Diethyl [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Apr 2015 - 11 Sep 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate

EC Number:

213-551-9

EC Name:

Diethyl [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate

Cas Number:

976-56-7

Molecular formula:

C19H33O4P

IUPAC Name:

diethyl (3,5-di-tert-butyl-4-hydroxybenzyl)phosphonate

Details on test material:

- Physical state: solid / white

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL) and 1% (v/v) amphotericine B (250 μg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitaland β-naphthoflavone induces rat liver S9 mix

Test concentrations with justification for top dose:

1st Experiment
4 hours exposure, 24 hours harvest time, without S9 mix 0; (3.13); 6.25; 12.50; 25.00; (50.00); (100.00) μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix 0; (3.13); 6.25; 12.50; 25.00; 50.00; (100.00) μg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix 0; (3.13); 6.25; 12.50; 25.00; (50.00); (100.00) μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix 0; 3.13; 6.25; 12.50; 25.00; (50.00); (100.00) μg/mL
Concentratons in parantheses were not evaluated.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, dimethyl sulfoxide (DMSO) was selected as vehicle, which had been demonstrated to be suitable in the in vitro micronucleus test and for which historical control data are available. The final concentration of the vehicle DMSO in culture medium was 1% (v/v).

Controls

Untreated negative controls:

yes

Remarks:

only for 24/24 hrs; culture medium w/o DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

without S9: EMS; with S9: CPP

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 hrs
- Expression time (cells in growth medium): 20 and 40 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 44 hrs

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) and propidium iodide

NUMBER OF REPLICATIONS:
Two independent experiments were carried out and at least 2 cultures were prepared per test group.

NUMBER OF CELLS EVALUATED:
At least 1000 cells per culture were evaluated for the occurrence of micronucleated cells.

DETERMINATION OF CYTOTOXICITY
- Method: cell count, proliferation index

Evaluation criteria:

A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data (95% control limit).
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions. If the results of this test were statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01 or S) have been printed in the tables.

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:

see below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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: No precipitation of the test substance in culture medium was observed.
- Other confounding effects: In the absence of metabolic activation cell attachment/morphology was adversely influenced (grade > 2) at 50 μg/mL and above in the 1st Experiment and at 25 μg/mL and above in the 2nd Experiment. Besides, in the presence of metabolic activation cell attachment/morphology was adversely influenced at 100 μg/mL in the 1st Experiment and at 50 μg/mL and above in the 2nd Experiment. No slides were prepared due to strongly reduced cell numbers at the highest applied test substance concentration of 100 μg/mL in all experimental parts. The slides were not scorable for cytogenetic damage due to strong cytotoxicity and/or poor quality in the 1st Experiment in the absence of S9 mix at 50 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
In the pretest the pH value was not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations tested. In addition, a solution of the test substance in the vehicle DMSO was obtained in the stock preparation (Test group: 2000 μg/mL). In culture medium test substance precipitation occurred at 250 μg/mL and above at the end of treatment under all experimental conditions. After 4 hours or 24 hours treatment in the absence and presence of S9 mix cytotoxicity indicated by reduced RPD of below 40 - 50% was observed at 62.5 μg/mL and above

COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.1 – 0.7% micronucleated cells) were close to the concurrent vehicle and negative control values (0.2 – 1.0% micronucleated cells) and clearly within the range of the 95% control limit of our historical negative control data (0.0 - 1.0% micronucleated cells).
The positive control substances EMS (without S9 mix; 400 or 500 μg/mL) and CPP (with S9 mix; 0.5 μg/mL) induced statistically significant increased micronucleus frequencies in both independently performed experiments. In this study, in the absence and presence of metabolic activation the frequency of micronucleated cells (2.1 – 4.9% micronucleated cells) was clearly above the range of our historical negative control data (0.1 - 1.5% micronucleated cells) and close to our historical positive control data range (2.3 – 13.8% micronucleated cells).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
RPD (relative population doubling): In both main experiments in the absence and presence of S9 mix cytotoxicity indicated by reduced RPD of below 50% of control was observed at least at the highest applied test substance concentration. These values were calculated based on cell numbers determined at the end of each experiment. In detail, in the absence of S9 mix reduced RPD was obtained from 50 μg/mL onward after 4 and 24 hours exposure, respectively (-20.3% or -59.5%, respectively). Besides, in the presence of S9 mix RPD was decreased after 4 hours exposure with 100 μg/mL (-174.4%) at 24 hours preparation interval in the 1st Experiment and from 50 μg/mL onward (-46.2%) at 44 hours preparation interval in the 2nd Experiment.
CBPI (cytokinesis-block proliferation index): Distinctly reduced proliferative activity indicated by elevated cytostasis of above 50% was not observed in any test group scored for the occurrence of micronuclei. In detail, in the 1st Experiment in the absence of metabolic activation the highest scorable concentration of 50 μg/mL led to a cytostasis of 43.6%. Due to strong cytotoxicity indicated by severe cell loss no slides could be prepared at the next higher concentration of 100 μg/mL. In the 2nd Experiment in the absence of S9 mix after 24 hours continous exposure the highest scored concentration led to a slight reduction of CBPI indicated by 8.6% cytostasis. In addition, in the presence of metabolic activation in the 1st Experiment at 24 hours preparation interval no cytostasis was observed after treatment up to 25 μg/mL (-0.4%). But, due to strong cytotoxicity the slides of the next higher concentration of 50 μg/mL were not scorable. Besides, in the 2nd Experiment in the presence of S9 mix the highest scorable concentration of 25 μg/mL led to a cytostasis of 40.2%. Due to severe cytotoxicity indicated by RPD at the next higher concentration of 50 μg/mL the slides were not scored.

