2,2-dioctyl-1,3,2-oxathiastannolan-5-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

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:

1 december 2009-2 march 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study (OECD 476)

Justification for type of information:

Read across from the source substance DOTE / DOTI to the target substance DOTTG is based on:
1.Structural Similarity: both belong to the category of di-n-octyltin compounds with a thio bond (COLLA 2018)
2.Common raw materials: DOTTG is always manufactured in the same step together with DOTE /DOTI and further processed as a 2.5-10 % solution in DOTE or DOTI based on the same raw materials
3.Hydrolysis: DOTTG as a pure substance hydrolysis under simulated conditions to Dioctyltin dichloride (DOTC). In DOTE solution the latter spontaneously reacts with the access of DOTE to form the monochloro mercapto acetic ester (DOTEC). So DOTTG in DOTE solution under simulated gastric conditions results in the same metabolite as DOTE, the monochloro mercapto acetic ester (DOTEC) which is stabilized in a cyclic structure that is very similar to the DOTTG structure.
4.Applicational fate: both substances react during their technical function with HCl released from PVC and degrade themselves to the to the same mono-chloro mercato acetic ester DOTEC.
5.Supply chain considerations: The solid DOTTG It is never isolated and marketed as a pure substance only as a 2.5-10 % solution in DOTE or DOTI
6.Hazard and risk considerations: Since DOTE is already classified in the highest hazard categories it seems not to appears to be inappropriate to conduct additional animal studies on DOTTG. The further regulatory fate of DOTTG, such as possible authorization / restriction, is closely linked to DOTE. The registrants have adopted the DOTE classification of the RAC 47 opinion as classification for DOTTG.
7.Further considerations: Source and target substance have been chosen by The Netherlands CA as members of a group of “disubstituted organotins with a thio bond (S-ligands)”. This group has been subject to a pilot project (COLLA March 2017–March 2018)
In 1999 the Scientific Committee on Food (SCF, 1999) evaluated octyltin compounds for food contact materials and established a group tolerable daily intake (TDI) for di-n­ octyltins (DOT) of0.0006 mg/kg The group entry covers in this context the two source substances DOTE and DOTI as well as DOTTG

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (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:

TK gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The chromosome number of these cells is 40 (stable aneuploid karyotype, 2n = 40).
The cells were stored as frozen stock cultures in liquid nitrogen.
Each new stock culture is checked for mycoplasma contamination, which was absent.
Cell culturing : The L5178Y cells were grown in culture medium consisting of RPMI 1640 medium (with HEPES and Glutamax-I) supplemented with heat-inactivated horse serum (10 % v/v for growing in flasks, and 20 % for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin.
On the day of exposure, the growth rate (doubling time of 9-14 h) and viability (>90 %; by trypan blue exclusion) of the cells were checked.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate

Test concentrations with justification for top dose:

FIRST test : Without S9 (24hr of treatment): 0.006-10 µg/ml ; With S9 (4 hr of treatment) : 0.01-20 µg/ml
SECOND test : Without S9 (4hr of treatment): 0.86-20 µg/ml; Without S9 (24hr) : 0.02-0.40µg/ml; With S9 : 1.6-60µg/ml
THIRD test : Without S9 (24hr of treatment): 0.007-0.5 µg/ml ; With S9 (4hr of treatment): 1.4-100 µg/ml
With S9 (Third test, 4 hr of treatment) : 1.4-100 µg/ml (11 concentrations)

Vehicle / solvent:

Ethanol.
The test substance was diluted in ethanol to concentrations ranging from 0.04 to 10 mg/ml DOTE. From these stock solutions serial dilutions in ethanol were made and from each of these 0.1 ml were added to a final volume of 10 ml culture medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(ethanol)

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(ethanol)

True negative controls:

no

Positive controls:

yes

Remarks:

with S9 mix

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 4 hours or 24 hours (37°C, 5% CO2)
- Exposure duration: 20-24, and 44-48 hours (37°C, 5% CO2)
- Expression time (cells in growth medium): doubling time of 9-14 hours
- Selection time (if incubation with a selection agent): no data

NUMBER OF REPLICATIONS: single culture for each dose, 2 for negative control group, 1 for positive control group

NUMBER OF CELLS EVALUATED: 2000/well

DETERMINATION OF CYTOTOXICITY
- Method: by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG)

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no

Evaluation criteria:

