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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:
March 15, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Japanese: Methods of Testing New Chemical Substances
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
other: Standard Concerning Testing Laboratories Implementing Tests for New Chemical Substances
Version / remarks:
2011
Deviations:
no
Qualifier:
equivalent or similar 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:
Lot No. PER2952

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Provided by Japan Bioassay Research Centre on August 7, 1997
Additional strain / cell type characteristics:
other: Histidine-dependent
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Provided by Japan Bioassay Research Centre on April 9, 1997
Additional strain / cell type characteristics:
other: Tryptophan-dependent
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Dose finding test: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μl/plate
Main test I and II: 313, 625, 1250, 2500 and 5000 μl/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water (Hikari Pharmaceutical Co., Ltd; Lot No. C23VS1)
- Justification for choice of solvent/vehicle: Able to dissolve 2-iso-butoxyethanol for the purpose of injection
Controls
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
benzo(a)pyrene
furylfuramide
other: 2-Aminoanthracene (Lot No. EPM0250; Purity: 96.3%)
Details on test system and experimental conditions:
- Method: Preincubation method by Matsuyama et al. (1980), based on Maron and Ames (1983)
- Composition of S9 mix (in 1 ml): S9 from 7-week old Sprague-Dawley male rat liver: 10 vol % (0.1 ml); 0.2 mole/l Na-Phosphoric acid buffer: 100 μmole/ml (0.5 ml); Coenzyme solution 0.38 ml; KCl: 33 μmole/ml; Glucose-6-phosphate: 5 μmole/ml; Nicotinamide-adenine dinucleotide (reduced form, disodium salt): 4 μmole/ml; Nicotinamide-adenine dinucleotide phosphate (reduced form, tetrasodium salt): 4 μmole/ml; 0.4 mole/l MgCl solution: 8 μmole/ml (0.02 ml)

EXPERIMENTAL PROCEDURE:
- Protocol: In the experimental condition without metabolic activation (S9 mix absence), 0.1 ml test substance solution, 0.5 ml 0.1 mole/l Na-phosphoric acid buffer (pH7.4) and 0.1 ml bacteria were gently mixed. In the experimental condition with metabolic activation (S9 mix presence), 0.1 ml test substance solution and 0.5 ml S9 mix were gently mixed. 20 minutes after incubation at 37 °C, 2 ml of top agar was added, mixed, and poured onto the surface of minimum glucose agar.
0.1 ml distilled water for injection and positive control solution were used as the negative and positive control, respectively. Cultivation spanned 48 hours at 37 °C. The number of mutant colonies was counted by visual observation and a Colony Analyser (CA-11 System Science). Presence of sediment was checked by visual observation. Growth inhibition and the status of bacterial flora on the surface of agar was determined by visual observation and a stereo microscope. The mean mutant colony value for the negative and positive control plates was calculated.
- Contamination check: 1 ml test substance solution and 0.5ml 0.1 mole/l Na-phosphoric acid buffer (pH7.4) (or 1 ml test substance solution and S9 mix 0.5 ml) were gently mixed and incubated at 37 °C for 20 minutes. The solution was then gently mixed with 2 ml top agar for S. typhimurium and poured onto the surface of a minimum glucose agar. Bacterial contamination in agar was checked 48 hours after cultivation at 37 °C. No bacterial contamination was detected in the test substance solution or S9 mix



Evaluation criteria:
2-Iso-butoxyethanol was considered to have mutagenicity when the average number of mutant colonies in the plates containing the test item was more than twice that of the negative control and where reproducibility or dose-dependency was detected.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
2-Iso-butoxyethanol did not inhibit growth or induce mutagenicity in Salmonella typhimurium TA 98, TA 100, TA 1535, and TA 1537, and Escherichia coli WP2 uvrA in the dose finding test, nor in the two main tests, with or without metabolic activation.

Any other information on results incl. tables

In all test bacteria, colony numbers in the test material conditions were less than double the colony numbers of the negative control in both the presence and absence of metabolic activation. No bacterial contamination was detected in the test substance solution or S9 mix. Mutagenicity of the positive control was detected in all of the test bacteria. Furthermore, the value of the positive and negative controls was within the range of background data (average ±3 x standard deviation).

Applicant's summary and conclusion

Conclusions:
Following a reverse mutation experiment in Salmonella typhimurium and Escherichia coli, it has been concluded that 2-iso-butoxyethanol does not possess mutagenic potential. Based on the results obtained under the conditions of this study, classification in line with CLP Regulation (EC) No. 1272/2008 is not required.
Executive summary:

A reverse mutation study was performed to test the effect of 2-iso-butoxyethanol in Salmonella typhimurium TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2 uvrA. The test procedure was according to Good Laboratory Practise (GLP) and the Japanese Methods of Testing New Chemical Substances (2011) and Japanese Standard Concerning Testing Laboratories Implementing Tests for New Chemical Substances (2011) without deviation. This methodology is considered to be similar / equivalent to OECD Guideline 471 (Bacterial Reverse Mutation Assay). The purpose of the experiment was to determine the mutagenic potential of 2-iso-butoxyethanol with and without metabolic activation (S9 mix) using the preincubation technique described by Maron and Ames (1983).

A dose finding test was performed at 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μl/plate of the registered substance. No inhibition of bacterial growth was observed. Two subsequent tests were performed as part of the main experiment using 313, 625, 1250, 2500, and 5000 μl/plate of 2-iso-butoxyethanol. 0.1 ml distilled water (solvent) was used as a negative control and 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine, benzo[a]pyrene, and 2-aminoanthracene were selected as positive controls. Cultivation was permitted for 48 hours at 37°C. Where the sum of mutated colonies exposed to 2-iso-butoxyethanol was more than twice that of the negative control and where reproducibility or dose-dependency was detected, the registered substance could be categorised as a mutagen.

Reverse mutation inS. typhimuriumandE. colistrains revealed consistently negative results whereby colony numbers at all concentrations were less than double those of the negative control with and without metabolic activation. No bacterial contamination was detected in the solution containing 2-iso-butoxyethanol, nor the S9 mix, and mutagenicity arising from exposure to the positive controls was detected in all bacteria. In addition, the value of the positive and negative controls was within the range of background data (average ±3 x SD). It can be concluded, therefore, that 2-iso-butoxyethanol does not have the potential for genetic toxicity under these experimental conditions.