N,N'-propane-1,3-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionamide]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

To clarify equivocal results an independent repetition experiment (without metabolic activation) with closer spaced concentrations and double cell cultures was performed on sponsor’s request.

GLP compliance:

yes

Type of assay:

other: HPRT assay in Chinese Hamster V79 cells

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-propane-1,3-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionamide]

EC Number:

274-157-0

EC Name:

N,N'-propane-1,3-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionamide]

Cas Number:

69851-61-2

Molecular formula:

C37H58N2O4

IUPAC Name:

N,N'-propane-1,3-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanamide]

Test material form:

solid: particulate/powder

Details on test material:

- Purity: 98.4%

Method

Target gene:

HPRT locus using V79 cells of the Chinese Hamster

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins BioPharma Product Testing Munich GmbH stock cultures
- Cell cycle length, doubling time or proliferation index:12 - 14 h doubling time; high cloning efficiency of untreated cells, usually more than 50%
- Methods for maintenance in cell culture if applicable: The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank at the testing facility.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: stored over liquid nitrogen (vapour phase); minimal essential medium (MEM)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (via PCR)
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver (S9)

Test concentrations with justification for top dose:

Toxicity test: 1, 2.5, 5, 10, 25, 50, 100, 250 µg/mL
Experiment I (without activation): 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100 and 200 µg/mL
Experiment I (with activation): 0.5, 1, 2.5, 5, 10, 25, 50, and 100 µg/mL
Experiment I repeat assay (without activation): 10, 25, 50, 60, 70, 80, 90 and 100 µg/mL
The selection of the concentrations was based on data from the pre-experiments.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the nature of the test item, it was not possible to prepare a solution of the test item with an appropriate solvent and with the recommended concentration of 2 mg/mL. By lowering the highest concentration to 0.25 mg/mL and based on the results of the solubility test, the best suited vehicle was DMSO (1% v/v).

Controls

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Seeding of the Cultures: Prior to use, cultures were cleansed of pre-existing cells. Two or three day-old exponentially growing stock cultures (more than 50% confluent) were trypsinised at 37°C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsine concentration for all subculturing steps was 0.05%. Approximately 10-20e6 cells per concentration, solvent/negative and positive control, were seeded in complete culture medium (MEM supplemented with 10% FBS) in a culture flask, respectively.

Treatment: Approximately 24 hours after seeding the cells were exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 hours exposure the cultures (single cell culture for experiment I and double cell culture for the repetition of experiment I without metabolic activation) were checked for precipitation and the treatment medium containing the test item was removed. The cells were washed twice with PBS, trypsinised and counted with a cell counter. During the following expression period most of the cells were subcultured in complete culture medium (MEM supplemented with 10% FBS) in a sufficient number of cells (at least 2e6 cells per treatment group). In addition, for determination of the relative survival (RS) two 25 cm2 flasks were seeded with approx. 200 cells in complete culture medium for each treatment group. After incubation for an appropriate time (6 - 7 days) colonies were fixed with methanol, stained with Giemsa and counted. Cytotoxicity (relative survival) was calculated based on the cloning efficiency of cells plated immediately after treatment adjusted by any loss of cells during treatment.

Selection: At the end of the expression period (i.e. after 7 to 9 days) for selection the mutants, about 4e5 cells for each treatment group were seeded in cell culture petri dishes (diameter 90 mm) with selective medium containing 11 µg/mL 6-thioguanine (TG) for further incubation (additional 9 - 11 days). The cloning efficiencies (CE) were determined in parallel to the selection of mutants. For each treatment group two 25 cm2 flasks were seeded with approximately 200 cells in complete culture medium to determine the cloning efficiencies after additional 6 - 8 days. After incubation for an appropriate time (9 - 11 days for mutant frequency and 6 - 8 days for CE) colonies were fixed with methanol, stained with Giemsa and counted. The mutant frequency was calculated based on the number of mutant colonies corrected by the cloning efficiency at the time of mutant selection.

Assessment of Cytotoxicity and Mutant Frequency: Cytotoxicity was evaluated by relative survival (RS). The cloning efficiency (CE) of cells plated immediately after treatment was adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in negative / solvent controls (assigned a survival of 100%). The mutant frequency (MF) is the cloning efficiency of mutant colonies in selective medium divided by the cloning efficiency in non-selective medium measured for the same culture at the time of selection