The following criteria were used to validate the data obtained in the gene mutation assay (Cole et al., 1990; Aaron et al. 1994; Clive et al., 1995):
a) the average cloning efficiency of the negative controls should not be less than 60 % or more than 140 %.
b) the average suspension growth of the negative controls should be between 8 and 32.
c) the average mutant frequency of the negative controls should fall within the range of 40-300 TFT-resistant mutants per 1,000,000 clonable cells.
d) the mutant frequency of the positive controls should be higher than 400 TFTresistant mutants per 1,000,000 clonable cells, and should be at least twice that of the corresponding negative control.
e) unless the test substance shows no cytotoxicity at the highest possible concentration (determined by its solubility, pH and osmolar effects), the highest concentration should result in a clear cytotoxic response. The RTG value of one of the data points should be between 10 and 20%, or one data point between 1 and 10% and another between 20 and 30%.

The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.
The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test substance concentrations.

Statistics:

no statistical analysis was performed.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

But increase at 72 µg/ml but this increase is not indicative for mutagenicity.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Results without S9 (see tables 1 and 2) :
In the absence of S9-mix three assays were performed in which mutagenicity was evaluated; two with 24 hours exposure and one with 4 hours exposure. In the first assay with 24 hours exposure the RTG at the highest concentration was 22% (not between 10 and 20%) and only six concentrations could be evaluated due to severe cytotoxicity. In the second assay with 24 hours exposure (results obtained in third test), sufficient cytotoxicity was observed at the highest concentration (RTG of 13%) and nine concentrations could be evaluated resulting in clear to hardly any cytotoxic response. In the assay with 4 hours exposure (second test) at and above 5 μg/ml no clear dose response in cytotoxicity was observed, however, sufficient toxicity (RTG between 10 and 20%) was observed for a valid test.
In none of the three assays performed in the absence of S9-mix any increase in mutant frequency (MF) by more than 88 or 126 mutants per 1,000,000 clonable cells, i. e. no equivocal or positive response, compared to the negative control was observed at any dose level.

Results with S9 (see table 3) :
In the presence of S9-mix two assays were performed in which mutagenicity was evaluated. In the first assay not sufficient cytotoxicity was observed at the highest concentration of 20 μg/ml (RTG was 38%; not between 10 and 20%). In the second assay (results obtained in third test), although relatively small intervals between the concentrations were used, none of the concentrations resulted in an RTG between 10 and 20%. Above 44 μg/ml no dose response in cytotoxicity was observed. The RTG at 85 μg/ml was 30% whereas at 72 μg/ml the RTG was 5%. In addition, the relative suspension growth (RSG) at 61 and 85 μg/ml were 24 and 21%, respectively. This means that already in this phase almost 80% cytotoxicity was observed, however, since the initial cell yield and cloning efficiencies at these concentrations were above 100% (which is very unusual) this resulted not in RTG values below 20%. In the presence of S9-mix at 72 μg/ml the mutant frequency was significantly increased by 238 mutants per 1,000,000 clonable cells compared to the negative control. Since relatively small intervals (0.85) were used and the increase was observed at a single concentration causing more than 90% cytotoxicity, it is concluded that this increase is not indicative for mutagenicity.

Colony sizing :
Colony sizing was performed in the third test at 2 concentrations in the presence of S9-mix, since a positive response in mutant frequency was observed. Both at 72 and 85 μg/ml relatively more small then large colonies were formed; 68% and 65% compared to 32% and 35%, respectively.
Since the increase in mutant frequency at 72 μg/ml was considered to be not relevant, no conclusions were drawn from these findings.

Positive and negative controls :
Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively; Ethanol served as negative control. The negative controls were within historical background ranges and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls.

RANGE-FINDING/SCREENING STUDIES:
Prior to the main study two dose range finding studies were performed; single cultures were exposed for 24 hours in the absence of S9-mix to 5 concentrations of DOTE ranging from 312 to 5000 μg/ml and 9.4 to 150 μg/ml, in the first and second study, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative controls were within historical background ranges and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: mouse lymphoma L5178Y cells

Table 1 : Summary of the results after 24 hours exposure in the absence of S9-mix.

Dose (µg/ml)	First assay (first test)		Dose (µg/ml)	First assay (first test)
	MF	RTG		MF	RTG
			0.36	115	13
			0.31	64	29
			0.26	63	36
0.2	89	22	0.22	93	60
0.1	72	81	0.16	115	79
0.05	76	95	0.11	59	100
0.03	57	84	0.079	93	99
0.01	74	96	0.055	80	108
0.006	58	81	0.027	72	110
0	93*	100*	0	74*	100*

*Mean of duplicate cultures.

