Dioctyltin oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed in compliance with GLP and in accordance with the standardised guidelines OECD 476, EU Method B.17 and US EPA OPPTS 870.5300. The study was performed to a high standard sufficient to assess the quality of the presented results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

Thymidine kinase, TK+/- locus

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

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:

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)].

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 formulated the dosing solutions.
- 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 wased 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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

Conclusions:

Interpretation of results (migrated information):
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. L51788Y TK^+/-mouse lymphoma cells were treated in vitro both in the presence and absence of a rat liver derived auxillary 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% S0) 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.

Reference 2

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:

Sep 2010 - Feb 2011

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The concentrations evaluated (in bold) in the different experiments were:
4-hour first experiment:
without S9-mix: 100p, 50p, 25p, 12.5p, 6.25, 3.13, 1.56, 0.78, 0.39 and
0.2 μg/mL.
with S9-mix: 100p, 50p, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 and
0.2 μg/mL.
6-hour second experiment:
without S9-mix: 25p, 12.5p, 6.25, 3.13, 1.56, 0.78, 0.39 and 0.2 μg/mL.
with S9-mix: 50p, 25p, 12.5, 6.25, 3.13, 1.56, 0.78 and 0.39 μg/mL.
p precipitation
In all cases, the highest concentration in the culture medium did not change the osmolality of
more than 50 mOsm/kg and did not change the pH value of more than 1.0 unit compared to the
concurrent negative control

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Ethyl methanesulfonate (-S9); 7,12-dimethylbenz[a]anthracene (+S9)

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

other: not mutagen

Cytotoxicity / choice of top concentrations:

other: not mutagen

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The purpose of this study was to evaluate the potential of TIB KAT 223 to induce forward mutation at the HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in V79 Chinese Hamster lung cells with and without metabolic activation by liver homogenate (supplemented with cofactors and salts, ie, S9-mix) obtained from rats pretreated with Aroclor. TIB KAT 223 was suspended and diluted in cell culture medium. Depending on the experiment, cells were exposed to TIB KAT 223 for 4 or 6 hours with and without S9-mix. At the end of the exposure period the cloning efficiency was evaluated in microtiter plates. After a phenotypic expression period of 3 to 4 days, cells were cloned for 7 days in 75 cm2 flasks containing the selection agent 6-thioguanine (TG) for the determination of mutant frequency and in nonselective medium to measure the cloning efficiency. In addition, the number of the mutant colonies was determined. In a solubility pre-test concentrations from 0.01 to 5 mg/mL were investigated using the unaided eye for solubility, homogeneity and precipitation evaluation of the test compound in cell culture medium. In this pre-test all concentrations (except 0.01 mg/mL) were found to be suspensions. However, at 0.1 mg/mL (100 μg/mL) we observed a homogenous distribution of the particles. Therefore, 100 μg/mL was chosen as top treatment concentration in accordance to the OECD 476 Guidline. The highest evaluated concentrations were limited by test article precipitation observed at 12.5 μg/mL and above without S9 mix and 50 μg/mL (Exp. 1) respectively 25 μg/mL (Exp. 2) and above with S9 mix.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, under the experimental conditions of the study, TIB KAT 223 was found negative
in the mutation assay with V79 Chinese Hamster cells at the HPRT (hypoxanthine-guanine
phosphoribosyl transferase) locus in the presence or absence of metabolic activation up to
concentrations exhibiting test article precipitation

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March 2002 to 10 April 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed in compliance with GLP and in accordance with the standardised guidelines OECD 471 and US EPA OPPTS 870.5100. The study was performed to a high standard sufficient to assess the quality of the presented results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine requirement in the Salmonella typhimurium strains
Tryptophan requirement in the Escherichia coli strain.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: Stored as nutrient broth cultures with 7.4 % DMSO at <60 °C.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Stored as nutrient broth cultures with 7.4 % DMSO at <60 °C.
- Properly maintained: 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:

Range finding test: 0, 0.3, 0.8, 2.3, 7, 21, 62, 185, 556, 1667 and 5000 µg/plate.
Definitive test: 0, 62, 185, 556, 1667 and 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: N-ethyl-N-nitrosourea and 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)
2 mL molten top agar (0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine.HCl/0.05 mM biotin for the S. typhimurium stains, and supplemented with 0.05 mM tryptophane for the E.coli WP2 uvrA strain), maintained at 46 °C. 0.1 mL of culture of the appropriate strain, 0.1 mL of the appropriate test substance solution, or the solvent or positive control substance and 0.5 mL S9-mix for the plates with metabolic activation or 0.5 mL sodium phosphate 100 mM (pH 7.4) for without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose).