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I (with and without activation): no growth inhibition observed. Exp. I repeat (without activation): relative survival <70%; relative survival for all concentrations except highest was within range 50-63% (slightly below non-toxic section)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test item was noted in the pre- and main experiment at concentration 100 µg/mL. In the repetition of experiment I precipitation occurred at concentration 80 µg/mL.
For experiment I without metabolic activation the mutant frequencies for the two highest concentrations (50 and 100 µg/mL) were found above the historical data of the test facility. As there was no statistically significant increase, these results needed to be clarified in an independent repetition experiment. In the repetition of experiment I without metabolic activation none of the observed mutant frequencies was significantly increased over those of the negative controls. No dose-response relationship was observed.
DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Any other information on results incl. tables

Experiment I – Toxicity, without metabolic activation
Dose Group	Concentration [µg/mL]	Number of cells at		Number of colonies per flask			CE [%]	Adjusted CE [%]	Relative Survival (RS) [%]
		beginning of treatment	endo of treatment	I	II	mean
NC1	0	10000000	12665000	140	165	153	76	97	120
NC2		10000000	12818000	159	172	166	83	106	132
S1	0	10000000	10642000	141	120	131	65	69	100
S2		10000000	10846000	166	170	168	84	91
1	0.05	10000000	10625000	147	150	149	74	79	98
2	0.1	10000000	9537000	157	176	167	83	79	99
3	0.25	10000000	10013000	174	177	176	88	88	109
4	0.5	10000000	9622000	144	158	151	76	73	90
5	1	10000000	10217000	141	147	144	72	74	92
6	2.5	10000000	10591000	146	152	149	75	79	98
7	5	10000000	9418000	177	195	186	93	88	109
8	10	10000000	9265000	178	218	198	99	92	114
9	25	10000000	8993000	166	170	168	84	76	94
10	50	10000000	8041000	159	164	162	81	65	81
11 P	100	10000000	8041000	191	180	186	93	75	93
12 P	200	10000000	7854000	202	188	195	98	77	95
EMS	300	10000000	13107000	181	168	175	87	114	142
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency EMS: Ethylmethanesulfonate

 

Experiment I – Mutagenicity, without metabolic activation
		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration [µg/mL]	Number of colonies per flask			CE              [%]	Number of colonies per flask							CE              [%]	Mutant Frequency per 10^6cells
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	185	183	184	92	18	11	17	13	14	14.6	2.6	0.0037	39.7
NC2		184	186	185	93	7	14	15	9	13	11.6	3.1	0.0029	31.4
S1	0	148	187	168	84	14	12	7	17	7	11.4	3.9	0.0029	34.0
S2		143	186	165	82	19	9	11	13	13	13.0	3.3	0.0033	39.5
7	5	182	164	173	87	16	13	14	15	11	13.8	1.7	0.0035	39.9
8	10	187	181	184	92	10	14	16	17	10	13.4	2.9	0.0034	36.4
9	25	185	149	167	84	11	15	7	12	16	12.2	3.2	0.0031	36.5
10	50	189	186	188	94	15	15	19	18	21	17.6	2.3	0.0044	46.9
11 P	100	183	160	172	86	12	8	14	26	11	14.2	6.2	0.0036	41.4
EMS	300	134	115	125	62	78	89	92	90	94	88.6	5.6	0.0222	355.8
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency EMS: Ethylmethanesulfonate

 

Experiment I – Toxicity, with metabolic activation
Dose Group	Concen-tration	Number of cells at the		Number of colonies per flask			CE     [%]	Adjusted CE [%]	Relative Survival (RS)
	[µg/mL]	beginning of treatment	end of treatment	I	II	mean			[%]
NC1	0	10000000	11288000	175	148	162	81	91	97
NC2		10000000	10863000	157	158	158	79	86	91
S1	0	10000000	11458000	143	179	161	81	92	100
S2		10000000	12257000	147	168	158	79	97
1	0.5	10000000	11237000	173	175	174	87	98	104
2	1	10000000	11424000	154	156	155	78	89	94
3	2.5	10000000	11900000	153	167	160	80	95	101
4	5	10000000	11730000	125	138	132	66	77	82
5	10	10000000	11305000	200	203	202	101	114	121
6	25	10000000	10744000	189	195	192	96	103	109
7	50	10000000	10591000	204	207	206	103	109	115
8:00 PM	100	10000000	9979000	177	160	169	84	84	89
DMBA	1	10000000	11441000	151	132	142	71	81	86
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency DMBA: 7,12-dimethylbenz(a)anthracene