MF = mutant frequency ; RTG = Relative total growth.

Table 2 : Summary of the results after 4 hours exposure in the absence of S9-mix.

Dose (µg/ml)	Second test
	MF	RTG
20	105	20
17	88	9
14	154	11
10	121	20
7.3	72	21
5.1	81	21
3.6	124	30
1.8	105	42
0.86	76	64
0	66*	100*

*Mean of duplicate cultures.

MF = mutant frequency ; RTG = Relative total growth.

Table 3 : Summary of the results after 4 hours exposure in the presence of S9-mix.

Dose (µg/ml)	First assay (first test)		Dose (µg/ml)	Second assay (third test)
	MF	RTG		MF	RTG
			85	143	30
20	117	38	72	318	5
14	105	49	61	89	39
9.8	83	60	44	111	35
6.9	80	80	32	140	42
3.4	73	88	22	71	57
1.6	75	93	11	103	85
0.8	69	101	5.4	83	89
0.4	87	84	2.7	95	83
0.2	78	93	1.4	100	71
0	86*	100*	0	80*	100*

*Mean of duplicate cultures.

MF = mutant frequency ; RTG = Relative total growth.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that under the conditions used in this study, the test substance DOTE is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.
The test substance DOTE was tested and evaluated for mutagenicity in both the absence and presence of metabolic activation (S9-mix). In the absence of S9-mix beside treatment for 4 hours, an extended treatment for 24 hours was used. In the three assays performed sufficient cytotoxicity was observed to validate the concentrations used and in none of the assays any indication for a mutagenic potential was observed.

Executive summary:

The test substance DOTE was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). Three tests were conducted; in the first and third test single cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively; in the second test cultures were treated for 24 hours and 4 hours in the absence of S9-mix and 4 hours in the presence of S9-mix. The test substance was dissolved in ethanol prior to testing.

The highest concentrations of DOTE evaluated for mutagenicity in the absence of S9-mix, were 20 μg/ml and 0.36 μg/ml at 4 and 24 hours exposure, respectively. In the presence of S9-mix and 4 hours exposure, the highest concentration evaluated was 85 μg/ml. The maximum concentrations were limited by cytotoxicity.

DOTE was cytotoxic in both the absence and presence of S9-mix. In the absence of S9-mix and 24 hours exposure cytotoxicity, resulting in a reduction in initial cell yield and/or suspension growth, was observed at and above 0.1 μg/ml. The relative total growth (RTG) at the highest concentration evaluated in the first and third test were 22% and 13%, respectively. In the absence of S9-mix and 4 hours exposure cytotoxicity was observed at all concentrations included (as from 0.86 μg/ml). The relative total growth (RTG) at the highest concentration evaluated in the second test was 20%.

In the presence of S9-mix cytotoxicity was observed at and above 1.4 μg/ml. The relative total growth (RTG) at the highest concentration evaluated in the first and third test were 38% and 30%, respectively. In the absence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. In the presence of S9-mix at 72 μg/ml the mutant frequency was significantly increased by 238 mutants per 1,000,000 clonable cells compared to the negative control. Since relatively small intervals (0.85) were used and the increase was observed at a single concentration causing more than 90% cytotoxicity compared to six concentrations causing 50-70% cytotoxicity which showed no increase in mutant frequency, it is concluded that this increase is not indicative for mutagenicity. It is very lakely a secondary effect to cytotoxicity and thus not indicative for mutagenicity.

Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and presence of the S9-mix, respectively; ethanol (vehicle) served as negative control. The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls.

It is concluded that under the conditions used in this study, the test substance DOTE isnot mutagenicat the TK-locus of mouse lymphoma L5178Y cells.

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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

not reported.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in accordance with generally accepted scientific principles. Justification of read-across: Dioctyltin bis (IOMA) and dioctyltinnbis (2-EHMA) are isomers of the same compound and are expected to be chemically and toxicologically equivalent.

