Ethyl 9,9-dioctyl-4,7,11-trioxo-3,8,10-trioxa-9-stannatetradeca-5,12-dien-14-oate

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:

27 April 2012 to 31 July 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed in compliance with GLP, in accordance with the standardised guidelines OECD 476, EU Method B.17 and US EPA OPPTS 870.5300 and was performed to a high standard. The test material DOTO (di-n-octyltin oxide) is in the same category of substances as the registration substance and it is therefore considered to be acceptable to use a read-across approach to address this endpoint.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dioctyltin oxide

EC Number:

212-791-1

EC Name:

Dioctyltin oxide

Cas Number:

870-08-6

Molecular formula:

C16H34OSn

IUPAC Name:

Stannane, dioctyloxo-

Test material form:

other: solid (unspecified)

Details on test material:

- Name of test material (as cited in study report): Di-n-octyltin oxide
- Physical state: white solid
- Storage condition of test material: room temperature in the dark

Method

Target gene:

Thymidine kinase, TK+/- locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with penicillin (100 units/mL), streptomycin (100 µg/mL), sodium pyruvate (1 mM), amphoterin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media)
- Properly maintained: Yes. A bank of L5178Y TK+/- cells were stored in a liquid nitrogen freezer. Following removal from liquid nitrogen, the cultures were kept at 37 °C under an atmosphere of 5 % CO2 in air
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Preliminary Toxicity Test - with and without S9 mix
0, 3.53, 7.05, 14.10, 28.20, 56.41, 112.81, 225.63, 451.25, 902.50 µg/mL

Experiment 1 - with and without S9 mix
0, 3.5, 7, 14, 28, 42, 56, 84, 112 µg/mL test material and 400 µg/mL of ethylmethanesulphonate (EMS) as a positive control in the absence of S9 mix and 2 µg/mL of cyclophosphamide (CP) as a positive control in the presence of S9 mix
acetone as solvent control

Experiment 2 - without S9-mix
0, 0.63, 1.25, 2.5, 5, 7.5, 10, 15, 20 µg/mL test material and 150 µg/mL of ethylmethanesulphonate (EMS) as a positive control
acetone as solvent control

With S9-mix
0, 20, 30, 40, 50, 60, 70, 80, 90 µg/mL test material and 2 µg/mL of cyclophosphamide (CP) as a positive control
acetone as solvent control

Experiment 3 - with S9-mix
0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 µg/mL test material and 2 µg/mL of cyclophosphamide (CP) as a positive control
acetone as solvent control

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
The test material was accurately weighed and formulated in acetone prior to serial dilutions being prepared. The test material was formulated within 2 hours of it being applied to the test system. Vehicle and positive controls were used in parallel with the test material. Test material dilutions were dosed at 0.1 mL per culture and positive control solutions were dosed at 0.2 mL per culture.

Exponentially growing suspension cultures of L5178Y cells were treated in duplicate with the solvent control, positive controls or a range of concentrations of the test material for 4 or 24 hours in the presence and/or absence of S9-mix. The cells were then cultured to allow any induced mutations to be expressed. During this expression time the growth rate was monitored and, where appropriate, the cells subcultured daily. At the end of the 48-hour expression time, samples were grown both in selective and non selective medium, and the results obtained used to determine the mutant frequency per viable cell.


DURATION
- Exposure duration: 4 hours (Experiment 1; Experiment 2 with S9 mix and Experiment 3); 24 hours (Experiment 2 without S9 mix)
- Expression time (cells in growth medium): The post-treatment cultures were incubated at 37 °C with 5 % CO2 in air for a 48 hour expression period. To maintain exponential growth during the expression time, each culture was counted and, where appropriate, diluted daily to give approximately 2 x 10^5 cells per mL.
- Selection time (if incubation with a selection agent): On day 2, for the assessment of mutants, a sample of each of the post-expression cultures was diluted to 1 x 10^4 cells per mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine in 96-well microtitre plates.

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT).

NUMBER OF REPLICATIONS: Duplicate

DETERMINATION OF CYTOTOXICITY
- Method: Survival was measured by relative total growth (RTG). RTG is a measure of growth of test cultures both during the two-day expression and cloning phases of the assay, relative to the vehicle control.