Experiment I – Mutagenicity, with metabolic activation
		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration [µg/mL]	Number of colonies per flask			CE  [%]	Number of colonies per flask							CE  [%]	Mutant Frequency per 10^6cells
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	199	215	207	104	21	17	21	19	11	17.8	3.7	0.0045	43.0
NC2		166	179	173	86	12	16	14	14	10	13.2	2.0	0.0033	38.3
S1	0	174	197	186	93	5	16	8	5	7	8.2	4.1	0.0021	22.1
S2		189	194	192	96	16	15	15	12	17	15.0	1.7	0.0038	39.2
4	5	157	173	165	83	15	15	12	14	12	13.6	1.4	0.0034	41.2
5	10	174	167	171	85	9	9	10	10	15	10.6	2.2	0.0027	31.1
6	25	157	154	156	78	10	13	15	10	12	12.0	1.9	0.0030	38.6
7	50	168	162	165	83	8	15	12	11	7	10.6	2.9	0.0027	32.1
8 P	100	157	173	165	83	8	14	8	8	10	9.6	2.3	0.0024	29.1
DMBA	1.0	134	142	138	69	93	101	104	78	81	91.4	10.4	0.0229	331.2
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency DMBA: 7,12-dimethylbenz(a)anthracene

 

Experiment I (repetition) – Toxicity, without metabolic activation
Dose Group	Concen-tration [µg/mL]	Number of cells at the		Number of colonies per flask			CE             [%]	Adjusted CE          [%]	Relative Survival (RS) [%]
		beginning of treatment	end of treatment	I	II	mean
NC1	0	10000000	9010000	73	73	73	37	33	78
NC2		10000000	10081000	92	93	93	46	47	111
S1	0	10000000	7633000	125	128	127	63	48	100
S2		10000000	8058000	86	91	89	44	36
1	10	20000000	13090000	67	65	66	33	22	51
2	25	20000000	11424000	81	68	75	37	21	51
3	50	20000000	10914000	100	84	92	46	25	60
4	60	20000000	11322000	69	80	75	37	21	50
5	70	20000000	11356000	90	97	94	47	27	63
6 P	80	20000000	12512000	104	126	115	58	36	86
7 P	90	20000000	13430000	142	131	137	68	46	109
8 P	100	20000000	12580000	120	141	131	65	41	98
EMS	300	10000000	11475000	139	134	137	68	78	187
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency EMS: Ethylmethanesulfonate

 

Experiment I (repetition) – Mutagenicity, without metabolic activation
		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration [µg/mL]	Number of colonies per flask			CE              [%]	Number of colonies per flask							CE              [%]	Mutant Frequency per 10^6cells
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	155	160	158	79	8	6	6	11	3	6.8	2.6	0.0017	21.6
NC2		147	144	146	73	13	15	13	12	10	12.6	1.6	0.0032	43.3
S1	0	133	130	132	66	7	5	8	9	8	7.4	1.4	0.0019	28.1
S2		152	151	152	76	7	11	11	14	9	10.4	2.3	0.0026	34.3
2	25	162	158	160	80	14	8	11	8	6	9.4	2.8	0.0024	29.4
3	50	179	165	172	86	6	12	12	5	12	9.4	3.2	0.0024	27.3
4	60	141	152	147	73	7	8	11	15	7	9.6	3.1	0.0024	32.8
5	70	150	140	145	73	12	12	15	5	6	10.0	3.8	0.0025	34.5
6 P	80	169	151	160	80	6	7	7	3	6	5.8	1.5	0.0015	18.1
EMS	300	138	152	145	73	91	80	96	80	86	86.6	6.2	0.0217	298.6
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency EMS: Ethylmethanesulfonate

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test item is non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The test substance was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster in accordance with OECD Guideline 476. The selection of the concentrations was based on data from the pre-experiments. Experiment I (with and without metabolic activation) and repetition experiment I (without metabolic activation) were performed as a 4 h short-term exposure assay. In consultation with the Sponsor and due to the equivocal results in experiment I, an independent repetition of experiment I without metabolic activation with different experimental conditions (closer spaced concentrations and double cultures) was conducted. The test item was investigated at the following concentrations: 5.0, 10, 25, 50 and 100 µg/mL (Experiment I, without and with metabolic activation) and 25, 50, 60, 70 and 80 µg/mL (Experiment I (repetition), without metabolic activation).

Precipitation of the test item was noted in the pre- and main experiment at concentration 100 µg/mL. In the repetition of experiment I precipitation occurred at concentration 80 µg/mL. No growth inhibition was observed in the experiment I without and with metabolic activation. A growth inhibition (relative survival < 70%) was observed in the repetition experiment without metabolic activation: the relative survival for all concentrations evaluated, with the exception of the highest concentration, was found slightly below the non-toxic section (within the range of 50-63 %). For experiment I without metabolic activation the mutant frequencies for the two highest concentrations (50 and 100 µg/mL) were found above the historical data of the test facility. As there was no statistically significant increase, these results needed to be clarified in an independent repetition experiment. In the repetition of experiment I without metabolic activation none of the observed mutant frequencies was significantly increased over those of the negative controls. No dose-response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. In conclusion, in the described mutagenicity test under the experimental conditions reported, the test substance is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.