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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:
experimental study
Adequacy of study:
key study
Study period:
1981-09-16 to 1981-09-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Remarks:
Study predates GLP
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)
EC Number:
931-276-9
Cas Number:
114959-46-5
Molecular formula:
See information in Section 1.2.
IUPAC Name:
Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)
Details on test material:
- Name of test material (as cited in study report): [CAS Number 114959-46-5]
- Physical state: Dark brown coloured oil
- Lot/batch No.: TN 104/81
- Code number: ALX 1344
- STL Ref No.: ST 81/003
- Stability under test conditions: Stable for the duration of the experiment
- Storage condition of test material: In the dark at ambient temperature

Method

Target gene:
No data
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix pretreated with Aroclor 1254
Test concentrations with justification for top dose:
zero, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000 µg/plate
Vehicle / solvent:
The test substance was formulated as an emulsion in sterile water because of its insolubility in water and suitable organic solvents.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
carrier oil formulated in same way as test substance emulsions
Negative solvent / vehicle controls:
yes
Remarks:
Water and Tween 80
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
20 ug/plate with and without S9 mix for TA 1538, TA 98 and TA 100
Untreated negative controls:
yes
Remarks:
carrier oil formulated in same way as test substance emulsions
Negative solvent / vehicle controls:
yes
Remarks:
Water plus Tween 80
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
5 ug/plate with and without S9 mix for TA 1535
Untreated negative controls:
yes
Remarks:
carrier oil formulated in same way as test substance emulsions
Negative solvent / vehicle controls:
yes
Remarks:
Water plus Tween 80
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: neutral red
Remarks:
20 ug/plate with and without S9 mix for TA 1537
Untreated negative controls:
yes
Remarks:
carrier oil formulated in same way as test substance emulsions
Negative solvent / vehicle controls:
yes
Remarks:
Water plus Tween 80
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
20 ug/plate with and without S9 mix for E coli WP2 and E coli WP2 uvr A
Details on test system and experimental conditions:
The test substance was formulated as an emulsion in sterile water. Emulsions containing 200 mg/mL or 250 mg/mL of the test substance were prepared by blending a weighed quantity of the test material with an equal quantity of Tween 80 using an Ultra-Turrax blender. Sterile water was slowly added with continuous blending. The resulting emulsion was diluted to the required volume. Satisfactory emulsions containing 300 mg/mL or more of the test substance could not be made by this procedure. Emulsions containing from 100 mg/mL down to 1.0 mg/mL of the test substance were prepared by serial dilution of the 200 mg/mL or 250 mg/mL emulsions. Blank formulations containing 200 mg/mL or 250 mg/mL of Tween 80 in sterile water were supplied when required. Chemical stability of the aqueous emulsions of test material and carrier oil were assessed by a panel of qualified chemists. The emulsions were then assigned an expiry date of the end of the day of formulation and were issued with instructions to shake well before use.

The mineral oil carrier for the test material was included in each assay formulated in the same way as the test substance and at concentrations equivalent to the concentration in test substance. The carrier oil emulsion was made by blending 0.76 g of carrier oil and 2.0 g of Tween 80 using an Ultra-Turrax blender. Sterile water was then slowly added with continuous blending to give a total volume of 10 mL of emulsion. The emulsion was assigned an expiry date of the end of the day of formulation and was issued with instructions to shake well before use.

Test and negative control (carrier oil) emulsions were diluted with water to give suspensions of concentration 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 or 200 mg/mL.

Twenty µL of each sample was added to top agar mix in the presence or in the absence of S9 mix to give test substance amounts of 31.25, 62.5, 125, 250, 500, 1000, 2000 or 4000 µg/plate. The cultures were incubated at 37 degrees Centigrade for 48 hours before the revertant colonies were counted. Postitive controls were included in each assay.
Evaluation criteria:
Results are expressed as a ratio: mean number of revertant colonies per treated plate / mean number of revertant colonies per control plate. A system of triplicate plating was used and values of 2.5 x control value or greater were considered to indicate a mutagenic response.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
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
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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

In a preliminary cytotoxicity assay, amounts of test material up to 4000 µg per plate showed no evidence of precipitation in the top agar nor any toxicity to Salmonella typhimurium TA 100. In the mutation assays, microscopic examination of the background lawn showed that the test material was not cytotoxic in any of the bacterial strains tested at amounts up to 4000 µg per plate.

The addition of test material to agar layer cultures in amounts up to 4000 µg per plate (with or without incorporation of a rat liver microsomal activation system) did not lead to an increase in reverse gene mutation frequency in any of the bacterial strains tested. Relative reverse mutation rates are shown in Table 1.1a (attached).

The activity of the S9 mix and the sensitivities of the strains TA 1538, TA 98 and TA 100 were monitored by treating cultures with a known positive control compound, benzo(a)pyrene, which requires metabolic activation before it is able to induce gene mutation. The sensitivity of TA 1537 was monitored by the indirect mutagen, Neutral Red; the sensitivities of the E. coli strains and TA 1535 were monitored by testing with the direct-acting mutagens 4 -nitroquinoline-N-oxide or sodium azide respectively.

Applicant's summary and conclusion

Conclusions:
Application of the test substance in concentrations up to 4000 µg/plate presented no evidence of cytotoxicity and did not increase the reverse mutation frequency of Escherichia coli WP2 or WP2 uvr , or Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100, in the presence or in the absence of rat liver S9 fraction.