DETERMINATION OF VIABILITY:
For the assessment of viability, cells were diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

DATA EVALUATION:
Cell growth in individual microwell plates was assessed after 10-14 days using a magnifying mirror box. The survival plates and viability plates were scored for the number of wells containing positive wells (wells with colonies) together with the total number of scorable wells (normally 96 per plate). The numbers of small (colonies less than 25 % of the average area of the large colonies)and large colonies (colonies covering ¼ to ¾ of the surface of the well and generally no more than 2 cells thick)were also recorded. To assist the scoring of the colonies 5-trifluorothymidine (TFT) mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for approximately 2 hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a blue/black colour, aiding visualisation of mutant colonies.

CALCUALTIONS:
Viability calculations were based on P(0), the proportion of wells in which a colony had not grown:

P(0) = number of negative wells/total wells plated

%V = -ln P(0)/number of cells per well

Relative suspension growth (RSG) is defined as the relative total two day suspension growth of the test culture compared to the total two-day suspension growth of the vehicle control. Relative total growth (RTG) is a measure of growth of test cultures both during the two-day expression and cloning phases of the assay, relative to the vehicle control. The RSG of each test culture was multiplied by the relative cloning efficiency of the test culture at the time of mutant selection and expressed relative to the cloning efficiency of the vehicle control. The highest concentration assayed was designed to reduce RTG to 10 %-20 % of the solvent control culture values unless limited by solubility, pH or osmolarity effects or a limit concentration of 5 µL/mL, 5000 µg/mL or 10 mM (whichever is the lowest).

MUTANT FREQUENCY (M.F.)
The mutant frequency for each culture was then calculated:

M.F. =-[ ( ln P(0) selective medium) / cells per well in selectibe medium) ] / surviving fraction in non-selective medium

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
A statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value by the Global Evaluation Factor of 126 x 10^-6, for the microwell method, is required. The assay must also demonstrate a positive linear trend.

CRITERIA FOR A NEGATIVE RESPONSE
A negative response is obtained when there is no reproducible statistically significant dose-related increase in mutant frequency. When a test material induces a modest reproducible increase in mutant frequency that do not exceed to GEF value then scientific judgement is required. 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.

CRITERIA FOR SCORING MUTATION PLATES
Each well of mutation plates was scored as containing either a small colony or a large colony
Small colony: an average area less than 25 % of the area of the well, usually observed to be more than two cells thick
Large colony: Average coverage ¼ to ¾ of the surface of the well and are generally no more than 2 cells thick
An empty well was one which contained no cell growth.

Statistics:

Mutant frequency experimental data was analysed using the Mutant 240C (York Electronic Research) which follows the statistical guidelines recommended by UKEMS.

The distribution of colony-forming units over the wells is described by the Poisson distribution, viability on day 2 was therefore calculated using the zero term of the Poisson distribution [P(0)].

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

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 material

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Test Material solubility: The molecular weight of the test material was 361, therefore, the maximum proposed dose level in the solubility test was initially set at 3610 µg/mL, the 10 mM limit dose. However, due to formulation difficulties, the maximum achievable dose level suitable for dosing was set at 1805 µg/mL. Acetone is toxic to L5178Y at dose volumes greater than 0.5 % of the total culture volume. Therefore, the test material was formulated at 180.5 mg/mL and dosed at 0.5 % to give a maximum achievable dose level of 902.5 µg/mL. The purity of the test material was accounted for when the dosing solutions were formulated.
- Precipitation: Precipitate of the test material was observed at and above 28.20 µg/mL in both the 4 hour exposure groups and at 56.41 µg/mL and above in the 24 hour exposure group of the preliminary toxicity test. In experiment 1, precipitate of the test material was observed at 28 µg/mL and above, at 70 µg/mL and above in experiment 2 and at 60 µg/mL and above in experiment 3.
- pH and osmolality: The effect of the test material on the pH and osmolarity of the treatment medium was investigated. There were no marked change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm in the solubility test.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period with and without metabolic activation (20 % S9-mix), and at 1.5 x 10^5 cells/mL using a 24 hour exposure period without metabolic activation. The dose range used in the preliminary toxicity test was 3.53 to 902.5 µg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10 medium, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 2 x 10^5 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 10^5 cells/mL. After a further 24 hours, the cultures were counted and discarded. The cell counts were used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post tratment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control was performed to give a percentage Relative Suspension Growth (%RSG) value. Results from the preliminary toxicity test were used in conjuction with evaluations of the solubility of the test material to determine the concentrations used in the mutagenicity tests.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Preliminary toxicity test: 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 material when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult. Precipitate of the test material was observed at and above 28.20 µg/mL in both of the 4 hour exposure groups, and at and above 56.41 µg/mL in the 24 hour exposure group. Based on the %RSG values observed, the maximum dose levels in the subsequent mutagenicity test were limited by test material-induced toxicity.