DURATION
- Exposure duration: 48-72 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was assessed as a reduction in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.

Evaluation criteria:

The mutagenicity study was considered valid if the mean colony counts of the control values of the strains were within acceptable ranges, if the results of the positive controls met the criteria for a positive response, and if no more than 5 % of the plates were lost through contamination or other unforseen events.
A test substance was considered to be positive if the increase in the mean number of revertant colonies on the test plates was concentration-related or if a reproducible two-fold or more increase was observed compared to that of negative control plates.
A test substance was considered to be negative in the bacterial gene mutation test if it produced neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Statistics:

No statistical analysis was performed.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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 applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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 applicable

Positive controls validity:

not valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance was found to precipitate at all concentrations

RANGE-FINDING/SCREENING STUDIES: A dose range finding test was performed with TA 98 in both the absence and presence of S9-mix with ten different concentrations of the test substance, ranging from 0.3-5000 µg/plate. Dioctyloxostannane (dioctyltin oxide) was not toxic at any concentration both in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY: A slightly more dense background lawn of the bacterial growth was observed at the dose levels at which the slight increases were observed; however, these occured alongside precipitation of the test substance.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 2: Results of range finding test

Dose µg/plate		TA 98
		Without S9-mix	With S9-mix
0		38	52
		36	46
		29	48
	Mean	34	49
	SD	5	3
0.3		46	57
		48	61
		40	46
	Mean	45	55
	SD	4	8
0.8		46	69
		44	57
		26	60
	Mean	39	62
	SD	11	6
2.3		52	52
		42	55
		34	57
	Mean	43	55
	SD	9	3
7		27	59
		42	59
		46	60
	Mean	38	59
	SD	10	1
21		31	53
		27	42
		36	47
	Mean	31	47
	SD	5	6
62		42	56
		33	47
		40	60
	Mean	38	54
	SD	5	7
185		45	58
		36	63
		39	46
	Mean	40	56
	SD	5	9
556		34	38
		39	45
		35	40
	Mean	36	41
	SD	3	4
1667		45	59
		44	57
		39	68
	Mean	43	61
	SD	3	6
5000		37	57
		41	52
		38	50
	Mean	39	53
	SD	2	4
Positive control		1170	1090
		1225	1051
		1201	1074
	Mean	1199	1072
	SD	28	20

*The positive controls employed are detail in table 1.

Table 2: Results of the definitive test

Dose µg/plate		TA 1535		TA 1537		TA 98		TA 100		E. coli
		Without S9-mix	With S9-mix	Without S9-mix	With S9-mix	Without S9-mix	With S9-mix	Without S9-mix	With S9-mix	Without S9-mix	With S9-mix
0		15	17	8	22	29	77	165	156	**	31
		21	28	13	6	32	51	186	189	23	26
		16	15	13	15	27	36	163	175	37	25
	Mean	17	20	11	14	29	55	171	173	30	27
	SD	3	7	3	8	3	21	13	17	10	3
62		15	20	5	15	16	60	151	170	40	46
		18	24	15	14	27	40	148	166	46	39
		20	22	12	8	24	51	173	198	40	42
	Mean	18P	22	11	12	22	50	153	178	42*	42
	SD	3	2	5	4	6	10	14	17	3	4
185		8	23	11	16	25	57	159	181	35	41
		23	24	6	4	28	55	149	161	36	34
		20	13	4	9	28	56	211	182	31	47
	Mean	17P	20	7	10	27	56	173	175	34*	41
	SD	8	6	4	6	2	1	33	12	3	7
556		17	20	9	9	31	38	183	144	49	37
		30	23	9	8	26	53	168	155	36	33
		14	25	8	11	29	35	170	159	30	49
	Mean	20P	23	9	9	29	42	174	153	38*	40
	SD	9	3	1	2	3	10	8	8	10	8
1667		20	11	5	6	48	31	146	162	33	50
		28	20	18	8	35	37	146	152	41	38
		22	31	18	12	36	85	150	170	67	27
	Mean	23P	21	14	9	40	51	147	161	47*	38
	SD	4	10	8	3	7	30	2	9	18	12
5000		31	20	8	11	31	40	153	166	46	49
		34	19	7	14	51	42	137	161	46	39
		30	28	11	15	26	48	131	161	42	41
	Mean	32*	22	9	13	36	43	140	163	45*	43
	SD	2	5	2	2	13	4	11	3	2	5
Positive control		442	365	1053	211	1105	634	744	1245	287	698
		444	345	863	212	1185	738	704	940	221	677
		427	374	913	216	1186	555	708	1012	242	715
	Mean	438	361	943	213	1159	642	719	1066	250	697
	SD	9	15	98	3	46	92	22	159	34	19
P: Precipitation of test substance * : Precipitation of test substance and slightly more dense background lawn of bacterial growth than in concomitant control plates. **: Not counted; no bacteria on the agar plate

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of the test, the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in the Eschericia coli strain WP2 uvrA, in both the absence and presence of metabolic activation (S9-mix), the test substance was not mutagenic.