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

						Cytotoxicity
Exp.	Exposure/ Preparation interval	Test groups	S9 mix	Prec.*	Genotoxicity Micro- nucleated cells** [%]	Proliferation index cytostasis (CBPI) [%]	Relative population doubling (RPD) [%]
1	4/24 hrs	Vehicle control^1	-	n.d.	0.4	0	100
		3.13 µg/mL	-	-	n.d.	n.d.	98.6
		6.25 µg/mL	-	-	0.7	-3.9	99.7
		12.50 µg/mL	-	-	0.3	-1.3	93.1
		25.00 µg/mL	-	-	0.2	-0.4	75.7
		50.00 µg/mL	-	-	n.s.	n.s.	-20.3
		100.00 µg/mL	-	-	n.p.	n.p.	-219.2
		Positive control^2	-	n.d.	2.1^S	1.6	91
2	24/24 hrs	Vehicle control^1	-	n.d.	0.3	0	100
		Negative control	-	-	1	26.8	109.9
		3.13 µg/mL	-	-	n.d.	n.d.	108.4
		6.25 µg/mL	-	-	0.2	5.3	106.4
		12.50 µg/mL	-	-	0.2	14	95.6
		25.00 µg/mL	-	-	0.1	40.2	75.6
		50.00 µg/mL	-	-	n.d.	n.d.	-59.5
		100.00 µg/mL	-	-	n.p.	n.p.	-239.4
		Positive control^2	-	n.d.	0.4	-26.2	122.6
		Positive control^3	-	n.d.	2.8^S	11.3	130.8
1	4/24 hrs	Vehicle control^1	+	n.d.	0.2	0	100
		3.13 µg/mL	+	-	n.d.	n.d.	89.9
		6.25 µg/mL	+	-	0.5	1.4	100.4
		12.50 µg/mL	+	-	0.6	5	99.3
		25.00 µg/mL	+	-	0.4	7.7	102.9
		50.00 µg/mL	+	-	0.3	43.6	75.4
		100.00 µg/mL	+	-	n.p.	n.p.	-174.4
		Positive control^4	+	n.d.	4.0^S	31.9	101.2
2	4/44 hrs	Vehicle control^1	+	n.d.	0.2	0	100
		3.13 µg/mL	+	-	0.4	1.1	94.3
		6.25 µg/mL	+	-	0.4	-3.5	64.1
		12.50 µg/mL	+	-	0.4	5	70.5
		25.00 µg/mL	+	-	0.5	8.6	17.6
		50.00 µg/mL	+	-	n.d.	n.d.	-46.2
		100.00 µg/mL	+	-	n.p.	n.p.	-308.7
		Positive control^4	+	n.d.	4.9^S	-19	92.3

*       Precipitation in culture medium at the end of exposure period (macroscopic)

**    Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

^S      Frequency statistically significant higher than corresponding control values

n.d. Not determined

n.p. No cytospin slides prepared due to strong cytotoxicity

n.s. Not scorable due to strong cytotoxicity

¹      DMSO 1% (v/v)

²      EMS 400 μg/mL

³      EMS 500 μg/mL

⁴     CPP 0.5 μg/mL

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions described, the test article is not considered to have a chromosome-damaging (clastogenic) potency or to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells.

Executive summary:

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, both with and without the addition of liver S9 mix from 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. The test groups printed in bold type were evaluated.

1st Experiment

4 hours exposure, 24 hours harvest time, without S9 mix 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL

4 hours exposure, 44 hours harvest time, with S9 mix 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 μg/mL

A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group. The vehicle/negative controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei. Cytotoxicity indicated by clearly reduced relative cell count or proliferation index (CBPI) was observed at least at the highest applied test substance concentration in all experimental parts of this study. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, the test article is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.