- Experiment 1: There was evidence of marked dose-related toxicity following exposure to the test material in both the absence and presence of S9 mix as indicated by the RTG, %RSG and %V values. The levels of toxicity observed were similar to those noted during the preliminary toxicity test. It should, however, be noted that the reductions were only observed at dose levels that had been excluded from the statistical analysis due to the toxicity exceeding the upper limit of 90 %. Excessive toxicity was noted at 112 µg/mL in both the absence and presence of metabolic activation resulting in this dose level not being plated for viability or 5-TFT resistance.

- Experiment 2: There was evidence of marked toxicity following exposure to the test material in the absence of metabolic activation, even more marked for the extended 24 hour exposure, as indicated by the RTG and %RSG values. The expected levels of toxicity were not achieved in the presence of metabolic activation and only modest levels of toxicity were achieved. It was considered that this may have been due to the lowering of the S9 concentration to 1 % in this experiment compared to the 2 % S9 concentration in the preliminary toxicity test and in experiment 1. The most marked reduction in the presence of metabolic activation was observed at the penultimate dose level and the RTG value appeared to increase slightly at the maximum dose level indicating that maximum exposure had been achieved due to the presence of precipitate effectively reducing exposure of the test material to cells. It was therefore considered appropriate to perform a confirmatory Experiment 3 using the same exposure conditions and a narrower dose interval in the presence of metabolic activation.There was no evidence of any reductions in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not 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. The excessive toxicity observed at and above 15 µg/mL in the absence of metabolic activation, resulted in these dose levels not being plated for viability or 5 -TFT resistance.

-Experiment 3: As was seen in Experiment 2, the levels of toxicity observed, as indicated by the RTG and %RSG values were very modest and differed from those of the preliminary toxicity test and Experiment 1 where 2 % S9 had been used. The levels of toxicity observed were very similar to those observed in the presence of 1 % metabolic activation in Experiment 2. The most marked toxicity was once again observed at the penultimate dose level and a much greater increase in %RSG and RTG was observed at the maximum dose level. This was taken to confirm that maximum exposure to the test material to the cells had been achieved due to the presence of precipitate effectively reducing exposure of the test material to the cells. This also confirmed that the difference in toxicity observed in the preliminary toxicity and Experiment 1 was caused by the lowering of the S9 concentration from 2 % to 1 %. The test material was therefore considered to have been adequately tested.

Any other information on results incl. tables

Mutagenicity Test

- Experiment 1:The test material did not induce any statistically significant or dose related increases in the mutant frequency x 10^-6 per viable cell either in the absence or presence of metabolic activation. A very modest but statistically significant dose related increase in mutant frequency was observed in the presence of metabolic activation, at one dose level, however, the GEF was not exceeded and there was no evidence of any marked increase in the absolute number of mutant colonies. In both the presence and absence of metabolic activation, a marked increase in mutant frequency was observed in the dose level that was plated out that exceeded the upper limit of acceptable toxicity. However, there was again no evidence of an increase in absolute numbers of mutant colonies, there was no evidence of a shift towards small colony formation that would have indicated a clastogenic response, and the viability values were low resulting in a falsely high mutant frequency value. The increases in mutant frequency were therefore considered to be due to cytotoxicity and not a true genotoxic response and were, therefore considered artefactual and of no toxicological significance.

Table 1: Experiment 1 Summary of Results

Treatment (µg/mL)	4 hours -S9			Treatment (µg/mL)	4 hours +S9
	% RSG	RTG	MF §		% RSG	RTG	MF §
0	100	1.00	129.11	0	100	1.00	134.75
3.5 Ø	96			3.5 Ø	88
7	102	1.02	145.06	7	99	1.05	145.87
14	88	0.80	048.54	14	83	0.90	156.85
28	73	0.78	134.95	28	68	0.86	129.35
42	78	0.77	143.34	42	61	0.66	175.58
56	34	0.35	146.99	56	34	0.30	215.14 *
84 X	9	0.01	540.45	84 X	15	0.04	318.87
112 Ø	2			112 Ø	5
Linear trend			NS	Linear trend			*
EMS (400)	77	0.49	1081.21	CP (2)	48	0.29	1833.97

-Experiment 2:The test material did no induce any statistically significant or dose related increases in the mutant frequency x 10^-6 per viable cell in ether the absence or presence of metabolic activation.

