oligomeric reaction product of 2-hydroxybenzaldehyde with 1-chloro-2,3-epoxypropane and phenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2015- 29 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-({2-[bis({2-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-({2-[bis({4-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-{[2-({2-[(oxiran-2-yl)methoxy]phenyl}({4-[(oxiran-2-yl)methoxy]phenyl})methyl)phenoxy]methyl}oxirane

EC Number:

942-639-6

IUPAC Name:

2-({2-[bis({2-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-({2-[bis({4-[(oxiran-2-yl)methoxy]phenyl})methyl]phenoxy}methyl)oxirane; 2-{[2-({2-[(oxiran-2-yl)methoxy]phenyl}({4-[(oxiran-2-yl)methoxy]phenyl})methyl)phenoxy]methyl}oxirane

Test material form:

solid

Details on test material:

Name: EPICLON EXA-7250
Batch/Lot number: B006
Appearance: Brown solid (in smaller amount colour is yellow)
Purity: >99%
Manufacture date: 02 October 2014
Expiry date: 02 March 2017
Storage condition: Room temperature 15-25°C, below 70 RH%
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face
mask) for unknown materials were applied to assure personnel
health and safety.

Method

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:

The S9 was prepared from the livers of 7-week old male Sprague-Dawley rats treated with phenobarbital (PB, 4 times at 24-hour intervals) and 5, 6-benzoflavone (once at a single i.p. dose of 80 mg/kg on the same day of the third PB injection)

Test concentrations with justification for top dose:

A preliminary test was performed at 5000 µg/plate as the maximum dose using 7 doses with a common ratio of 4. As the results, the number of revertant colonies treated with the test substance was greater than 2-fold that treated with the negative (solvent) control at the following dose levels.

Without S9 mix; 5000 µg/plate (TAlOO)
1250-5000 µg/plate (TA1535)

With S9 mix; 1250-5000 µg/plate (TAlOO)
313-5000 µg/plate (TA1535)
1250-5000 µg/plate (WP2uvrA)

Based on the results of the preliminary test, the main test I was performed at 5000 µg/plate as the maximum dose using 5-9 doses with a common ratio of 2. As the results, the number of revertant colonies treated with the test substance was greater than 2-fold that treated with the negative (solvent) control at thefollowing dose levels.

Without S9 mix; 5000 µg/plate (TAlOO)
1250-5000 µg/plate (TA1535)

With S9 mix; 625-5000 µg/plate (TAlOO)
156-5000 µg/plate (TA1535)
1250-5000 µg/plate (WP2uvrA)

The main test II was performed at using the same doses as the main test I.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2-AA) and 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylarnide (AF-2)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

Details on test system and experimental conditions:

METHOD OF APPLICATION: This study was performed using the pre-incubation method with and without S9 mix.

After pre-culture, bacterial density of each strain was measured by a digital photometer (TAITEC Co., Ltd. Mini photo 518R), and then the bacterial suspension for counting the number of cells was prepared by the stepwise dilution method. The suspension was overlaid to a minimal glucose agar plate and incubated at 37°C for 48 hours. The number of cells was calculated by counting the number of colonies on the plate. The bacterial suspension for the reverse mutation test was stored at room temperature.

Preparation of liquid growth medium
Oxoid Nutrient Broth No.2 (Oxoid Ltd., Lot No. 1239615) 2.5 g was dissolved in 100 mL of purified water. The medium was autoclaved for 15 minutes at 121°C and stored in a cold storage.

Preparation of soft agar
Bacto-agar (Becton, Dickinson and Company, Lot No.3220297) 0.6 g and sodium chloride (Wako Pure Chemical Industries, Ltd.) 0.5 g were added to 100 mL of purified water. This mixture was autoclaved for 15 minutes at 121°C and then was stored at 45°C until use.

Preparation of top agar
Each of the following each amino acid solution was added to the soft agar at a volume ratio of 1 to 10 just before use. The top agar was kept at 45°C until use.

Salmonella typhimurium: 0.5 mmol/L D-biotin and 0.5 mmol/L L-histidine solution mixture
Escherichia coli: 0.5 mmol/L L-tryptophan solution

The S9 was prepared from the livers of 7-week old male Sprague-Dawley rats (body weight: 204 to 244 g) treated with phenobarbital (PB, 4 times at 24-hour intervals at i.p. doses of 30, 60, 60 and 60 mg/kg, respectively) and 5, 6-benzoflavone (once at a single i.p. dose of 80 mg/kg on the same day of the third PB injection).

