3,9-bis(2,4-di-tert-butylphenoxy)-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

No indepedent repeat experiment for incubation with S9 mix. Dose with S9 mix should have been slightly higher. It resulted only in 24% viability instead of the required 10 - 20% viability.

GLP compliance:

yes (incl. QA statement)

Remarks:

The report includes GLP certificates from the German, Dutch and Indian Authorities. All certificates are valid for the period of the study.

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,9-bis(2,4-di-tert-butylphenoxy)-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane

EC Number:

247-952-5

EC Name:

3,9-bis(2,4-di-tert-butylphenoxy)-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane

Cas Number:

26741-53-7

Molecular formula:

C33H50O6P2

IUPAC Name:

3,9-bis(2,4-di-tert-butylphenoxy)-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane

Method

Target gene:

tk - thymidinkinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Arochlor 1254 induced rat live

Test concentrations with justification for top dose:

17, 34, 68 and 136 µg/ml (3h, with S9)
55, 110, 220, 440 µg/ml (3h and 24h, without S9)

Vehicle / solvent:

DMSO

The results of the solubility test of Study No. 04733 for the same compound indicated that the test item forms a workable suspension with DMSO at the required concentration of 250000 /lg/mL. Also, DMSO is one of the organic solvents compatible with the test system. Hence, DMSO was selected as the vehicle to prepare the stock and dilutions ofthe test item as weil as the positive controls.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h and 24h
- Expression time (cells in growth medium): 48h

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT) at 4 mug/mL

NUMBER OF REPLICATIONS: tests were done in duplicate

DETERMINATION OF CYTOTOXICITY
- Method: cell growth

Evaluation criteria:

There are several criteria for determining a positive result, such as a concentration related, or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response. A test item, for which the results do not meet the above criteria is considered non mutagenic in this system.
Positive results for an in vitro mammalian cell gene mutation test indicate that the test substance induces gene mutations in the cultured mammalian cells
used. A positive concentration response that is reproducible is most meaningful. Negative results indicate that, under the test conditions the test
substance does not induce gene mutations in the cultured mammalian cells used.

Statistics:

The number of negative contraI values are assumed to follow Poisson Distribution. Test of significance of difference between negative control values over different dose groups and the positive control was carried out using a method of analysis suggested by M.R.Thomas (Cole and Arlett, 1984), based on chi2. All analysis and significance tests were evaluated at 5% level of signiftcance (P<0.05).

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

RTG was 24%

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Moderatetyl cytotoxic: RTG was 29 (24h) and 39 (3h). The next highest concentration was found to be non scorable based on precipitates upon centrifugation in pretests.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the presence of metabolie activation at the end of exposure to treatment, the pH of the test medium ranged between 7.05 to 7.22 with 7.10 in the DMS0 control. Similarly, in the absence of metabolic activation, the pH of the treatment medium ranged between 7.05 and 7.20 Wilh 7.21 in the DMSO control at the end of exposure to treatment.
- Precipitation: from 800 µg/ml onward, in presence and absence of metabolic activation

RANGE-FINDING/SCREENING STUDIES:
Preliminary test results of Study No. 04733 for the same test item indicated heavy precipitation of the test item at and above 2400 µg/ml. This precipitation interfered with the observation of the cells. Hence, the test item was checked fo it precipitation in the treatment medium (TM) up to the maximum concentration of 1600 µg/ml of the medium. At and abave 800 µg/mL, the test item precipitated In the form of a pellet in the centrifuge tubes both in the presence and absence of metabolic activation in the 3-hour as weil as in the 24-hour exposure regime. Hence cell counts could not be determined. Based on these observations, it was decided to test up to 136 µg/mL in the presence of metabolie aetivation for the 3-hour exposure and up to 440 µg/mL for the 3-hour as well as the 24-hour exposure in the absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, solvent and positive control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the preliminary toxicity test, the test item was toxic to the cells at and above 200 µg/mL when exposed for 3 hours in the presence of metabolic activation resulting in dead and disfigured cells. There was evidence of growth inhibition as relative total growth (RTG) at 100 µg/mL. However, this growth inhibition was not significant. In the 3-hour as well as the 24-hour exposure in the absence of metablic activation, there was evidence of growth inhibition as relativ total growth (RTG) at 400 µg/mL. However, this growth inhibition was not significant.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Presence of Metabolic Activation (Experiment 1)

Group	Test item concn. (µg/mL)	Non-selective medium			Selective medium     P(0) individual plates			M.F x 10-6	M.F/survivor x 10-6	Induced M.F x 10-6	RTG
		P(0)	C.E.	RCE	R1	R2	Total
G1	DMSO (200 µI)	16/96	90	100	78/96	83/96	161/192	88	0.98	-	100
G2	17	18/96	84	93	79/96	84/96	163/192	82	0.98	-	69
G3	34	16/96	90	100	86/96	81/96	167/192	70	0.78	-	60
G4	68	20/96	78	88	89/96	80/96	169/192	64	0.81	-	43
G5	136	22/96	74	82	79/96	87/96	166/192	73	0.99	-	24
G6	CPA 12	23/96	71	80	23/96	36/96	59/192	590+	8.26	7.28	43

Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Absence of Metabolic Activation (Experiment 2)

Group	Test item concn. (µg/mL)	Non-selective medium			Selective medium     P(0) individual plates			M.F x 10-6	M.F/survivor x 10-6	Induced M.F x 10-6	RTG
		P(0)	C.E.	RCE	R1	R2	Total
G1	DMSO (200 µI)	14/96	96	100	78/96	84/96	162/192	85	0.88	-	100
G2	55	16/96	90	93	81/96	85/96	166/192	73	0.81	-	80
G3	110	16/96	90	93	83/96	80/96	163/192	82	0.91	0.03	62
G4	220	18/96	84	87	78/96	79/96	157/192	101	1.20	0.32	51
G5	440	18/96	84	87	76/96	78/96	154/192	110	1.32	0.44	39
G6	MMS 20	21/96	76	79	42/96	33/96	75/192	470+	6.18	5.30	50

Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Absence of Metabolic Activation (Experiment 3)

Group	Test item concn. (µg/mL)	Non-selective medium			Selective medium     P(0) individual plates			M.F x 10-6	M.F/survivor x 10-6	Induced M.F x 10-6	RTG
		P(0)	C.E.	RCE	R1	R2	Total
G1	DMSO (200 µJ)	15/96	93	100	80/96	83/96	163/192	82	0.88	-	100
G2	55	15/96	93	100	79/96	81/96	160/192	91	0.98	0.10	82
G3	110	18/96	84	90	86/96	76/96	162/192	85	1.01	0.13	74
G4	220	19/96	81	87	77/96	80/96	157/192	101	1.24	0.36	44
G5	440	20/96	78	85	81/96	84/96	165/192	76	0.97	0.08	27
G6	MMS20	20/96	78	85	36/96	32/96	68/192	519+	6.62	5.74	29

M.F = Mutant Frequency

C.E = Cloning Efficiency

RCE = Relative Cloning Efficiency

RTG = Relative Total Growth

+ = Significantly higher than control (p ≤ 0.05)

Applicant's summary and conclusion

Conclusions:

The study indicated that the test item does not have the potential to cause gene mutation at the tk locus at the concentrations tested and under the conditions of testing.