for the vehicle and positive controls for each strain..

 

ADDITIONAL INFORMATION ON CYTOTOXICITY:

 

A marked toxicity was observed in all strains at dose-levels ≥ 150 μg/plate.

 

Applicant's summary and conclusion

Conclusions:
Under our experimental conditions, the test item CECAJEL 210 (batch No. 9194) did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella
Executive summary:

The objective of this study was to evaluate the potential of the test item CECAJEL 210

(batch No. 9194) to induce reverse mutation inSalmonella typhimurium. The study was performed according to the international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

 

Methods

A preliminary toxicity test was performed to define the dose-levels of CECAJEL 210 to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were 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 minutes, 37°C). Five strains of bacteriaSalmonella 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 37°C, 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 CECAJEL 210 was dissolved in dimethylsulfoxide (DMSO).

 

The dose-levels of the positive controls were as follows:

without S9 mix:

• 1 μg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

• 50 μg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

• 0.5 μg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

• 0.5 μg/plate of Mitomycin C (MMC): TA 102 strain.

 

with S9 mix:

• 2 μg/plate of 2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,

• 10 μg/plate of 2-Anthramine (2AM): TA 102 strain.

 

Results

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

 

Experiments without S9 mix:

The selected treatment-levels were:

2.34, 4.69, 9.38, 18.75, 37.5 and 75 μg/plate, for all the strains in the first experiment,

1.25, 2.5, 5, 10 and 20 μg/plate, for all the strains in the second experiment.

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. A moderate to marked toxicity was noted in all strains at dose-levels ≥ 10 μg/plate. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

 

Experiments with S9 mix:

The selected treatment-levels were:

18.75, 37.5, 75, 150 and 300 μg/plate, for the TA 98, TA 1537 and TA 102 strains in the

first experiment,

9.38, 18.75, 37.5, 75 and 150 μg/plate, for the TA 100 and TA 1535 strains in the

first experiment,

6.25, 12.5, 25, 50 and 100 μg/plate, for all the strains in the second experiment.

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. A marked toxicity was observed in all strains at dose-levels ≥ 150 μg/plate. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

 

Conclusion

Under our experimental conditions, the test item CECAJEL 210 (batch No. 9194) did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella