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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a reverse gene mutation assay in bacteria (Ames Test) and in a mutagenicity assay in CHO cells (HPRT Assay), no mutagenicity was observed with or without metabolic activation. No evidence of a dominant lethal effect was observed in the progeny of male mice. The test article was also not carcinogenic in lifetime feeding studies.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

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

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

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.

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.

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'.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames Test

In a reverse gene mutation assay in bacteria, four tester strains (S. typhimurium TA1535, TA1537, TA100, TA98) were exposed to the test substance at concentrations of 0.2, 2, 20, 200, 2000 µg/plate in the presence and absence of mammalian metabolic activation. Each concentration was tested in triplicate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background. The test substance was not mutagenic in the S. typhimurium strains TA1535, TA1537, TA100 and TA98 under the experimental conditions with or without metabolic activation (Batelle, 1978).

HPRT-Assay

In a GLP-compliant mammalian mutagenicity assay following OECD guideline 476, 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 dose rage used was 0 -100 µg/ml without, and 0 -40 µg/ml with S9 mix.

The test substance did not increase the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were close to the range of the concurrent vehicle control values and within the range of the historical negative control data. The mutation frequencies of the vehicle control groups were within the historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. 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. To summarize, in the absence and the presence of metabolic activation, the test article is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Dominant Lethal study

In an in vivo dominant lethal test, 20 male mice (Tif: MAGf) were orally treated with the test substance at doses of 0, 1000 and 3000 mg/kg bw. They were then mated to untreated females from the same strain over a period of six weeks. At the end of each week the females were replaced by new ones (2 females/male/week). The experiment was done to evaluate any cytotoxic or mutagenic effects on the male germinal cells as expressed by the loss of pre-implementation zygotes as well as by the rate deaths of post-implementation stages of embryonic development. The females mated to males which had been treated with the test substance did not differ significantly from the females mated to control, neither in mating ratio nor in the number of implantations and embryonic deaths (resorptions). No evidence of a dominant lethal effect was observed in the progeny of male mice treated with the test substance.

Assessment

The test article was not mutagenic in vitro and in vivo. In addition, when tested in life time feeding studies in rats, no evidence of carcinogenic activity was reported. Therefore, the test article is considered to be not genotoxic. No further testing is required.

Justification for selection of genetic toxicity endpoint
Evaluation of genetic toxicity was done based on the complete data set available.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available in vitro and in vivo studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance does not need to be classified and labelled for mutagenic toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EC.

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental in vitro and in vivo test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled for mutagenic toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation EC No 605/2014.