Executive summary:

The mutagenicity of the test substance was assessed in a bacterial reverse mutation assay (Ames Test) in accordance with the OECD guideline 471 and EPA OPPTS 870.5100 and to GLP. The cultures were exposed to 0, 62, 185, 556, 1667 and 5000 µg/plate. Precipitation of the test substance was observed at a number of the concentrations tested. In some of the plates a slight dose-dependant increase in the number of revertants were noticed, however this was accompanied by an increased background lawn and precipitation. This was therefore not attributed to the mutagenic potential of the test substance. Under the conditions of the test, the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in the Eschericia coli strain WP2 uvrA, in both the absence and presence of metabolic activation (S9-mix), the test substance was not mutagenic.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun 2010 - Feb 2011

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

n.a.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from livers of male rats (Sprague Dawley strain)

Test concentrations with justification for top dose:

The concentrations tested and evaluated (in bold) in the different experiments were:
First main experiment 3h treatment without S9-mix: 25p, 12.5p, 6.25, 3.13, 1.56, 0.78, 0.39 and (Reported as experiment I) 0.2 μg/mL
First main experiment 3h treatment with S9-mix: 100p, 50p, 25p, 12.5, 6.25, 3.13, 1.56 and (Reported as experiment I) 0.78 μg/mL
Second main experiment 20h treatment without S9-mix: 25p, 12.5p, 6.25, 3.13, 1.56, 0.78, 0.39 (Not evaluated/reported due because the cells did and 0.2 μg/mL not fulfill the quality criteria for the evaluation of the metaphases)
Third main experiment 20h without S9: Not treated because these cells did not fulfill the quality criteria for a treatment.
Fourth main experiment 20h without S9: 25p, 12.5p, 6.25, 3.13, 1.56, 0.78, 0.39 and 0.2 μg/mL
(Reported as experiment II)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: ethylmethane sulfonate (-S9), cyclophosphamide (+S9)

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

see enclosed study report

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, TIB KAT 223 was found negative in the chromosome aberration test in V79 Chinese hamster cells in the presence and in the absence of metabolic activation, when tested up to concentrations exhibiting test article precipitation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 October 2003 to 11 December 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted in accordance with the standardised testing guideline OECD 474 and in line with GLP with no deviations thought to affect the quality of the presented data. The study was reported to a high standard, sufficient to assess the reliability of the data presented.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adult
- Weight at study initiation: Approximately 34 g
- Fasting period before study: 2 hours and 45 minutes in the range finding test and 3 hours and 30 minutes in the main test.
- Diet: ad libitum
- Water: Tap water, ad libitum delivered in polypropylene bottles
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): At least 30 % not exceeding 70 %
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark.

DOSE-RANGE FINDING TEST IN-LIFE DATES: From: 4 November 2003 To: 7 November 2003
MAIN TEST IN-LIFE DATES: From: 25 November 2003 To: 28 November 2003

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 100, 50 and 25 mg/mL
- Amount of vehicle: 20 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The day before dosing, the test substance was suspended in corn oil at a stock concentration of 100 mg/mL and stirred overnight on a magnetic stirrer. The two lower concentrations of 50 and 25 mg/mL were prepared prior to dosing on day 0.

Duration of treatment / exposure:

One treatment

Frequency of treatment:

A single exposure was used

Post exposure period:

At 24 hours after treatment, 5 animals of each dose level of the test substance, 5 negative control animals and 5 positive control animals were sacrificed by cervical dislocation. After 48 hours the remaining animals of the high dose and and the negative control group were sacrificed.

Remarks:

Doses / Concentrations:
0 (A), 500 (B), 1000 (C) and 2000 (D) mg/kg bw
Basis:
nominal conc.
Main test

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
Range finding test

No. of animals per sex per dose:

In the range finding test, 2 males and 2 females were used in each dose group to determine the toxicity of the test substance.
In the main test, 10 male animals were used in each of the high dose group and the negative control group and 5 in all other dose groups and the positive control.