1mL of S9 was added to 9 mL of the cofactor mix to give the proper ratio for the S9 mix. The S9 mix was freshly prepared just prior to use in each test and was kept in an ice bath until use. The ingredients and their contents per mL of the S9 mix preparation were as follows:

S9
Magnesium chloride
Potassium chloride
D -Glucose 6 -phosphate
13-NADPH
13-NADH
Sodium phosphate buffer (pH 7.4)

(1) The test substance was weighed and dissolved in DMSO with vortex mixing and ultrasonic wave treatment to make a 50 mg/mL solution.
(2) A portion of this solution was diluted stepwise with DMSO to make the test substance solutions of each concentration.
(3) All procedures, including those for weighing the test substance, dilution, dispensation and treatment of the solutions, were performed under a yellow light at room temperature.

The storage period and temperature after preparation of the test substance solutions to the time of use were as follows;

Preliminary test: 50 minutes at room temperature (23°C)
Main test I: 1 hour and 50 minutes at room temperature (23°C)
Main test II:- 1 hour and 20 minutes at room temperature (23°C)

As the results of the preliminary test, the number of revertant colonies treated with the test substance was greater than 2-fold that treated with the negative (solvent) control at the following dose levels.

Without S9 mix; 5000 µg/plate (TAlOO)
1250-5000 µg/plate (TA1535)

With S9 mix; 1250-5000 µg/plate (TAlOO)
313-5000 µg/plate (TA1535)
1250-5000 µg/plate (WP2uvrA)

Based on these results, the main test I and main test Il were performed 5000 µg/plate as the maximum dose using 5-9 doses with a common ratio of 2

Pre-incubation method
(1) For each treatment, 0.1 mL of the test substance solution, negative (solvent) control or positive control solution was added into a sterilized test tube with aluminum cap.
(2) For assays without S9 mix, 0.5 mL of 0.1 mol/L sodium phosphate buffer (pH 7.4) was mixed and 0.1 mL of the bacterial suspension was added to this mixture.
(3) For assays with S9 mix, 0.5 mL of S9 mix was mixed and 0.1 mL of the bacterial suspension was added to this mixture.
(4) The mixture was incubated with gentle shaking (Shaking frequency: 120 times/minute) for 20 minutes at 37°C (pre-incubation).
(5) After pre-incubation, 2 mL of the molten top agar was added to this mixture and then poured onto a minimal glucose agar plate.
(6) After the overlaid agar had solidified, the plates were incubated for 48 hours at 37°C.

Preliminary test: 1 plate/dose (2 plates/dose for the negative (solvent) and positive controls)
Main test I: 3 plates/dose
Main test II: 3 plates/dose

Evaluation criteria:

Sterility test
In the preliminary and two main tests, bacterial contamination was examined using one plate for each of the highest dose of the test substance solution, S9 mix, and the 0.1 mol/L sodium phosphate buffer.
(1) 0.1 mL of the highest dose of the test substance solution or 0.5 mL of the S9 mix orthe 0.1 moVL sodium phosphate buffer was mixed with 2 mL of the molten top agar.
(2) The mixture was poured onto a minimal glucose agar plate.
(3) After the overlaid agar had solidified, the plates were incubated for 48 hours at 37°C and then checked for bacterial contamination macroscopically.

Observation
Precipitation: Precipitation was judged by observation of the plate surface macroscopically.
Microbial growth inhibition: Background lawn of the bacterial cells that have amino-acid requirement was observed by a stereoscopic microscope (Nikon, SMZ-10), and microbial growth inhibition was judged by the relationship between the test substance treated plates and the negative (solvent) control plate.

Measurement of colony number
Revertant colonies were measured using an automatic colony counter (System Science Co., Ltd., CA-1lD) or manual counting. Revertant colonies in positive control treatment groups, in TAlOO of test substance treatment groups without precipitation, and in the test substance treatment groups in which many revertant colonies were observed were measured using an automatic colony counter and the others measured by manual counting. Correction of the measuring area was employed using instrumental analysis.

Statistics:

No statistical analysis was performed with the test results in this study.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Any other information on results incl. tables

Maintest I

Number of revertant colonies: The number of revertant colonies treated with the test substance was greater than 2-fold that treated with the negative (solvent) control at the following dose levels.

