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Toxicological information

Genetic toxicity: in vitro

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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:
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


Cross-reference
Reason / purpose:
read-across source

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
-Purity : 96.3%
-Appearance : colourless liquid
-Batch number: ESOC21.20091
Expiry date : 13 november 2010

Method

Target gene:
TK gene
Species / strain
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.
Controlsopen allclose all
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.

Results and discussion

Test resultsopen allclose all
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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.