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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-10-12 to 2007-11-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Substance type: Borated Glycol Ether
- Physical state:Clear colourless liquid
- Analytical purity: 100%
- Isomers composition: Not applicable
- Purity test date: Not provided
- Lot/batch No.: DEG4131078
- Expiration date of the lot/batch: 2009-06-05
- Stability under test conditions: Room temperature (20°C)

Method

Target gene:
Defective excision repair gene in Salmonella thyphymirium strains (uvrB) and Escherichia coli strain (uvrA).
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells:
TA 98: University of California, Berkeley Divison of Biochemistry & Molecular Biology, CA 94720 USA
E. coli WP2 (uvrA), TA 1537, TA 1535 and TA 100: TRINOVA BIOCHEM GMBH, Kerkrader Stra e 10, D-35394 Giessen
- Suitability of cells: cells selected according to guidelines
Metabolic activation:
with and without
Metabolic activation system:
S9 (CCR; batch Nos. 250507 and 200707)
Test concentrations with justification for top dose:
1st study (plate incorporation method): 5.0, 1.6, 0.5, 0.16 and 0.05 mg/plate (factor root 10)
2nd study (preincubation method): 5.0, 2.3, 1.1, 0.5 and 0.23 mg/plate (factor root 5)
Vehicle / solvent:
distilled water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
benzo(a)pyrene
other: 2-amino-anthracen, ICR 191, 4-nitro-o-phenylendiamine, nitrofurantoine
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- plate incorporation:
2.0 ml top agar with 0.5 mM Histidin/Biotin (or L-Tryptophan for E.coli) for plates without S9
1.5 ml top agar with 0.5 mM Histidin/Biotin (or L-Tryptophan for E.coli) for plates with S9
0.5 ml S9 mix for plates with metabolic activation
0.1 ml bacteria (overnight culture)

- preincubation:
0.35 ml test/reference item solution and double distilled water
1.75 ml buffer solution or S9-mix
0.35 ml bacteria (overnight culture)
After preincubation for 20 min. At 33-37°C: 0.7 ml of the precincubated solution and 1.5 ml top agar with 0.5 nM Histidin/Biotin or L-Tryptophan (for E.coli) solution

DURATION
- Preincubation period: 20 minutes (33-37°c)
- Exposure duration: 48 hours (36.2-37.7°C)

NUMBER OF REPLICATIONS: 3 per concentration level and control (Titer control replicates were prepared 2-fold)
Evaluation criteria:
The rate of induced revertant His+ colonies was derermined.
In addition, genotypes were evaluated. Plates of the mutagenicity test were inspected for present and reduced background lawn after an incubation time of 48h. Plates with colonies which correspond not with the typical shape and colour of Salmonella thyphimuium or Escherichia coli were regarded as contaminated and were not included in calculations.
Statistics:
Arithmetic mean values and standard deviations were calculated based on colonies per plate of three replicates. For evaluation of the results, the induction rate of the mean values was calculated (revertant colonies test item/revertant colonies control). If there is a concentration effect relationship over at least 3 concentrations and/or induction rate equal to or greater than 2, then the test item is classified as mutagenic.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- There were no confounding factors

RANGE-FINDING/SCREENING STUDIES: No

COMPARISON WITH HISTORICAL CONTROL DATA: No

ADDITIONAL INFORMATION ON CYTOTOXICITY: No
Remarks on result:
other: plate incorporation & preincubation

Any other information on results incl. tables

Table 1: Mutagenic and cytogenic effects of the test item in the Ames test

Strain

S9

Tested concentration range (mg/plate)

Lowest mutagenic concentration

Lowest cytotoxic concentration (mg/plate)

1st study

2nd study

(mg/plate)

1st study

2nd

study

TA 1537

-

0.05 – 0.5

0.23 -5.0

none

not cytotoxic
up to 5.0

not cytotoxic 
up to 5.0

+

TA 98

-

+

TA 100

-

+

TA 1535

-

+

E coli
WP2 (uvrA)

-

+

Applicant's summary and conclusion

Conclusions:
The test item is regarded to be not mutagenic under the test conditions.
Executive summary:

The mutagenic effect of B-TEGME was determined in a bacterial reverse mutation test according to OECD No. 471 in two independent study parts (plate incorporation and preincubation method). Test systems were the Salmonnella thyphimurium strains TA 1537, TA 98, TA100 and TA 1535 (uvrB) and Escherichia coli WP2 (uvrA) strains with (+) and without (-) the metabolic system S9 (from male Wistar rats), respectively. Positive and negative conrols were included in each study. B-TEGME was dissolved in double distilled water and applied once at the start of the experiments with the concentration ranges: 0.05 – 0.5 mg/plate in the 1st experiment and 0.23 -5.0 mg/plate in the second experiment. The exposure duration was 48h.

Mutagenic and cytotoxic effects were absent up to the limit dose of 5 mg/plate.