Justification for type of information:

Read across from the source substance DOTE / DOTI to the target substance DOTTG is based on:
1.Structural Similarity: both belong to the category of di-n-octyltin compounds with a thio bond (COLLA 2018)
2.Common raw materials: DOTTG is always manufactured in the same step together with DOTE /DOTI and further processed as a 2.5-10 % solution in DOTE or DOTI based on the same raw materials
3.Hydrolysis: DOTTG as a pure substance hydrolysis under simulated conditions to Dioctyltin dichloride (DOTC). In DOTE solution the latter spontaneously reacts with the access of DOTE to form the monochloro mercapto acetic ester (DOTEC). So DOTTG in DOTE solution under simulated gastric conditions results in the same metabolite as DOTE, the monochloro mercapto acetic ester (DOTEC) which is stabilized in a cyclic structure that is very similar to the DOTTG structure.
4.Applicational fate: both substances react during their technical function with HCl released from PVC and degrade themselves to the to the same mono-chloro mercato acetic ester DOTEC.
5.Supply chain considerations: The solid DOTTG It is never isolated and marketed as a pure substance only as a 2.5-10 % solution in DOTE or DOTI
6.Hazard and risk considerations: Since DOTE is already classified in the highest hazard categories it seems not to appears to be inappropriate to conduct additional animal studies on DOTTG. The further regulatory fate of DOTTG, such as possible authorization / restriction, is closely linked to DOTE. The registrants have adopted the DOTE classification of the RAC 47 opinion as classification for DOTTG.
7.Further considerations: Source and target substance have been chosen by The Netherlands CA as members of a group of “disubstituted organotins with a thio bond (S-ligands)”. This group has been subject to a pilot project (COLLA March 2017–March 2018)
In 1999 the Scientific Committee on Food (SCF, 1999) evaluated octyltin compounds for food contact materials and established a group tolerable daily intake (TDI) for di-n­ octyltins (DOT) of0.0006 mg/kg The group entry covers in this context the two source substances DOTE and DOTI as well as DOTTG

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

A preliminary toxicity study was conducted to obtain suitable dose levels for the micronucleus test. In the micronucleus test the test material was prepared in 1% methylcellulose and administered by oral gavage to 25 male and 25 female CFLP strain mice. The test material was administed in 2 equal administrations separated by an interval of 24 hours. Following the second dose the animals were observed for a further 6 hours before being sacrificed and samples of the femur bone marrow taken. The bone marrow smear was plated onto a microscope slide and prepard for examination, the incidence of micronucleated cells per 2000 polychromatic and 2000 normochromatic to polychromatic erythrocytes was determined for each animal.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: CFLP

Sex:

male/female

Details on test animals or test system and environmental conditions:

Breeder: Anglia Laboratory, Alconbury, Huntingdon, UK
Initial body weights: 19-23 g
Acclimitisation: one week
Housed in plastic disposable cage (5/cage)
Environmental conditions: maintained at 21 ± 2 ºC and 50 ± 5% relative humidity with 20 changes of air/hour
Photoperiod: illuminated by artificial light for 12 hours per day
Diet: Grain Harvester Anglia Lab animal diet and tap water ad libitum
Fasted overnight before treatment
Mice (CFLP) were pretested to determine target dose for micronucleus test.

Route of administration:

oral: gavage

Vehicle:

1% methylcellulose (also used as negative control)

Details on exposure:

Total dose (in 2 administrations) were 2250, 4500, 1900 mg/kg; positive control mitomycin C = 14 mg/kg

EXPERIMENTAL DESIGN
Group Material Conc mg/mL Dose mL/10g Dose mg/kg Total (mg/kg) M F
1 control (veh) - 0.1 - - 5 5
5 ZK 30434 112.5 0.1 1125 2250 5 5
6 ZK 30434 225 0.1 2250 4500 5 5
7 ZK 30434 450 0.1 4500 9000 5 5
8 control (pos) 0.7 0.1 7 14 5 5

Duration of treatment / exposure:

30 hours (doses at 0 and 24 hours, sacrifice 6 hours after last dose)

Frequency of treatment:

2 doses separated by an interval of 24 hours.

Post exposure period:

6 hours

Remarks:

Doses / Concentrations:
2250, 4500, and 9000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5/sex/dose (10)

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C in physiological saline by IP injection of 14 mg/kg (total of 2 doses)

Tissues and cell types examined:

Animals were sacrificed at 6 hours after the last dose by cervical dislocation. Femurs were dissected out from each animal, cleared of tissue and one epiphysis removed from each bone

Details of tissue and slide preparation:

A direct bone marrow smear (from epiphysis from each femur) was made and placed on a methanol-cleaned slide. Smears were air dried and fixed in methanol overnight. After fixation the smears were air-dried and placed in buffered distilled water (pH 6.8) for 10 minutes prior to staining in Giemsa (dilution factor, 1 part Giemsa:9 parts buffered distilled water) for 10 minutes. After rinsing in buffered water, slides were air-dried, then defatted in xylene for 10 minutes and mounting in DPX. Stained smears were examined by light microscopy.