Without S9 mix; 5000 µg/plate (TAIOO)

1250-5000µg/plate(TA1535)

With S9 mix;      625-5000 µg/plate(TAlOO)

156-5000 µg/plate (TA1535)

1250-5000 µg/plate (WP2uvrA)

 Mutation activities: The mutation activities were calculated in the following dose, the tester strain. (The mutation activities were adopted the maximum in the main test I)

Without S9 mix; 5000 µg/plate (TAlOO): 31.0 number ofrevertant colonies/mg

WithS9mix;      625 µg/plate (TAlOO): 309 number ofrevertant colonies/mg

 

Microbial growth inhibition: Microbial growth inhibition was not observed in all tester strains with or without S9 mix.

Precipitation: Precipitation of the test substance was observed at the following dose levels.

Without S9 mix; 313 µg/plate or more (all tester strains)

With S9 mix;     1250µg/plateormore(alltesterstrains)

Maintest II

Number of revertant colonies: The number of revertant colonies treated with the test substance was greater than 2-fold that treated with the negative (solvent) control at the following dose levels.

Without S9mix;5000 µg/plate (TAIOO)

1250-5000µg/plate(TA1535)

WithS9mix;      625-5000 µg/plate(TA!OO)

156-5000 µg/plate (TA1535) 1250-5000 µg/plate (WP2uvrA)

 Mutation activities: The mutation activities were calculatedinthe following dose, the tester strain. (The mutation activities were adopted the maximum in the main testII)

WithoutS9mix;5000µg/plate(TAI00):34.6numberofrevertantcolonies/mg

WithS9mix;      625µg/plate(TAIOO):312numberofrevertantcolonies/mg

 Microbial growth inhibition: Microbial growth inhibition was not observedinall tester strains with or without S9 mix.

Precipitation: Precipitation of the test substance was observed at the following dose levels.

Without S9mix;313 µg/plate or more (all tester strains) WithS9mix;      1250µg/plateormore(alltesterstrains)

 

Applicant's summary and conclusion

Conclusions:

In the main test I, the number of revertant colonies induced by the test substance was great r than 2-fold of that of the corresponding negative (solvent) control value in TAlOO, TA1535 without S9 mix, in TAlOO, TA1535 and WP2uvrA with S9 mix. The same result was obtained also in the main test II, and the reproducibility of the test results was confmned. The maximum mutation.activities was 312 at 625 µg/plate doses in TAlOO with S9 mix of main test II. Furthermore, both tests were performed accurately because the acceptable criteria were satisfied.

From the results described above, it was concluded that EPICLON EXA-7250 was mutagenic under the conditions employed in this study.

Executive summary:

Mutagenicity of EPICLON EXA-7250 was assessed in the bacterial reverse mutationtest using five strains,Salmonella typhimuriumTAlOO, TA1535, TA98 and TA1537, and Escherichia coli WP2uvrA.The test was conducted by the pre-incubation method with and without S9mix.

 

A preliminary test was performed at 5000 µg/plate as the maximum dose using 7 doses with aco=onratio of 4. As the results, the number of revertant colonies treated withthe test substance was greater than 2-fold that treated with the negative (solvent) control at the following doselevels.

Without S9 mix; 5000 µg/plate (TAlOO)

1250-5000 µg/plate (TA1535)

WithS9mix;      1250-5000µg/plate(TAlOO)

313-5000 µg/plate (TA1535)

1250-5000 µg/plate(WP2uvrA)

Microbial growth inhibition of the test substance was not observed in all tester strains with or without S9 mix.

Precipitation of the test substance was observed at 313 µg/plate or more in all tester strains without S9 mix, at 1250 µg/plate or more in all tester strains with S9 mix.

 

Based on the results of the preliminary test,the main test I was performed at 5000µg/plate as the maximum dose using 5-9 doses with a common ratio of 2.

As the results,the number of revertant colonies treated with the test substance was greater than 2-fold that treated with the negative (solvent) control at the following dose levels.

Without S9 mix; 5000 µg/plate (TAlOO)

1250-5000 µg/plate (TA1535) WithS9mix;     625-5000µg/plate(TAlOO)

156-5000 µg/plate (TA1535)

1250-5000 µg/plate(WP2uvrA)

Microbial growth inhibition of the test substance was not observed in all tester strains with or without S9 mix.

Precipitation of the test substance was observed at 313 µg/plate or more in all tester strains without S9 mix, at 1250 µg/plate or more in all tester strains with S9 mix.

 

The main test II was performed at using the same doses as the main test I. The results of the main test II were same as the main test I.

 

From the results described above, it was concluded that EPICLON EXA-7250 was mutagenic under the conditions employed in this study.