Ethylene bis[3,3-bis(3-tert-butyl-4-hydroxyphenyl)butyrate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Aug - 9 Sept 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hostanox O 3

IUPAC Name:

Hostanox O 3

Constituent 2

Reference substance name:

Bis-[3,3-bis-(4'-hydroxy-3'-tert.butylphenyl)-butanacid]-glycolester

IUPAC Name:

Bis-[3,3-bis-(4'-hydroxy-3'-tert.butylphenyl)-butanacid]-glycolester

Constituent 3

Reference substance name:

Ethylene bis[3,3-bis(3-tert-butyl-4-hydroxyphenyl)butyrate]

EC Number:

251-073-2

EC Name:

Ethylene bis[3,3-bis(3-tert-butyl-4-hydroxyphenyl)butyrate]

Cas Number:

32509-66-3

Molecular formula:

C50H66O8

IUPAC Name:

2-{[3,3-bis(3-tert-butyl-4-hydroxyphenyl)butanoyl]oxy}ethyl 3,3-bis(3-tert-butyl-4-hydroxyphenyl)butanoate

Method

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase locus on the X-chromosome of the V79 cells

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

without S9 mix: 1, 2.5, 5, 10 and 15 µg/mL
with S9 mix: 10, 25, 50, 75 and 100 µg/mL

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Ethyl methane sulfonate, 9,10-Dimethyl-1,2-benzanthracene

Details on test system and experimental conditions:

- Cell culture
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permiting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters. Thawed stock cultures are kept at approx. 37°C and approx. 4% CO2 in 175 cm2 plastic flasks. Seeding is carried out with about 5x10(exp)6 cells per flask in 30 mL of MEM-medium supplement with approx. 10% (v/v) FCS containing approx. 2 mM L-Glutamine and aprox. 0.1% (w/v) Neomycinsulfate. The cells were subcultured twice a week. For the selection of mutants the medium was supplemented with approx. 11 µg/mLThioguanine.

- Toxicity experiments and dose range finding
A preliminary cytotoxcity test was undertaken in order to select apprpriate dose levels for the mutation assay. In the test a wide range of dose levels of the test compound (1 - 250 µg/mL; determined by the solubility) was used. Cell cultures were subjected to the same treatment conditions as in mutation assays, and the survival of the cellss was subsequently determined. Treatments were performed both in the presence and absence of S9 metabolic activation system; a single cell culture was used at each point.

- Test procedure
In preliminary experiments approx. 4500 cells were seeded in each well of a microtiter plate, allowed to attach overnight and exposed to the test and control compoiund for four hours. For each concentration at least 6 wells were used. Approx. 24 h after treatment, the cells were fixed and stained with crystal violet. Survival was determined by measurement of the crystal violet extinction.
In the main experiments the cultures were prepared and treated with the test compound in the same way as for the preliminary experiment. 24 h after seeding of approx. 4500 cells per well in a microtiter plate, the medium was replaced with serum-reduced (5% v/v) medium containing the test compound, either without S9-mix or with S9-mix. After 4 h the treatment medium was replaced with normal medium after rinsing twice with medium. The cultures were stained with crystal violet and survivla was determined after an incubation period of approx. 24 h.

- Rationale for dose selection
For non-toxic, freely soluble test compounds, the top dose is 10 mM or 5000 µg/mL according to the current test guideline. For non-toxic, poorly soluble test compounds, the top dose is the highest non-precipitating dose.
For toxic compounds a percentage survival rate relative to the solvant contrl was calculated for each treatment. The dose level which results in a predicted survival of about 30% was estimated from the results obtained. This dose was chosen as the highest dose level. At least three lower levels are included in the treatment series.

- Mutagenicity test
Two-day old, exponentially growing cultures which were more than 50% confluent were trypsinated and a single cell suspension was prepared. The Trypsin concentration was approx. 0.25% (v/v) in Ca-Mg-free salt solution. Subsequently the cells were replated for mutagenicity testing and for determination of plating efficiency.
The treatment schadule of the mutagenicity test is described below:
Day 1: Subculturing of an exponentially growing culture
a) Approx. 4500 cells, in each well of a microtiterplate for determination of the plating efficiency
b) 6x10(exp)6 cells in 175 cm2 flaks with 30 mL medium for the mutagenicity test, one flask per experimental point.
Day 2: Treatment of a) and b) with the test compound in presence and absence of S9-mix (final protein concentration approx. 0.3 mg/mL) for 4 h
Day 3: Fixation and staining of the cells in a) microtiter plate for the determination of the plating efficiency
Day 5: Subculturing of b) in 175 cm2 flasks
Day 9: Subculturing of b) in five 75 cm2 flasks with medium containing 6-Thioguanine:
Mutant selection (about 300000 cells/flask);
subculturing of b) in two 25 cm2 flasks for plating efficiency (about 400 cells per flask)
Day 16: Fiaxation and staining of colonies of b) - from subcultures seeded on Day 9
All incubations were carried out at approx. 37°C and 4% CO2. Staining was performed with approx. 10% (v/v) Methylene blue in approx. 0.01% KOH solution. Omly colonies with more than 50 cells were counted.