Evaluation criteria:

Incidence of micronucleated cells per 2000 polychromatic and 2000 normochromatic ertyhrocytes per animal was deterimned for each animal.

Statistics:

Ratios were analyzed by Barlett's test < 0.001 using non-paraetric methods based on Kruskal-Wallis mean ranks. These methods are more than robust against inequality of variance than classical variance methods. Postive control groups dosed with mitomycin C and the groups dosed with ZK 30 434 were compared to vehicle control using the non-parametric equivalent of the least significant differences.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

bone marrow depression

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Under the conditions studied, ZK 30 434 failed to show any evidence of mutagenic potential when administered orally in this test procedure. However, evidence of bone marrow depression was observed.

Group mean number of micronucelaetd cells per 2000 normochromatic erythrocytes and 2000 polychromatic erythrocytes per animal and the group mean ratio.

		Normochromatic (N)		Polychromatic (P)		Ratio N:P erythrocytes
Material	Total dose (mg/kg)	Mean	Range	Mean	Range	Mean	Range
Vehicle control	-	2.2	0-6	2.8	1-6	1.09	0.93-1.22
ZK 30 434	2250	2.3	0-6	2.5	0-5	1.38	1.19-1.69
ZK 30 434	4500	2.4	0-5	2.4	0-6	1.72**	1.47-2.09
ZK 30 434	9000	2.3	0-6	2.6	1-5	3.93***	2.56-5.39
Positive control	14	11.4***	8-15	89.5***	74-115	10.46***	7.75-15.38

** p<0.01

*** p< 0.001

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions studied, ZK30434 failed to show any evidence of mutagenic potential when administered orally in this test procedure.
However, evidence of bone marrow depression was observed, which is an indication that the test item reached the bone marrow.

Executive summary:

The effect of ZK 30 434 on the incidence of micronucleated polychromatic erythrocytes from the bone marrow of mice was tested with doses of 2250, 4500, and 9000 mg/kg bodyweight administered by oral gavage in two equal doses separated by an interval of 24 hours. Negative control (1% methycellulose by oral gavage) and postive control group (14 mg/kg mitomycin C by injection) were run concurrently. After 6 hours of the second dose bone marrow smears were examined for the presence of micronuclei in 2000 polychromatic and 2000 normochromatic erythorcytes per mouse.

At all doses of ZK 30 434 both the group mean micronucleated cell counts were comparable with the concurrent control values. The ratio of normochromatic to polychromatic cells was comparable to controls for 2250 mg/kg group and significantly higher for 4500 and 9000 mg/kg groups (by Kruskal-Wallis method). Postive control produced expected increase in micronucleated cell count and ratio. It was concluded that ZK 30 434 failed to show any evidence of mutagenic potential when adminsitered orally in the test procedure. However, evidence of bone marrow depression was observed, which is an indication that the test item reached the bone marrow.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The genetic toxicity endpoint is addressed with data from an in vitro gene mutation study in bacteria which was conducted on the registered substance together with an in vitro study in bacteria, an in vitro gene mutation study in mammalian cells and an in vivo cytogenicity test which were all conducted with the read across substance, dioctyltin bis-(isooctylthioglycolate). All studies were performed under GLP and in accordance with standardised guidelines; they were all performed and reported to a high standard. As such, the study with the registered substance was assigned a reliability score of 1 according to the criteria of Klimisch (1997) and the three studies which were performed with the read across substance, dioctyl tin oxide, were assigned a reliability score of 2. When considered together, the data are considered suitable for assessment as an accurate reflection of the registered substance.

 

In vitro Gene Mutation Study in Bacteria

In the key study (Thompson, 2013), the potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material in two independent experiments, using either the plate incorporation or pre-incubation method, at five dose levels, both with and without metabolic activation. The dose levels assessed were 50, 150, 500, 1500 and 5000 µg/plate. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains. The test material was considered to be non-mutagenic under the conditions of this test.

 

 

In vitro Gene Mutation in Mammalian Cells

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. 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 up to a maximum concentration of 902.50 µg/mL.

 

In vivo Cytogenicity Test in Mammalian Cells

The key study, Hossack (1980), was performed in accordance with the standardised guideline OECD 474 and in line with GLP. The test material 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.

 

The available data are considered to be complete and the conclusion, non-mutagenic, was taken forward for risk assessment.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the 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.