O,O'-dioctadecylpentaerythritol bis(phosphite)

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:

21 March 2013 to 11 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP and reported with a valid certificate.

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:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

O,O'-dioctadecylpentaerythritol bis(phosphite)

EC Number:

223-276-6

EC Name:

O,O'-dioctadecylpentaerythritol bis(phosphite)

Cas Number:

3806-34-6

Molecular formula:

C41H82O6P2

IUPAC Name:

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

Test material form:

other: granular solid

Details on test material:

CAS Number: 3806-34-6

Method

Target gene:

thymidine kinase, TK +/-, locus.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the
MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ~g/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ~g/ml) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (RO) are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The dose range of the test item was selected following the results of a preliminary toxicity test and was 19.53 to 625 μg/ml in both the absence and presence of metabolic activation in Experiment 1. In Experiment 2 the dose range was 1.22 to 156.25 μg/ml in the absence of metabolic activation, and 19.53 to 625 μg/ml in the presence of metabolic activation.

Vehicle / solvent:

acetone

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Preparation of Test and Control Items
Following solubility checks performed in-house, the test item was accurately weighed and dissolved in acetone prior to serial dilutions being prepared. The test item was considered to be a UVCB product therefore a purity value was not applied. Due to increased toxicity in previous tests using acetone as a vehicle the dosing volume was reduced to 0.5% and the maximum achievable dose of 2500 μg/mL was used. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et a/1991) in the solubility test.
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Solvent and positive controls were used in parallel with the test item. Solvent (RO) treatment groups were used as the negative controls. Ethylmethanesulphonate (EMS) Sigma batch BCBJ1417V at 400 μg/mL and 150 μg/mL for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0302605 at 2 μg/mL was used as the positive control in the presence of metabolic activation.

Microsomal Enzyme Fractions
PB/ßNF S9 was prepared in-house on 14 April 2013 from the livers of male Sprague-Dawley rats weighing approximately 250g. These had each received, orally, three consecutive daily doses of phenobarbital/ß-naphthoflavone (80/1 00 mg per kg per day) prior to S9 preparation on the fourth day. The S9 was stored at approximately -196°C in a liquid nitrogen freezer.
S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCI (33 mM) and MgCI2 (8 mM) in R0.
20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1, and 10% S9-mix (i.e. 1% final concentration of S9) was added to the cultures of the Experiment 2.

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5E+5 cells/ml, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5E+55 cells/ml using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 9.77 to 2500 μg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2E+5 cells/ml.
The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2E+5 cells/ml. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
-Maximum recommended dose level, 5000 μg/mL or 10 mM.
-The presence of excessive precipitate where no test item-induced toxicity was observed.
-Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Mutagenicity Test
Experiment 1
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1E+6 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test item (19.53 to 625 μg/mL in the absence and presence of metabolic activation), vehicle and positive controls. To each universal was added 2 ml of S9-mix if required, 0.1 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to bring the total volume to 20 ml.
The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1E+6 cells/ml in 10 ml cultures in R10 medium for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3E+6 cells/ml in 10 ml cultures were established in 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at up to eight dose levels of the test item (1.22 to 156.25 μg/mL in the absence of metabolic activation, and 19.53 to 625 μg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 ml of S9-mix if required, 0.1 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to give a final volume of 20 ml (R10 is used for the 24-hour exposure group).
The treatment vessels were incubated at 37° with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

Measurement of Survival, Viability and Mutant Frequency
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2E+5 cells/ml. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days by counting and diluting to 2E+5 cells/ml.
On Day 2 of the experiment, the cells were counted, diluted to 10E+4 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/Ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days' incubation at 37°C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of thizolyl blue tetrazolium bromide (MTT) solution (2.5 mg/ml in phosphate buffered saline) was added to each well of the mutation plates. The plates were incubated for approximately two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

Evaluation criteria:

The normal range for mutant frequency per survivor is 50 – 170 E+6 for the TK+/- locus in L5178Y cells at this laboratory.
For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The Global Evaluation Factor (GEF) value was set at 126E+6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126E+6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Statistics:

UKEMS statistical package.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range of the test item used in the preliminary toxicity test was 9.77 to 2500 μg/mL.
In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The toxicity was more prominent in the 24 hour exposure group. It was considered that there was a plateau of toxicity observed in both of the 4 hour exposure groups from 625 μg/mL and the increased precipitate above this dose level effectively reduces the exposure to the test item. In the 24 hour exposure group the toxicity curve was steep with maximum toxicity occurring above 78.13 μg/mL. It should be noted that the toxicity was unaffected by the precipitate in the 24 hour exposure group. A precipitate of the test item was observed at and above 156.25 μg/mL in all three exposure groups. Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item induced toxicity and the incidence of excessive precipitate effectively reducing the exposure.

