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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:
23 July 1997 - 05 August 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD, EC and US EPA test guidelines and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japan Ministry of Health and Welfare (1989) Notification Yakushin 1 No 24: Guidelines for Toxicity Studies of Drugs, 4 I, Bacterial Reverse Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzoflex 2-45 (Diethylene glycol dibenzoate DEGDB)
- Physical state: Clear colourless liquid
- Analytical purity: Diethylene glycol dibenzoate - 89.9% (w/w)
- Impurities (identity and concentrations):
Diethylene glycol monobenzoate - 5.85% (w/w)
Dipropylene glycol dibenzoate - 1.50% (w/w)
Ethylene glycol dibenzoate - 0.19% (w/w)
Triethylene glycol dibenzoate - 0.15% (w/w)
Unidentified component 1 - 0.10% (w/w)
Unidentified component 2 - 0.10% (w/w)
Unidentified component 3 - 0.54% (w/w)
- Lot/batch No.: 56625030
- Expiration date of the lot/batch: 19 May 1998
- Storage condition of test material: Room temperature.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
First test - 5000, 1500, 500, 150, 50, 15, and 5 µg/plate plus a vehicle control.
Second test - 5000, 1500, 500, 150, and 50 µg/plate, plus a vehicle control.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was confirmed by the sponsor to be poorly soluble in water (solubility 51 ppm at 25°C). The solubility at 50 mg/mL was assessed in DMSO, ethanol, methanol, and acetone and the test substance was found to be fully soluble in each
solvent tested. DMSO was selected as the solvent following confirmation by the sponsor. This solvent is the most commonly used solvent within the
testing laboratory when water is not suitable.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
5 µg/plate for TA1535, 3 µg/plate for TA100, and 2 µg/plate for CM891

Migrated to IUCLID6: In the absence of S9 mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537

Migrated to IUCLID6: In the absence of S9 mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
1 µg/plate for TA98

Migrated to IUCLID6: In the absence of S9 mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
In the presence of S9 mix. 2 µg/plate for TA1535; 10 µg/plate for CM891.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5µg/plate for TA1537, TA98, and TA100

Migrated to IUCLID6: In the presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation (second test only)


DURATION
- Preincubation period: 30 minutes (second test only)
- Exposure duration: 3 days


SELECTION AGENT (mutation assays): Histidine / tryptophan deficient agar


NUMBER OF REPLICATIONS: 3 replicate plates per concentration per strain


DETERMINATION OF CYTOTOXICITY
- Method: observation of the number of revertant colony counts and the presence or absence of the background bacterial lawn


Evaluation criteria:
The number of revertant colony counts for each test or positive control plate was determined and compared to the relevant concurrent vehicle control.

Results and discussion

Test resultsopen allclose all
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
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: In the first test, at 5000 µg and 1500 µg/plate when the test substance solution was mixed with the top agar, a cloudy solution formed; slight cloudiness was observed at 500 µg/plate and at 150 µg/plate there was no observable cloudiness. In all cases the appearance was similar once the agar had set on the plates.
In the pre-incubation assay, a cloudy solution formed with a few small globules at 5000 µg/plate, a cloudy solution with no globules was observed at 1500 µg and 500 µg/plate; a slightly cloudy solution was observed at 150 µg/plate, and at 50 µg/plate no cloudiness was observed. In all cases, the appearance was similar following the pre-incubation period.


COMPARISON WITH HISTORICAL CONTROL DATA: The mean revertant colony counts for the solvent controls were within the historical range.


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):
negative

It was concluded that DEGDB showed no evidence of mutagenic activity in this bacterial system.
Executive summary:

A bacterial reverse mutation assay was performed to assess the potential of the test material DEGDB to cause gene mutation. The study was conducted according to OECD, EC, US EPA, and Japanese test guidelines and in compliance with GLP.

DEGDB was tested against four strains of Salmonella typhimurium and one strain of Escherichia coli, both in the presence and in the absence of metabolic activation (provided by S9 mix). Relevant positive controls were tested concurrently and provided positive results, demonstrating the sensitivity of the assay.

No increases in the number of revertant colonies relative to the concurrent vehicle controls were observed for the test material in any of the strains tested. It was concluded that DEGDB showed no evidence of mutagenic activity in this bacterial system.