2,2'-methylenebis(6-nonyl-p-cresol)

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:

11 September 2012 - 5 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-methylenebis(6-nonyl-p-cresol)

EC Number:

232-092-5

EC Name:

2,2'-methylenebis(6-nonyl-p-cresol)

Cas Number:

7786-17-6

Molecular formula:

C33H52O2

IUPAC Name:

2-[(2-hydroxy-5-methyl-3-nonylphenyl)methyl]-4-methyl-6-nonylphenol

Test material form:

liquid: viscous

Details on test material:

- Physical state: Clear orange/amber sticky high viscous liquid.
- Stability under storage conditions: Stable.
- Stability under test conditions: The stability of the test material was confirmed when used in the test system.
- Storage condition of test material: At room temperature in the dark.

Method

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Several different mediums were used; basic medium, growth medium, exposure medium, selective medium and non-selective. The compositions of which can be seen in the field "Any other information on materials and methods incl. tables".
- Properly maintained: Yes, Stock cultures of the cells were stored in liquid nitrogen (-196°C). Cell density was preferably kept below 1 x 106 cells/mL.
- Periodically checked for Mycoplasma contamination: Yes.
- Periodically "cleansed" against high spontaneous background: Yes.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 µg/mL.

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Based on the findings of a dose range finding test the definitive study was performed as two independent experiments.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
> Experiment 1: 3 hours with and without S9-mix (8% v/v).
> Experiment 2: 24 hours without S9-mix and 3 hours with S9-mix (12% v/v).
- Expression time: Cells were cultured for a further 2 days. During this culture period at least 4 x 10^6 cells (where possible) were subcultured every day in order to maintain log phase growth.
- Selection time: 11 or 12 days.

SELECTION AGENT: Selective medium (TFT-selection), see the field “Any other information on materials and methods incl. tables” for information on composition.
STAIN: 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Cultures were stained for 2 hours.

NUMBER OF REPLICATIONS:
> Exposure cultures were performed as a single replicate.
> Solvent and positive controls were performed in duplicate.

INCUBATION CONDITIONS: All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 – 100% (actual range 45 – 98%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 35.2 – 37.9°C). Cultures for the 3 hour incubations (10^6 cells/mL) were places in 30 mL centrifuge tubes, and incubated in a shaking incubator at 145 rpm. The cell cultures for the 24 hour incubation (1.25 x 10^5 cells/mL) were places in sterile 72 cm² culture flasks.

DOSE RANGE FINDING TEST
- Method: The dose range finding test was conducted according to the same method employed for the definitive test, where cells were exposed to the test material for 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix.
- Concentrations: 1, 3, 10, 33 and 100 µg/mL.
- Cytotoxicity determination: The surviving cells of the 3 hours treatment were subcultured twice. After 24 hours of subculturing, the cells were counted and subcultured again for another 24 hours, after which the cells were counted. The surviving cells of the 24 hours treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 10^5 cells/mL were counted no subculture was performed.
- Determination of dosing range: The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.

Evaluation criteria:

DATA EVALUATION
> The test material is considered positive (mutagenic) in the mutation assay if it induces a MF (mutation frequency) of more than the control + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
> The test material is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
> The test material is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

In addition to the criteria stated above, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

ACCEPATBILITY CRITERIA
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS (methylmethanesulfonate) should not be below 500 per 10^6 survivors, and for CP (cyclophosphamide) not below 700 per 10^6 survivors.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test material was tested beyond the limit of solubility to obtain adequate mutagenicity data.
- Precipitation: The test material precipitated in the exposure medium at concentrations of 33 μg/mL and above. It was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test material concentration for the dose range finding test was 100 μg/mL.

RANGE-FINDING/SCREENING STUDIES: No toxicity in the relative suspension growth was observed up to and including the highest concentration of 100 μg/mL compared to the suspension growth of the solvent control, in cultures exposed for 3 hours (with and without metabolic activation) and 24 hours (with metabolic activation).

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity was observed in any of the dose levels evaluated, both in the absence and presence of metabolic activation.

MUTAGENICITY IN THE DEFINITIVE STUDY
The numbers of small and large colonies in treated cultures were comparable to the numbers in the solvent controls.
Both in the presence and absence of metabolic activation (S9-mix), the test material did not induce a significant increase in the mutation frequency in the first experiment. These results were confirmed by the second experiment.

CONTROLS
- Solvent control: The growth rate over the two-day expression period for cultures treated with ethanol was between 14 and 21 (3 hours treatment) and 80 and 85 (24 hours treatment).
- Positive controls: Mutation frequencies in cultures treated with positive control chemicals were increased by 13- and 11-fold for MMS in the absence of S9-mix, and by 8.1- and 11-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay, with the exception of mutation frequency of the positive control in the presence of S9-mix (second experiment). However the value of 121% was just above the upper limit of the range (120%). Therefore this deviation in the absolute cloning efficiency had no effect on the results of the study.