Mutagenicity Test
Experiment 1
There was evidence of marked dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values. There was evidence of modest reductions in viability (%V) in the presence of metabolic activation only therefore indicating that residual toxicity had occurred. Based on the RTG and %RSG values observed, it was considered that optimum levels of toxicity were not achieved in either the absence or presence of metabolic activation. The toxicity observed in the presence of metabolic activation at 625 μg/ml was considered to be approaching the optimum level (0.34 RTG). However it was considered that the test item was tested to its maximum potential considering the plateau of toxicity observed in the preliminary toxicity test above this dose level. Acceptable levels of toxicity were seen with both positive control substances.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 E-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test item did not induce any statistically significant or dose related (linear-trend) increase in the mutant frequency E-6 per viable cell in the absence or presence of metabolic activation. A precipitate of the test item was observed at and above 78.13 μg/ml in the absence of metabolic activation and at and above 156.25 μg/mL in the presence of metabolic activation.

Experiment 2
As was seen previously, there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values. There was evidence of a marked reduction in viability (%V) in the absence of metabolic activation only therefore indicating that residual toxicity had occurred. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the absence of metabolic activation only. The excessive toxicity observed at 156.25 μg/mL in the absence of metabolic activation, resulted in this dose level not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.
The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item.
Neither of the vehicle control mutant frequency values ·were outside the acceptable range of 50 to 170 E-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test item did induce a small but statistically significant dose related (linear-trend) increase in the mutant frequency E-6 per viable cell in the absence of metabolic activation. However the highest mutant frequency value observed would be considered acceptable for a vehicle control and in addition the GEF was not exceeded. Therefore the small response observed was considered to be of no toxicological significance. A precipitate of the test item was observed at and above 39.06 μg/mL in the absence of metabolic activation and at and above 156.25 μg/mL in the presence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (μg/mL)	%RSG (-S9) 4-Hour Exposure	%RSG (+S9) 4-Hour Exposure	%RSG (-S9) 24-Hour Exposure
0	100	100	100
9.77	83	99	26
19.53	78	103	15
39.06	86	91	32
78.13	80	88	9
156.25	80 P	80 P	0 P
312.5	88 P	81 P	0 P
625	53 P	42 P	0 P
1250	52 P	47 P	0 P
2500	57 P	53 P	0 P

P = Precipitate

 

Key to Tables

$	=	Cell counts (x10^5cells/mL). Set up on previous day to 2 x 10^5cells/mL unless otherwise stated in parenthesis.
SG	=	Suspension Growth
%RSG	=	Relative Suspension Growth
RTG	=	Relative Total Growth
%V	=	Viability Day 2
§ or #	=	Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis
A,B	=	Replicate cultures
CP	=	Cyclophosphamide
EMS	=	Ethylmethanesuplphonate
MF§	=	5-TFT resistant mutant/10^6viable cells 2 days after treatment
NP	=	Not plated due to toxicity or surplus to requirements
Ø	=	Not plated for viability or 5-TFT resistance
Nv	=	Number of wells scored, viability plates
Yv	=	Number of wells without colonies, viability plates
Ym	=	Number of wells without colonies, mutation plates
Nm	=	Number of wells scored, mutation plates
**	=	P<0.01
NS	=	Not significant
P	=	Precipitate of test item

 

Summary of Results

Experiment 1

Treatment (μg/ml)	4-Hour -S-9			Treatment (μg/ml)	4-Hour +S-9
	%RSG	RTG	MF§		%RSG	RTG	MF§
0 19.53 39.06 78.13 153.25 312.5 625	100 97 94 90 94 74 63	1.00 1.00 1.06 0.89 0.95 0.67 0.69	127.35 138.76 128.84 116.43 158.90 147.25 147.01	0 19.53 39.06 78.13 156.25 312.5 625	100 100 89 85 83 65 43	1.00 1.16 0.90 0.89 0.82 0.69 0.34	141.47 124.91 138.76 132.42 98.97 132.42 145.49
Linear trend			NS	Linear trend			NS
EMS 400	83	0.61	866.65	CP 2	64	0.39	859.31

