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:
2017-07-28 to 2017-09-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted July 21, 1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- provided by the sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light and under nitrogen
- Solubility of the test substance in the solvent/vehicle:
test substance formed a clear solution in ethanol at a concentration of approximately 500 mg/mL with sonication at 37.0ºC for 20 minutes
- Stability of the test substance in the solvent/vehicle:
stable in ethanol, at concentrations of 0.0314 and 59.0 mg/mL, at room temperature for at least 3.5 hours, and at a concentration of 99.1 mg/mL for at least 3.1 hours.


Method

Target gene:
histidine locus of several Salmonella typhimurium strains
tryptophan locus of Escherichia coli strain WP2 uvrA
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix
Test concentrations with justification for top dose:
preliminary toxicity assay: 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 µg per plate
initial mutagenicity assay 5.00, 15.0, 50.0, 150, 500, 1500 and 3000 µg per plate (tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation, and TA100 in the presence of S9 activation) and 1.50, 5.00, 15.0, 50.0, 150, 333 and 500 µg per plate (tester strains TA100 in the absence of S9 activation, and TA1535 in the presence and absence of S9 activation).
retest of mutagenicity assay: 5.00, 15.0, 50.0, 150, 500, 1500, 3000, and 5000 µg per plate with tester strain TA1535 in the presence of S9 activation
protocol criteria for top dose or toxicity were met
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility pretest
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2 aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DETERMINATION OF CYTOTOXICITY
- Method: thinning of the microcolony lawn

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: main test was performed in triplicates

DETERMINATION OF CYTOTOXICITY
- Method: thinning of the microcolony lawn
Rationale for test conditions:
The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two consecutive increasing concentrations of test substance as specified below:

Data sets were judged positive if the increase in mean revertants at the peak of the dose-response was equal to or greater than 3.0-times (Strains TA1535 and TA1537) respectively equal to or greater than 2.0-times (Strains TA98, TA100 and WP2 uvrA) the mean vehicle control value and above the corresponding acceptable vehicle control range.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation observed up to the limit dose
- Other confounding effects: no
- Sterility Results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.


RANGE-FINDING/SCREENING STUDIES:
A range-finding study was conducted in all tester strains. Toxicity was observed beginning at 100, 333, 667, 1000, and 3333 µg per plate with all conditions.

Main test:
Toxicity was observed beginning at 150, 333, 1500, and 3000 µg per plate with all conditions except tester strain TA1535 in the presence of S9 activation
No precipitate was observed.

TA1535:
In the retest of mutagenicity assay, the dose levels tested were 5.00, 15.0, 50.0, 150, 500, 1500, 3000, and 5000 µg per plate with tester strain TA1535 in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 1500 µg per plate. No positive mutagenic responses were observed for the test substance.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: given in report
- Negative (solvent/vehicle) historical control data: given in report

Any other information on results incl. tables

Preliminary Toxicity Assay

Tester Strains

Without metabolic activation

(µg per plate)

With metabolic activation

(µg per plate)

Toxicity

Toxicity

TA98

≥ 1000

≥ 3333

TA100

≥ 333

≥ 1000

TA1535

≥ 100a

≥ 667

TA1537

≥ 667

≥ 3333

WP2uvrA

≥ 1000

≥ 3333

aToxicity was observed as a reduction in revertant counts.

Toxicity levels in main test

Tester Strains

Without metabolic activation

(µg per plate)

With metabolic activation

(µg per plate)

Toxicity

Toxicity

TA98

≥ 1500

3000

TA100

≥ 150

≥ 1500

TA1535

≥ 333

-

TA1537

≥ 1500

3000

WP2uvrA

≥ 1500

3000

In order to achieve the protocol criteria for top dose or toxicity, the test condition TA1535 in the presence of S9 activation was repeated. 

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, CGE-PMDA adduct did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9. All criteria for a valid study were met as described in the protocol.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrAwere exposed to CGE-PMDA adduct in ethanol in concentrations of 0 (control), 5.00, 15.0, 50.0, 150, 500, 1500 and 3000 µg per plate (tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation, and TA100 in the presence of S9 activation) and 1.50, 5.00, 15.0, 50.0, 150, 333 and 500 µg per plate (tester strains TA100 in the absence of S9 activation, and TA1535 in the presence and absence of S9 activation). In the retest of mutagenicity assay, the dose levels tested were 5.00, 15.0, 50.0, 150, 500, 1500, 3000, and 5000 µg per plate with tester strain TA1535 in the presence of S9 activation.The assay was performed using the plate incorporation method.

 

The test substance was tested up to cytotoxic concentrations.Toxicity was observed beginning at 150, 333, 1500 and 3000 µg per plate with all conditions except tester strain TA1535 in the presence of S9 activation. In order to achieve the protocol criteria for top dose or toxicity, the test condition TA1535 in the presence of S9 activation was repeated.Toxicity was then observed beginning at 1500 µg per plate.Precipitation was not observed. The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

 

There was no evidence of an increase in the number of revertant colonies that was equal to or greater than 3.0-times the mean vehicle control value in strains TA1535 and TA 1537 or exceeded twice the background in the tester strains TA98, TA100 and WP2 uvrA examined at the peak of the dose-response. Therefore, test substance was considered to be non-genotoxic (nonmutagenic) in Salmonella tester strains TA98, TA100, TA1535, TA1537 andEscherichia coli strain WP2 uvrA under the conditions employed (plate incorporation assay).

 

Under the conditions of the study, the test substance was negative for mutagenic potential.