Evaluation criteria:

Critieria for a positive response
The evaluation of the results was performed as follows:
- the test compound is classified as mutagenic if it reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than ths spontaneouss mutant frequency in this experiment.
the test copmound is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered indepently from the enhancement factor for induced mutants.
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

The biometry of the results was performed off-line with the Mann-Whitney-U-Test.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the highest tested concentration sowed cytotoxic effects

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Solubility and toxicity
In a preliminary experiment for solubility Hostanox O 3 was studied with respect to its solubility in cell culture medium. The highest concentration at which no visible precipitation was observed, was found to be 100 µg/mL.
Accordingly, the preliminary study for cytotoxicity was carried out using a maximum concentration of 250 µg/mL and a wide range of lower dose levels.
Following treatment in the absence of S9 metabolic activation, high cytotoxicity was obeserved. Survival declined in a dose-related manner reaching 4.7% of solvent control value at 25 µg/mL and 47.7% at 10 µg/mL. On the basis of these results, a concnetration of 15 µg/mL was used for the main assays and four lower doses were included in the treatment series.
In the presence of S9 metabolic activation survival declined steeply between the four highest concentrations. At a concentration of 250 µg/mL survival was reduced to 9.0% of the solvent control value.
Based on these results 100 µg/mL was determined in the presence of S9 mix as maximum dose level for mutagenicity testing in the main assays and four lower concentrations were included in the treatment series. Before treatment, the pH values and osmolarity of the treament media were determined. The addition of test compound solutions did not have any obvious effect on the osmolarity or pH of the treatment medium.

- Survival after treatment
In the absence of S9 metabolic activation in both experiments a dose-related decrease in survival was observed reaching 10.7 and 25.6% of the solvent control value at the highest dose tested. In the presence of S9 metabolic activation survival decreased in a dose-related manner reaching 16.0 and 5.5% of the solvent control value after treatment at the dose level of 75 µg/mL.

-Mutation results
Hostanox O 3 was assessed for its mutagenic potential in vitro in the HPRT-test in two independent experiment without and with metabolic activation.
In the presence of metabolic activation a slight but statistically significant increase of the mutant colonies was observed in the second experiment at the concnetration of 10 µg/mL and 50 µg/mL. These enhancements were induced by a low solvent control value and neither reproducible nor dose dependent. Therfore, this effect was considered to be without biological relevance.
In the absence of metabolic activation a slight but statistically significant increase in the mutant frequency was observed at the concentrations of 1.0 and 2.5 µg/mL, but only in the first experiment. These findings were not reproducible and not dose dependent and therefore also of no biological relevance.
The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1a: Mutagenicity Data (Main experiment; without S9 mix)

	Dose/[µg/mL]	Mean number of mutant colonies
Negative control	0.0	3.4
Solvent control (DMSO)	0.0	5.4
Positive control (EMS)	1000.0	117.2*
Hostanox O 3	1.0	8.8*
	2.5	7.8*
	5.0	0.8
	10.0	1.0
	15.0	2.2

 

Table 1b: Mutagenicity Data (Main experiment; with S9 mix)

	Dose/[µg/mL]	Mean number of mutant colonies
Negative control	0.0	3.6
Solvent control (DMSO)	0.0	4.6
Positive control (DMBA)	7.7	23.2*
Hostanox O 3	10.0	5.2
	25.0	3.4
	50.0	2.0
	75.0	3.4
	100.0	3.6

 

Table 2a: Mutagenicity Data (Repeat experiment; without S9 mix)

	Dose/[µg/mL]	Mean number of mutant colonies
Negative control	0.0	1.6
Solvent control (DMSO)	0.0	1.2
Positive control (EMS)	1000.0	166.4*
Hostanox O 3	1.0	2.0
	2.5	2.8
	5.0	1.0
	10.0	0.4
	15.0	0.0

 

Table 2b: Mutagenicity Data (Repeat experiment; with S9 mix)

	Dose/[µg/mL]	Mean number of mutant colonies
Negative control	0.0	3.4
Solvent control (DMSO)	0.0	1.6
Positive control (DMBA)	7.7	36.2*
Hostanox O 3	10.0	2.4*
	25.0	1.6
	50.0	4.6*
	75.0	1.4
	100.0	2.4

 

^*          Statistically significant (p ≤ 0.05)

Applicant's summary and conclusion

Conclusions:

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

Hostanox O 3 is not mutagenic in the HPRT-test with cells of the V79 Chinese hamster cell line.

Executive summary:

The potential of Hostanox O 3 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in vitro was investigated. The assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activation (S9 mix). The test article was dissolved in DMSO and tested at the following concentrations:

without S9 mix: 1, 2.5, 5, 10 and 15 µg/mL

with S9 mix: 10, 25, 50, 75 and 100 µg/mL

The concentration ranges were based on the results of preliminary testing for solubility and toxicity. The highest concentration showed cytotoxic effects with and without metabolic activation. Up to the highest investigated dose no relevant increase in mutant colony numbers was obtained in two independent experiments. Appropriate reference mutagens used as positive controls showed a distinct increase in induced colonies, thus indicating the sensitivity of the assay.

In conclusion, Hostanox O 3 does not induce gene mutations in the HPRT-test with V79 Chinese hamster cell, either in the presence or in the absence of a metabolic activation system, under the experimental conditions described.

Hostanox O 3 is therefore considered to be non-mutagenic in this HPRT-assay.