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Route of administration: Intraperitoneal injection
- Doses / concentrations: 0.75 mg/kg bw (10 mL/kg bw) in saline

Tissues and cell types examined:

From each mouse, the bone marrow cells of both femurs were immediately collected into foetal calf serum and processed into glassdrawn smears. Two smears per animal were prepared.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dose levels and sex chosen for the main test were based on the clinical signs observed during the dose range finding test. The range-finding test demonstrated an absence of toxicity and no sex difference, therefore, the main test was carried out with male mice only and graded dose levels up to the limit dose of the guideline were used.

DETAILS OF SLIDE PREPARATION: The smears were air-dried and fixed in methanol. One smear per animal was stained with May-Grünwald Giemsa solution. The pther slide was stored as a reserve slide.

METHOD OF ANALYSIS: The slides were read by moving from the begining of the smear to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear.
The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE were scored for the presence of micronuclei until a total number of 2000 PE had been scored. The incidence of MPE was therefore recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.

Evaluation criteria:

The study was considered to be valid if the positive controls gave a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls were in the historical range.
A response was considered positive if the mean number of MPE/2000 PE was statistically significantly higher in comparison to the vehicle controls.
The test substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus, if a clear dose-related increase in the mean numbers of MPE/2000 PE was observed, when compared to the vehicle controls.
A test substance was considered to be negative if it produced no positive response at any of the doses and time points analysed.
The test substance or its metabolites was considered to have reached the general circulation and thereby the bone marrow, if the test substance statistically reduced the mean number of PE/E or caused systemic toxicity.
Both statistical significance and biological relevance were considered when evaluating the responses.

Statistics:

At 24 hours, data on MPE and PE were evaluated using One Way Anova with a factor group (A, B, C and D). If the Anova demonstrated a significant effect (p<0.05), pooled error variance t-tests were performed, or if the variances were not homogenous, separate variance t-tests. These t-tests were applied to the negative control group (A) versus treatment groups B (500), C (1000) and D (2000). Furthermore, the positive control group E and the negative control group A were compared using the same process. A linear trend test (orthogonal contrasts) was also applied across groups A-D.
At 48 hours after administration, for treatment groups A and D, data on MPE and PE were analysed using pooled error variance t-tests, or if variances were not homogenous, separate variance t-tests.
All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000 and 2000 mg/kg bw, dosed as 20 mL/kg bw
- Clinical signs of toxicity in test animals: Female 3 died 24 hours post dosing due to a failed oral administration. No severe clinical signs were observed as a results of test substance administration.

RESULTS OF DEFINITIVE STUDY
- Statistical evaluation: At both sacrifice times, the two way ANOVA did not demonstrate a statisitically significant effect for MPE and PE. There was no observable genotoxicity or clastogenicity up to a dose of 2000 mg/kg bw. At 24 hours, the incidence of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) was statistically significantly different (P<0.001) from the vehicle control. The test system was therefore considered to be valid.

Table 1: Results of range finding test

Test substance concentration	1 h post administration	4 h post administration	24 h post administration	48 h post administration	Animal number and sex
500 mg/kg bw		sl; se; br	sl; se; p; br; †		Male 2
					Male 4
					Female 1
					Female 3
1000 mg/kg bw					Male 6
					Male 8
					Female 5
					Female 7
2000 mg/kg bw					Male 10
					Male 12
					Female 9
					Female 11
Empty cells = no clinical signs observed sl = Sluggishness se = slit-eyes p = piloerection br = irregular breathing † = animal died

 