ANALYSIS OF TEST SOLUTIONS
> Experiment 1 (with S9-mix): The concentrations analysed in the low and high formulations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
Stability analysis suggested that formulations were not stable over a 4 hour period; therefore an additional experiment was performed.
No test substance was detected in the vehicle.
> Experiment 1 (without S9-mix): The concentrations analysed in the high formulations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
For the low formulation applied in experiment 1 (without S9-mix) the mean accuracy was below the target concentration (i.e. 89% of target). However this was just below the upper limit of the range (90%). Furthermore the high dose was in agreement with the nominal concentration, and this was used for the preparation of the lower dose level. Therefore this deviation is not considered to have affected the reliability of the test results.
Formulations at the entire range were determined to be stable when stored at room temperature under normal laboratory light conditions for at least 4 hours. Since the test solutions were added to the test system within one hour and the stability in the second measurement was stable after 0.5, 1 and 4 hours. It can be concluded that the test substance formulations were stable when used in the test system.
No test substance was detected in the vehicle.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment 1 – Cytotoxic and Mutagenic Response

Dose (µg/mL)	RSG (%)	CEday2 (%)	RSday2 (%)	RTG (%)	TotalMutation Frequency per 10^6 survivors (small/large)
3 hour exposure, without metabolic activation
SC1	100	121	100	100	50 (27/21)
SC2		102			61 (27/32)
0.03	121	108	97	118	64 (28/34)
0.1	107	125	112	119	60 (37/21)
0.3	111	116	104	115	67 (28/36)
1	119	116	104	123	41 (14/27)
3	131	102	91	120	56 (23/32)
10	127	115	102	130	48 (26/20)
33*	124	118	105	131	46 (20/25)
100*	123	135	120	148	40 (19/20)
MMS	80	78	70	56	705 (378/235)
3 hour exposure, with 8 % (v/v) metabolic activation
SC1	100	113	100	100	101 (29/68)
SC2		101			98 (25/69)
0.03	99	76	71	70	129 (48/74)
0.1	103	104	97	100	83 (23/57)
0.3	105	78	73	77	130 (40/84)
1	101	74	69	69	134 (41/88)
3	98	107	100	98	100 (21/75)
10	99	72	68	67	121 (28/89)
33*	95	97	90	86	92 (29/59)
100*	105	84	78	82	122 (29/88)
CP	45	49	46	20	811 (160/594)

 

Table 2: Experiment 2 – Cytotoxic and Mutagenic Response

Dose (µg/mL)	RSG (%)	CEday2 (%)	RSday2 (%)	RTG (%)	TotalMutation Frequency per 10^6 survivors (small/large)
24 hour exposure, without metabolic activation
SC1	100	86	100	100	54 (30/23)
SC2		95			52 (29/21)
0.03	107	80	88	95	46 (22/22)
0.1	101	89	98	99	58 (35/22)
0.3	100	97	106	107	58 (33/23)
1	99	104	114	113	47 (21/26)
3	141	89	98	138	62 (44/17)
10	169	95	105	178	55 (30/23)
33*	124	85	94	116	41 (20/20)
100*	104	90	99	104	40 (22/16)
MMS	108	70	77	84	605 (357/190)
3 hour exposure, with 12 % (v/v) metabolic activation
SC1	100	83	100	100	69 (31/36)
SC2		98			58 (27/30)
0.03	91	99	110	101	47 (24/22)
0.1	88	94	104	92	55 (26/27)
0.3	85	83	91	78	62 (40/21)
1	103	98	109	112	49 (25/23)
3	102	93	103	105	63 (29/32)
10	93	70	78	73	68 (29/38)
33*	93	95	105	98	53 (27/25)
100*	92	98	109	100	54 (34/18)
CP	66	52	58	38	699 (436/207)

 

Note all calculations were made without rounding off.

RSG = Relative Suspension Growth; CE = CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = ethanol; MMS = Methylmethanesulfonate; CP = Cyclophosphamide.

* = precipitated in the exposure medium.

Applicant's summary and conclusion

Conclusions:

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

Under the conditions of the test, no significant increase in the mutation frequency at the TK Locus was observed after treatment with the test material. Cultures were exposed to the test material up to and including the precipitating concentration of 100 µg/mL, with and without metabolic activation (S9-mix). Accordingly the test material was determined to be non-mutagenic in the TK mutation test system.

Executive summary:

The genotoxic potential of the test material was determined in an in vitro gene mutation study using L5178Y mouse lymphoma cells. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 476, EU Method B.17 and IWGT recommendations. Based on the findings of a dose range finding test the definitive study was performed as two independent experiments within the dose range 0.03 to 100 µg/mL. In experiment 1, cultures were exposed to the test material for 3 hours, with and without metabolic activation (8 % v/v S9-mix). In experiment 2, cultures were exposed for 3 hours without S9-mix, and for 24 hours with 12 % v/v S9-mix. The test material precipitated in the exposure medium at concentrations of 33 μg/mL and above. It was tested beyond the limit of the solubility to obtain adequate cytotoxicity and mutagenicity data.

The numbers of small and large colonies in treated cultures were comparable to the numbers in the solvent controls. Both in the presence and absence of metabolic activation (S9-mix), the test material did not induce a significant increase in the mutation frequency in the first experiment, results that were confirmed by the second experiment. Therefore under the conditions of the test, the test material was determined to be non-mutagenic.