Experiment 2

Treatment (μg/ml)	24-Hour -S-9			Treatment (μg/ml)	4-Hour +S-9
	%RSG	RTG	MF§		%RSG	RTG	MF§
0 1.22 2.44 4.88 9.77 19.53 39.06 78.13 156.25 Ø	100 86 75 68 68 42 47 10 9	1.00 1.06 0.89 0.82 0.75 0.51 0.56 0.15	145.53 131.08 100.00 122.27 109.76 124.18 148.66 192.41	0 19.53 39.06 78.13 156.25 312.5 625	100 101 96 90 65 62 59	1.00 0.94 0.92 0.91 0.67 0.73 0.64	107.52 93.11 110.80 117.68 99.98 94.67 123.59
Linear trend			**	Linear trend			NS
EMS 150	67	0.51	1120.17	CP 2	55	0.29	1112.09

 

Cell and 96-Well Plate Counts: Experiment 1 (-S9) 4-Hour Exposure

Treatment (μg/mL)		Cell counts $			Viability § after day 2 2 cells/well				Resistant mutants § after day 2 2000 cells/well
		0h	24h	48h
0   19.53   39.06   78.13   153.25   312.5   625	A B A B A B A B A B A B A B	8.82 8.89 9.14 9.30 9.67 9.04 9.30 9.50 9.05 9.33 8.34 8.80 8.06 8.39	7.84 7.59 7.33 6.67 6.22 6.25 6.35 5.81 6.50 5.71 5.74 5.23 4.68 5.51	8.43 8.89 8.76 9.08 9.44 9.58 9.18 9.45 9.67 10.05 8.74 9.87 8.93 8.86	78 79 82 84 85 82 78 81 77 77 78 76 82 83	82 66 72 78 81 78 79 74 80 80 74 72 78 80	82 79	79 80	15 16 22 17 18 20 17 16 23 15 16 23 27 23	22 15 22 21 23 22 19 16 30 23 22 16 24 17	27 12	20 21
Positive Control EMS (μg/mL)
400	A B	9.19 8.82	6.10 6.67	8.73 8.32	67 68	67 60			64 59	71 58

 

Statistical Analysis: Experiment 1 (-S9) 4-Hour Exposure

Treatment (μg/mL)	SG	%RSG	%V	RTG	MF§
0 19.53 39.06 78.13     P 153.25  P 312.5     P 625        P	16.70 15.61 14.82 14.16 15.05 12.76 11.33	100 97 94 90 94 74 63	84.05 86.56 94.51 83.70 85.11 75.99 91.99	1.00 1.00 1.06 0.89 0.95 0.67 0.69	127.35 138.76 128.84 116.43 158.90 147.25 147.01
Positive Control EMS
Treatment (μg/mL)	SG	%RSG	%V	RTG	MF§
400	13.61	83	61.61	0.61	866.65

Test for linear trend

Slope	3.595E-008
Variance	9.648E-016
b^2/Sb	1.340

 

Large and Small Colonies Analysis: Experiment 1 (-S9) 4-Hour Exposure

Treatment (μg/mL)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0   19.53   39.06   78.13   153.25   312.5   625   400 EMS	A B A B A B A B A B A B A B A B	78 79 82 84 85 82 78 81 77 77 78 76 82 83 67 68	92 66 72 78 81 78 79 74 80 80 74 72 78 80 67 70	82 79	79 80	5 10 13 9 13 9 6 6 9 9 8 12 12 11 29 21	10 10 10 11 15 11 10 10 9 9 14 7 16 12 36 18	15 7	11 9	10 6 9 8 5 11 11 10 14 6 8 11 15 12 35 38	12 5 12 10 8 11 9 6 21 14 8 9 8 5 35 40	12 5	9 12

Mutation frequencies

Treatment (μg/mL)	Viable		Small colonies			Large colonies			Proportion small colony mutants
	Yv	Nv	Mutants		MF§	Mutants		MF§
			Ym	Nm		Ym	Nm
0 19.53 39.06 78.13 153.25 312.5 625 400 EMS	143 68 58 72 70 84 61 112	768 384 384 384 384 384 384 384	691 341 336 352 348 343 333 280	768 384 384 384 384 384 384 384	62.9 68.6 70.6 52.0 57.8 74.3 77.5 256.3	697 345 349 348 329 348 344 236	768 384 384 384 384 384 384 384	57.7 61.9 50.6 58.8 90.8 64.8 59.8 395.1	0.52 0.52 0.58 0.47 0.40 0.53 0.56 0.41

 

 

