Hexamethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

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:

23 Jan 2015 - 23 Apr 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

EC Number:

252-346-9

EC Name:

Hexamethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

Cas Number:

35074-77-2

Molecular formula:

C40H62O6

IUPAC Name:

6-{[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoyl]oxy}hexyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate

Details on test material:

- Physical state: Solid, white to off-white
- Storage condition of test material: room temperature

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericine B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

1st Experiment
without S9 mix: 0; 6.3; 12.5; 25.0; 50.0; 100.0 μg/mL
with S9 mix: 0; 6.3; 12.5; 25.0; 50.0; 100.0 μg/mL
2nd Experiment
without S9 mix: 0; 5.0; 10.0; 20.0; 40.0; (80.0) μg/mL
with S9 mix: 0; 5.0; 10.0; 20.0; 40.0; (80.0) μg/mL
Numbers in parantheses were discontinued

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, acetone was selected as vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 2 weeks

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures were used for each of the two experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10exp6 clonable cells) or isolated statistically significant increases without a doseresponse relationship may indicate a biological effect but are not regarded as sufficien evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantl increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced by test substance treatment
- Effects of osmolality: not influenced by test substance treatment
- Precipitation: In the 1st Experiment in the absence and presence of S9 mix, test substance precipitation was observed in culture medium at the end of treatment at 50 μg/mL and above and in the 2nd Experiment at 40 μg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
In the pretest the parameters pH value and osmolarity were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, suspension of the test substance in the vehicle acetone was observed at 2600 μg/mL and above. In culture medium, test substance precipitation occurred by the end of treatment at concentrations of 40.6 μg/mL and above in the absence and presence of S9 mix. After 4 hours treatment in the presence and absence of S9 mix, cytotoxicity was not observed as indicated by a reduced relative cloning efficiency of about or below 20% relative survival.

COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments after 4 hours treatment with the test substance the values for the corrected mutation frequencies were clearly within the range of our historical negative control data. The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies were clearly within our historical positive control data
range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective vehicle control values were observed in both experiments in the absence and presence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results

Exp.	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr.[per 106 cells]	Cytotoxicity***
						CE1 [%]	CE2 [%]
1	4	Vehicle control^1	-	n.d.	3.57	100.0	100.0
		6.3	-	-	4.48	94.8	98.2
		12.5	-	-	0.95	99.3	95.7
		25	-	-	0	100	91.2
		50	-	+	0.88	89.3	96.7
		100	-	+	1.19	94.4	97.6
		Positive control^2	-	n.d.	89.32	101.6	79.8
2	4	Vehicle control^1	-	n.d.	4.28	100.0	100.0
		5	-	-	2.35	107.5	104.6
		10	-	-	0	96.1	104.8
		20	-	-	3.02	106.6	102.1
		40	-	+	0.71	102.9	94.3
		80	-	+	n.c.^1	98.8	n.c.1
		Positive control^2	-	n.d.	148.58	96.1	89.5
1	4	Vehicle control^1	+	n.d.	1.02	100.0	100.0
		6.3	+	-	1.67	92.8	103.9
		12.5	+	-	1.91	99.5	108.2
		25	+	-	0	108.2	103.9
		50	+	+	1.67	103.4	101.7
		100	+	+	3.13	101.5	108.3
		Positive control^3	+	n.d.	104.61	106	88.9
2	4	Vehicle control^1	+	n.d.	5.86	100.0	100.0
		5	+	-	0	95.8	110.3
		10	+	-	4.05	95.8	103.2
		20	+	-	2.37	103.1	112.6
		40	+	+	0	97.6	107.6
		80	+	+	n.c.^1	99.8	n.c.1
		Positive control^3	+	n.d.	233.23	89.5	99.1

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

n.c.¹ Culture was not continued since a minimum of only four analysable concentrations are required

n.d. Not determined

¹ Acetone 1% (v/v)

² EMS 400 μg/mL

³ DMBA 1.25 μg/mL

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested.

Test groups printed in bold type were evaluated in this study:

1st Experiment

without S9 mix: 0; 6.3; 12.5; 25.0; 50.0; 100.0 μg/mL

with S9 mix: 0; 6.3; 12.5; 25.0; 50.0; 100.0 μg/mL

2nd Experiment

without S9 mix: 0; 5.0; 10.0; 20.0; 40.0; 80.0 μg/mL

with S9 mix: 0; 5.0; 10.0; 20.0; 40.0; 80.0 μg/mL

Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6-thioguaninecontaining medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation, no cytotoxicity was observed up to the highest tested concentration evaluated for gene mutations. Based on the results of the present study, the test substance either did not cause any relevant increase in the mutant frequencies without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.