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Genetic toxicity in vitro

Description of key information

A reverse mutation experiment was undertaken in line with the Maron and Ames (1983) pre-incubation technique to determine the mutagenic potential of 2-iso-butoxyethanol in Salmonella typhimurium TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2 uvrA. No significant increase in revertant colony frequency was observed with and without metabolic activation (S9 mix) in any bacterial strain, at any concentration. Under the experimental conditions, 2-iso-butoxyethanol was established not to be a mutagen. This conclusion has been supported by an in vitro mammalian chromosome aberration experiment with hamster lung (CHI/IU) cells, in which no significant inhibition of cell proliferation, nor significant structural aberration or polyploidy, was observed in chromosomal DNA with and without metabolic activation following 6- and 24-hour exposure to 2-iso-butoxyethanol. Classification is not required (CLP Regulation (EC) No. 1272/2008).

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Referenceopen allclose all

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
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
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
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.
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.

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).

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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 27, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
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
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Chromosome
Species / strain / cell type:
other: Hamster lung (CHI/IU) cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from the liver of 7-week old male Sprague-Dawley rats
Test concentrations with justification for top dose:
Dose finding concentrations: 0.0188, 0.0375, 0.0750, 0.150, 0.300, 0.600, 1.200 mg/ml.
Although cell proliferation decreased to 79% in the 24h continous test, cell proliferation inhibition did not exceed 50% in all conditions with 1.2 mg/ml. Therefore the following test concentrations were used for the abberration test: 0.15, 0.30, 0.60, 1.20 mg/ml
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
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:

- Procedure: The test consisted of a 6-hour experiment with and without metabolic activation (S9 mix) and a 24-hour continuous experiment. Four concentration groups from 0.15, 0.30, 0.60, and 1.2mg/ml (common ratio: 2) were analysed at all treatment conditions. Four dishes were prepared per each concentration group, with two dishes for chromosome sampling and the rest for cell proliferation rate.
- Positive control: Mitomycin as a positive control for a 6-hour treatment without S9 mix and for a 24-hour continuous treatment at 15 µl/dish (final concentration:0.1 µl/ml) and 12.5 µl/dish (final concentration:0.05 µl/ml), respectively. Cyclophosphamide was used as a positive control for the 6-hour treatment with the presence of S9 mix at 30 µl/dish (final concentration: 10 µl/ml). It is known that these concentrations of mitomycin and cyclophosphamide induce structural abnormality of chromosome. Only two dishes were prepared for chromosome sampling and the culture solution was replaced by 10%CS/MEN or S9.
- Negative control: Distilled water for injection was used as a negative control and added at the level of 10 vol% per dish.

Other
- 10%CS/MEN: Culture solution containing calf serum (Lot No. 522123, Biological Industries; 990250, GIBCO) at the level of 10 vol% of Eagle’s Minimal Essential Medium (EMEM, Nissui Pharmaceutical Co. Ltd.).
- S9 mix: S9 (Lot No. RAA-653 and RAA-655, Kikkoman Corp.) was prepared from the liver of 7-week old male Sprague-Dawley rats into which phenobarbital and 5,6-benzoflavone were injected. The final S9 mixture contained 5 vol% S9, 0.83mmol/L glucose-6-phosphate, 0.67mmole/L β-NADP+, 0.83mmol/L MgCl2, 5.5 mmol/L KCl, and 0.67mmol/L HEPES.
Evaluation criteria:
Type and number of structural abnormalities were examined in the 200 metaphase cells (chromosome number: 23 - 27) per group (100 cell/dish, 25 cell/observer). Polyploid cell numbers were counted in the 800 metaphase cells (chromosome number: 38 or more than 38) per group (400 cell/dish, 40 cell/observer). From the results, an appearance ratio was calculated of cells with structural abnormality against those deemed to be polyploid. Inducibility of chromosome abnormality was comprehensively evaluated by referring to the Fisher’s exact test result (see Statistics) and from the biological perspectives.
Statistics:
A significance test (Fisher’s exact test, one-sided) of the appearance ratio was conducted for the test substance treatment, positive control, and negative control for 6-hour and 24-hour tests. Cochran-Armitage's trend test, one-sided was performed for 6-hour tests with and without metabolic activation.
Key result
Species / strain:
other: Hamster lung (CHI/IU) cells
Remarks:
6-hour treatment
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:
other: Hamster lung (CHI/IU) cells
Remarks:
24-hour treatment
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Following chromosome analysis, no statistically significant increase in the number of polyploid cells or the number of cells with structural abnormalities was reported.

Examination for the inhibition of cell proliferation revealed a decline in cell proliferation to 79 % in the 24-hour continuous treatment. However, cell proliferation inhibition did not exceed 50 % in all experimental conditions with 1.2 mg/ml test substance. At the start and end of the treatment, precipitation was not identified with bare eyes in the culture solution in all test substance groups. Based on the obtained result, chromosome analysis was carried out for the conditions described below:

 

- Short-term treatment without the S9 mix: 0.15, 0.30, 0.60, 1.2mg/ml (common ratio: 2)

- Short-term treatment with the S9 mix: 0.15, 0.30, 0.60, 1.2mg/ml (common ratio: 2)

- 24-hours of continuous treatment: 0.15, 0.30, 0.60, 1.2mg/ml (common ratio: 2).