Table 2: Results of the main test

Group	Bodyweight (g)	Mouse no.	Observation time (h)	PE	NE	MPE	MNE
A	36.1	2	24	87	113	2	0
	32.2	4	24	74	126	2	0
	35.7	6	24	93	107	3	0
	37.6	8	24	113	87	1	0
	32.7	10	24	79	121	2	0
	34.5	12	48	98	102	1	1
	36.0	14	48	96	104	2	0
	35.6	16	48	93	107	2	0
	32.8	18	48	79	121	4	0
	35.1	20	48	74	126	2	0
	34.83 ± 0.56	Mean ± S.D.	24	89.2 ± 15.2		2.0 ± 0.7
			48	88.0 ± 10.8		2.2 ± 1.1
B	33.1	22	24	101	99	3	0
	34.7	24	24	63	137	1	0
	33.8	26	24	89	111	2	0
	35.6	28	24	81	119	4	0
	34.7	30	24	92	108	3	0
	34.38 ± 0.43	Mean ± S.D.		85.2 ± 14.3		2.6 ± 1.1
C	34.6	42	24	77	123	2	0
	32.8	44	24	90	110	2	0
	35.7	46	24	109	91	2	0
	33.9	48	24	77	123	2	0
	33.6	50	24	102	98	1	0
	34.12 ± 0.49	Mean ± S.D.		91.0 ± 14.4		1.8 ± 0.4
D	36.3	62	24	66	134	2	0
	34.7	64	24	111	89	2	0
	31.5	66	24	81	119	1	0
	33.6	68	24	99	101	1	1
	33.7	70	24	103	97	1	0
	37.8	72	48	83	117	1	0
	33.8	74	48	77	123	2	0
	33.2	76	48	73	127	2	0
	33.3	78	48	74	126	2	0
	34.3	80	48	69	131	0	0
	34.22 ± 0.55	Mean ± S.D.	24	92.0 ± 18.2		1.4 ± 0.5
			48	75.2 ± 5.2		1.4 ± 0.9
E	33.8	82	24	81	119	50	0
	34.1	84	24	78	122	64	0
	32.3	86	24	66	134	57	1
	34.4	88	24	74	126	40	0
	33.9	90	24	86	114	41	0
	33.70 ± 0.36	Mean ± S.D.		77.0 ± 7.6		50.4 ± 10.3*
A = Vehicle control (corn oil) B = 500 mg/kg bw test substance C = 1000 mg/kg bw test substance D = 2000 mg/kg bw test substance E = Mitomycin C 0.75 mg/kg bw in saline i.p. injection Body weight = Bodyweight prior to dosing PE = number of PE scored per 200 E scored NE = number NE scored per 200 E scored MPE = number of MPE scored per 2000 PE scored MNE = number MNE scored per number of NE scored * P<0.001 (t-tests) n = 5

 

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of the test, the test substance was found not to produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow cells of mice.

Executive summary:

The potential genotoxicity and clastogenicity of the test substance to the bone marrow cells of Swiss male mice in vivo was assessed in a study conducted in accordance with OECD 474 and to GLP. Up to an oral dose of 2000 mg/kg bw (the limit dose recommended by the guideline), no chromosomal damage or damage to the mitotic spindle apparatus was noted. Under the conditions of the test the test substance was found to be non-genotoxic and non-clastogenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro Gene Mutation Study in Bacteria

The key study, Krul (2010) was submitted to fulfil the data requirement for in vitro mutagenicity in bacteria. The study was performed in accordance with standardised guidelines, OECD 471 and EPA OPPTS 870.5100 and in accordance with GLP. The study was both performed and reported to a high standard. As such it was deemed acceptable to assign the study a reliability score of 1 in accordance with the criteria for assessing data quality as outlined in Klimisch (1997). Under the conditions of the test, the test substance did not induce an increase in revertants in the bacterial strains selected for the study.

In vitro Gene Mutation

The key study, Flanders (2012), was performed in accordance with standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300 and in line with GLP. The study was both performed and reported to a high standard. As such it was deemed acceptable to assign the study a reliability score of 1 in accordance with the criteria for assessing data quality as outlined in Klimisch (1997). 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.

In vivo Cytogenicity Test:

The key study, de Vogel (2004), was performed in accordance with the standardised guideline OECD 474 and in line with GLP. The study was reported to a high standard and was sufficient to assess the genotoxic and clastogenic potential of the substance. In accordance with the criteria for assessing data quality as outlined in Klimisch (1997), the study was assigned a reliability score of 1. The test substance was found not to induce chromosome damage, or damage to the mitotic spindle apparatus under the conditions of the test, and was concluded to be negative for genotoxicity and clastogenicity.

Justification for selection of genetic toxicity endpoint
As multiple studies are presented to address genetic toxicity, no one study was selected as the key study as they represent different types of genetic toxicity and are therefore not comparable.

Short description of key information:
In vitro Gene Mutation Study in Bacteria
Key study:- Krul (2010), OECD 471 and EPA OPPTS 870.5100; Negative up to precipitating concentrations.

In vitro Gene Mutation
Key study:- Flanders (2012), OECD 476, EU Method B.17, EPA OPPTS 870.5300; Negative up to precipitating concentrations

In vivo Cytogenicity Test
Key study:- de Vogel (2004), OECD 474; Negative up to 2000 mg/kg bw (limit concentration)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with with criteria for classification and labelling as defined in Regulation (EC) No 1272/2008 (CLP) and Directive 67/548/EEC (DSD), the test material does not require classification for genetic toxicity.