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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:
2011-12-13 to 2012-01-30
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
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): 2-(2-naphthoxy)ethanol
- Physical state: solid
- Analytical purity: ≥98.5%
- Lot/batch No.: E00173-186
- Expiration date of the lot/batch: July 2013
- Storage condition of test material: Room temperature, stored protected from light without desiccant

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary Test

6.7, 10, 33, 67, 100, 333, 667, 1000, 333 and 5000 µg/plate

Mutagenicity Assay

15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen as solvent as choice based on the solubility of the test article and compatibility with the target cells.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene (with metabolic activation)
Details on test system and experimental conditions:
Solubility Test

A solubility test was conducted to determine the vehicle. The test was conducted, using
water and DMSO, to determine the vehicle, selected in order
of preference, that permitted preparation of the highest soluble or workable stock
concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic
solvents.


Preliminary Toxicity Assay

The preliminary toxicity assay was used to establish the dose-range over which the test article
would be assayed. Vehicle control and a minimum of eight dose levels of the test article were
plated, one plate per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and
WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat
liver S9.


Mutagenicity Assay

The mutagenicity assay was used to evaluate the mutagenic potential of the test article. A
minimum of five dose levels of test article along with appropriate vehicle control and positive
controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA
in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test article,
vehicle control and positive controls were plated in triplicate.


Plating and Scoring Procedures

The test system was exposed to the test article via the plate incorporation methodology
originally described by Ames et al. (1975) and updated by Maron and Ames (1983).

On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V),
was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final
concentration of 50 µM each. Top agar not used with S9 or Sham mix was supplemented with
25 mL of water for each 100 mL of minimal top agar. For the preparation of media and
reagents, all references to water imply sterile, deionized water. Bottom agar was Vogel-Bonner
minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom
agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented
with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner
salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).

Each plate was labeled with a code system that identified the test article, test phase, dose level,
tester strain and activation, as described in detail in BioReliance's Standard Operating
Procedures.

One-half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain (cells seeded) and 25 µL of
vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C.
After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar.
When plating the positive controls, the test article aliquot was replaced by a 50 µL aliquot of
appropriate positive control. After the overlay had solidified, the plates were inverted and
incubated for approximately 48 to 72 hours at 37±2°C. Plates that were not counted
immediately following the incubation period were stored at 2-8°C until colony counting could
be conducted.

The condition of the bacterial background lawn was evaluated for evidence of test article
toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination
without magnification. Toxicity and degree of precipitation were scored relative to the vehicle
control plate using the codes shown in attachment 1.

Revertant colonies for a given tester strain and activation condition, except for positive controls,
were counted either entirely by automated colony counter or entirely by hand unless the assay
was the preliminary toxicity assay or the plate exhibited toxicity.
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 article 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 increasing concentrations
of test article.

Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean
revertants at the peak of the dose response was greater than or equal to 3.0-times the mean
vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged
positive if the increase in mean revertants at the peak of the dose response was greater than or
equal to 2.0-times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets
the criteria for evaluation as positive. This could be a dose-responsive increase that does not
achieve the respective threshold cited above or a non-dose responsive increase that is equal to or
greater than the respective threshold cited. A response was evaluated as negative, if it was
neither positive nor equivocal.

Results and discussion

Test results
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:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Solubility Test

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions and the S9 and Sham mixes.

Tester Strain Titer Results

Experiment TA98 TA100  TA1535  TA1537  WP2 uvrA 
 B1 0.9 0.5  0.8  1.1 
 B2 0.6 

Titer Value (x 109cells per ml)

Preliminary Toxicity

Assay The results of the preliminary toxicity assay are presented in Tables 1 and 2 of attachment 2. In the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 µL plating aliquot. The dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg per plate. No precipitate was observed. Toxicity was observed beginning at 1000 or 3333 µg per plate. Based on the findings of the toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 µg per plate.

Mutagenicity Assay

The results of the mutagenicity assay are presented in Tables 3 through 6 (Attachment 3: Table 3 and 4. Attachment 4: Table 5 and 6). These data were generated in Experiments B1 and B2. In Experiment B1 (Mutagenicity Assay), no positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 and WP2 uvrA in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate. Precipitate was observed at 5000 µg per plate. Toxicity was observed beginning at 500, 1500 or at 5000 µg per plate. Due to numerous intermediate colonies that interfered with evaluation of the assay plates, tester strain TA1537 was not evaluated for mutagenicity but was retested in Experiment B2.

In Experiment B2 (Retest of the Mutagenicity Assay), no positive mutagenic responses were observed with tester strain TA1537 in either the presence or absence of S9 activation. An increase in revertant counts (3.0-fold maximum increase) was observed with tester strain TA1537 in the absence of S9 activation. However, the increase was not considered indicative of mutagenic activity because (1) the vehicle control value was on the low end of the acceptable range and (2) the plate counts for all treated dose levels, except for the high dose which was toxic, were within the historical vehicle control range. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 µg per plate. Precipitate and toxicity were observed at 5000 µg per plate.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, EC 202-228-8 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. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because no unique metabolism requirements were known about the test article and because no equivocal responses were observed in the assay that would suggest further testing was warranted.

At the request of the Sponsor, dosing formulation analysis for concentration and stability was not conducted. Due to the lack of dose formulation analysis, the interpretation of the study data was based on the nominal dose levels as documented in the study records and not on the actual formulated test article concentrations as confirmed by analytical analysis. Nevertheless, precipitate and toxicity in the assay demonstrated that the test system was dosed up to the regulatory-required level.
Executive summary:

The study was conducted in accordance with OECD guidelines 471. All critieria for a valid study were met. Under the condition of this study EC 220-228-8 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. The study was concluded to be negative. The interpretation of the study data was based on the nominal dose levels due to lack of dose formulation analysis for concentration and stability. Nevertheless, precipitate in the assay demonstrated the test system was dosed up to the regulatory-required level.