 

At the start and end of the treatment, precipitation was not identified in the culture solution in the all test substance groups. However, precipitation was identified with bare eyes during the experiment involving the negative control and 0.15 mg/ml groups of short-term treatment without the S9 mix. The experiment of the short-term treatment without the S9 mix was cancelled and re-examination was conducted as below.

 

- Short-term treatment with the S9 mix (re-examination): 0.15, 0.30, 0.60, 1.2 mg/ml (common ratio: 2)

 

At the start and end of the treatment, precipitation was not identified with bare eyes in the culture solution in all test substance groups. Mitotic index analysis indicated that the highest analysable concentration was 1.2 mg/ml in the experiment. Therefore, the groups described below were used for chromosome analysis.

 

- Short-term treatment without the S9 mix: 0.30, 0.60, 1.2mg/ml

- Short-term treatment with the S9 mix: 0.30, 0.60, 1.2mg/ml

- 24-hours of continuous treatment: 0.30, 0.60, 1.2mg/ml

Conclusions:
An in vitro mammalian chromosome aberration experiment using hamster lung (CHI/IU) cells found that 2-iso-butoxyethanol did not bring about significant inhibition of cell proliferation, nor were abnormalities in the chromosomal DNA such as structural aberration and polyploid cell reported from exposure to 2-iso-butoxyethanol. The registered substance can be concluded to be non-mutagenic under the experimental conditions and it does not require classification (CLP Regulation (EC) No. 1272/2008).
Executive summary:

To establish the potential genetic toxicity of 2-iso-butoxyethanol, an in vitro mammalian chromosome aberration experiment with hamster lung (CHI/IU) cells was performed according to Japanese Methods of Testing New Chemical Substances (2011) and the Japanese Standard Concerning Testing Laboratories Implementing Tests for New Chemical Substances (2011), which is similar / equivalent to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test). The test aimed to establish whether 2-iso-butoxyethanol had mutagenic potential.

An intial test to examine the potential cell proliferation inhibition at 0.019 - 1.2 mg/ml 2-iso-butoxyethanol was used to determine the test concentrations . Four test doses ranging from 0.15 mg/ml to 1.2 mg/ml (common ratio: 2) were used in the chromosomal aberration test. Three conditions were arranged, including a 6-hour treatment with and without metabolic activation and a 24-hour continuous treatment without metabolic activation. The S9 mix was derived from the liver of 7-week old Sprague-Dawley male rats. Mitomycin was selected as a positive control for the short-term and 24-hour treatment without S9 mix at 15 µl/dish (final concentration:0.1µl/ml) and 12.5µl/dish (final concentration:0.05 µl/ml) respectively. Cyclophosphamide was selected as a positive control to the short-term treatment with S9 mix at 30 µl/dish (final concentration: 10 µl/ml). Distilled water for injection was added at a level of 10 vol% per dish in the negative control.

A significance test (Fisher’s exact test, p<0.01, one-sided) of the appearance ratio, calculated from the frequency of cells with structural abnormalities and polyploid cell, was conducted among the test substance, positive control, and negative control groups. The potential of 2-iso-butoxyethanol to induce chromosome abnormality was comprehensively evaluated by referring to the Fisher’s exact test result and from biological perspectives.

Cell proliferation inhibition was found not to exceed 50% in all experimental conditions at up to 1.2 mg/ml 2-iso-butoxyethanol. In addition, there was no statistically significant increase in the number of cells that exhibited structural abnormalities or polyploidy. The positive and negative controls were valid. Subsequently, it has been determined conclusively that 2-isobutoxyethanol did not inhibit growth nor micronucleus abnormality in CHL/IU hamster cells and, therefore, the registered substance is non-mutagenic under the experimental conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

A Klimisch score of 1 (reliable without restriction) has been assigned to the endpoint conclusion attained from the bacterial chromosomal aberration experiment (Reference 1). This reliability level has been recommended as the study was performed according to the Japanese Methods of Testing New Chemical Substances (2011) and Standard Concerning Testing Laboratories Implementing Tests for New Chemical Substances (2011) without deviation, which is similar / equivalent to Good Laboratory Practise (GLP) and OECD Guideline 471 (Bacterial Reverse Mutation Assay). The experiment detailed in Reference 2 was performed in line with Japanese Methods of Testing New Chemical Substances (2011) and Standard Concerning Testing Laboratories Implementing Tests for New Chemical Substances (2011), which is similar / equivalent to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test). No deviation from the protocol was reported. As the test was not compliant with Good Laboratory Practise (GLP), the endpoint conclusion is suitable for regulatory use with some restriction and has been assigned a Klimisch score of 2.

Justification for classification or non-classification