Cell and 96-Well Plate Counts: Experiment 1 (+S9) 4-Hour Exposure

Treatment (μg/mL)		Cell counts $			Viability § after day 2 2 cells/well				Resistant mutants § after day 2 2000 cells/well
		0h	24h	48h
0   19.53   39.06   78.13   156.25   312.5   625	A B A B A B A B A B A B A B	10.14 9.85 10.07 10.65 9.01 10.37 9.63 9.13 9.44 9.24 8.76 8.63 6.61 7.53	6.60 6.90 6.39 6.65 6.90 6.28 5.81 6.19 5.77 5.78 5.32 4.85 4.30 4.64	9.67 9.27 9.22 9.71 9.19 8.62 10.14 9.20 10.09 9.57 9.83 9.07 8.55 8.87	77 69 81 77 72 70 75 82 84 75 81 64 74 61	62 75 72 83 74 80 72 71 76 58 80 75 69 58	82 74	79 71	12 22 17 18 18 18 25 11 23 8 16 20 19 11	24 17 20 18 19 16 19 15 13 7 13 21 18 11	12 18	18 19
Positive Control CP (μg/mL)
2	A B	10.35 10.15	5.16 5.17	7.33 8.19	55 49	65 56			66 36	65 37

 

Statistical Analysis: Experiment 1 (+S9) 4-Hour Exposure

Treatment (μg/mL)	SG	%RSG	%V	RTG	MF§
0 19.53 39.06 78.13 156.25  P 312.5     P 625        P	15.98 15.43 14.67 14.51 14.19 12.01 9.73	100 100 89 85 83 65 43	72.82 84.40 73.67 75.99 71.99 75.99 57.33	1.00 1.16 0.90 0.89 0.82 0.69 0.34	141.47 124.91 138.76 132.42 98.97 132.42 145.49
Positive Control CP
Treatment (μg/mL)	SG	%RSG	%V	RTG	MF§
2	10.02	64	44.09	0.39	859.31

Test for linear trend

Slope	7.575e-009
Variance	1.301E-015
b^2/Sb	0.044

 

Large and Small Colonies Analysis: Experiment 1 (+S9) 4-Hour Exposure

Treatment (μg/mL)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0   19.53   39.06   78.13   156.25   312.5   625   2 CP	A B A B A B A B A B A B A B A B	77 69 81 77 72 70 75 82 84 75 81 64 74 61 55 49	62 75 72 83 74 80 72 71 76 58 80 75 69 58 65 56	82 74	79 71	7 9 7 10 7 5 11 7 8 4 7 8 7 4 51 30	13 8 14 5 11 8 6 6 7 3 5 13 5 3 56 36	8 8	11 14	6 13 10 8 11 13 14 4 15 4 9 12 12 7 15 6	11 9 6 13 8 8 13 9 6 4 8 8 13 8 9 1	4 10	7 5

Mutation frequencies

Treatment (μg/mL)	Viable		Small colonies			Large colonies			Proportion small colony mutants
	Yv	Nv	Mutants		MF§	Mutants		MF§
			Ym	Nm		Ym	Nm
0 19.53 39.06 78.13 156.25 312.5 625 2 CP	179 71 88 84 91 84 122 159	768 384 384 384 384 384 384 384	690 348 353 354 362 351 365 211	768 384 384 384 384 384 384 384	73.5 58.3 57.1 53.5 41.0 59.1 44.3 679.1	703 347 344 344 355 347 344 353	768 384 384 384 384 384 384 384	60.7 60.0 74.7 72.4 54.5 66.7 95.9 95.5	0.55 0.49 0.44 0.43 0.43 0.47 0.32 0.85

 

Applicant's summary and conclusion

Conclusions:

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

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method 817 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

 

Methods

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at up to eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of the test item was selected following the results of a preliminary toxicity test and was 19.53 to 625 μg/ml in both the absence and presence of metabolic activation in Experiment 1. In Experiment 2 the dose range was 1.22 to 156.25 μg/ml in the absence of metabolic activation, and 19.53 to 625 μg/ml in the presence of metabolic activation.

 

Results

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity and by the incidence of a test item precipitate effectively reducing exposure to the cultures. A precipitate of the test item was observed at and above 78.13 μg/ml in the absence of metabolic activation and at and above 156.25 μg/ml in the presence of metabolic activation in Experiment 1. In Experiment 2 a precipitate of the test item was observed at and above 39.06 μg/ml in the absence of metabolic activation and at and above 156.25 μg/ml in the presence of metabolic activation. The negative (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

 

Conclusion

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.