Table 2: Experiment 2 Summary of Results

Treatment (µg/mL)	4 hours -S9			Treatment (µg/mL)	4 hours +S9
	% RSG	RTG	MF §		% RSG	RTG	MF §
0	100	1.00	175.05	0	100	1.00	162.07
0.63	90	0.92	151.31	20 Ø	89
1.25	77	0.75	189.17	30 Ø	94
2.5	69	0.88	152.31	40	99	0.91	149.64
5	38	0.59	150.95	50	87	0.73	172.61
7.5	20	0.36	142.04	60	81	0.71	171.65
10	17	0.29	158.60	70	80	0.63	159.50
15 Ø	3			80	57	0.46	174.73
20 Ø	0			90	59	0.55	139.04
Linear trend			NS	Linear trend			NS
EMS (150)	49	0.52	1260.54	CP (2)	64	0.34	2212.67

-Experiment 3:The test material did not induce any statistically significant or dose related increases in the mutant frequency x 10 ^-6 per viable cell in the presence of metabolic activation. precipitate of the test material was observed at and above 60 µg/mL.

Table 3: Experiment 3 Summary of Results

Treatment (µg/mL)	4 hours +S9
	% RSG	RTG	MF §
0	100	1.00	126.04
20 Ø	104
30 Ø	94
40 Ø	104
50 Ø	85
60	82	0.91	112.86
70	71	0.71	139.88
80	78	0.77	137.90
90	62	0.63	139.56
100	52	0.54	150.21
110	69	0.88	101.91
Linear trend			NS
CP (2)	75	0.46	1429.48

In all of the Experiments, none of the vehicle control mutant frequency values were outside the acceptable range and all 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.

Key to tables

%RSG = Relative Suspension Growth

RTG = Relative Total Growth

CP = cyclophosphamide

EMS = ethylmethanesulphonate

MF§ = 5-TFT resistant mutants/ 10^6 viable cells 2 days after treatment

Ø = not plated for viability or 5-TFT resistance

X = treatment excluded from test statistics due to toxicity

NS = Not Significant

* = P < 0.05

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation

Under the conditions of the assay, the test material was determined not to be mutagenic in L5178Y TK+/- cells treated in vitro in either the presence or absence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.

Executive summary:

The potential of the test material to cause gene mutation or clastogenic effects in mammalian cells was determined in accordance with standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300. L5178Y TK +/- mouse lymphoma cells were treated in vitro both in the presence and absence of a rat liver derived auxiliary metabolic system (S9 mix). Large and small mutant colonies were scored for all cultures in each experiment.

Initially, two independent experiments were performed. In Experiment 1, cells were treated with the test material at eight dose levels, in duplicate, together with vehicle 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 test material 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. However, due to a marked difference in toxicity in the 4-hour exposure groups in the presence of metabolic activation between Experiment 1 and 2, and an apparent maximum exposure being achieved at the penultimate dose level in Experiment 2, a confirmatory Experiment 3 was performed using a 4-hour exposure group at ten dose levels in the presence of metabolic activation (1 % S9) only.

 

The dose range of the test material was selected following the results of a preliminary toxicity test and was 3.5 to 112 µg/mL in both the absence and presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 0.63 to 20 µg/mL in the absence of metabolic activation, and 20 to 90 µg/mL in the presence of metabolic activation. In Experiment 3 the dose range was 20 to 110 µg/mL in the presence of metabolic activation only.

 

Under the conditions of the test, the maximum dose levels used in the mutagenicity test were limited by test material-induced toxicity. Overall, precipitate of test material was observed at and above 28 µg/mL in the mutagenicity test. The vehicle controls had mutant frequency values that were considered acceptable 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 material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first, second or third experiment. The test material is therefore considered to be non-mutagenic to L5178Y cells under the conditions of this assay.