Dimethylbis(oleoyloxy)stannane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read-across to a mixture of DMTC (Dimethyltin dichloride CAS No. 753 -73 -1) and MMTC (Monomethyltin chloride CAS No. 99-16-8)

- In vitro gene mutation studies in bacteria (Holmes 1990a & 1990b)

The test material was not considered to be mutagenic when tested according to the procedures used in these assays.

- In vitro chromosome aberration study in mammalian cells (Blachman 1990)

Under the conditions of the study it was concluded that the test material induced chromosomal aberrations in cultured human peripheral lymphocytes in the presence but not in the absence of metabolic activation.

- In vitro gene mutation study in mammalian cells (Bakke 1990)

The test material did not induce a concentration-dependent increase in mutant frequency either without or with S9 and was evaluated as negative under both activation conditions.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 April 1990 - 19 July 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Percentage of DMTC is only 72%. 28% is MMTC.

Reason / purpose for cross-reference:

other: read across: target

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine auxotrophs

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: histidine auxotrophs

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9

Test concentrations with justification for top dose:

Range finding assay: 10, 50, 100, 500, 1000, and 5000 µg/plate
First mutagenicity assay: 10, 50, 100, 500, 1000, and 5000 µg/plate
Second mutagenicity assay: 5, 10, 50, 100, 500, and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionized water for the test material; DMSO for the positive control substances.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Deionized water for the test material; DMSO for the positive control substances

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: The cultures are allowed to sit unshaken for 2 to 4 hours, then gently shaken (100 rpm) for 11 to 14 hours at 37 °C.
- Exposure duration: After the top agar has set, the plates are incubated at 37°C for about 48 hours.
- Expression time (cells in growth medium): set, the plates are incubated at 37°C for about 48 hours. The histidine-independent revertant colonies are counted following the incubation period; however, if the plates cannot be immediately evaluated, they are refrigerated at 4°C until they can be counted.

NUMBER OF REPLICATIONS: with three plates per dose level, both with and without a mammalian liver metabolic activation.

NUMBER OF CELLS EVALUATED: 10^8

DETERMINATION OF CYTOTOXICITY
Toxicity is evidenced by several phenomena: a substantial decrease in the number of revertant colonies on the test plates compared with the controls, the clearing or absence of the background lawn growth, or the formation of pinpoint nonrevertant colonies.

Evaluation criteria:

The histidine-independent revertant colonies are counted using an automated colony counter. When accurate counts cannot be obtained (e.g., because of precipitation on the plates), the colonies are counted manually using an electric probe colony counter. The actual numbers of revertant colonies observed and the condition of the background lawn growth are presented in the attached tables. No designation or "-7" indicates a normal background lawn. Toxicity is designated by "-5" for background lawn thinning, "-4" for pinpoint colonies, and "-3" for absence of bacterial growth.

CRITERIA FOR INTERPRETATION
Positive - A test material is considered a mutagen when it induces a reproducible, dose-related increase in the number of revertants in one or more strains. This increase should occur for at least three consecutive dose levels.

Negative - A test material is considered a nonmutagen when no dose-related increase in the number of revertants is observed in at least two independent experiments.

Inconclusive - When a test material cannot be identified clearly as a mutagen or nonmutagen, the results are classified as inconclusive.

Statistics:

The results are a tabulation of the number of colonies appearing on the plates. Mean and standard deviation values were, determined for the number of revertants at each dose level.

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:

cytotoxicity

Remarks:

≥ 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The Ames Salmonella/microsome assay was used to evaluate the ability of mixes of methyltin compounds to induce genetic damage in S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100. The assays were performed using the standard plate incorporation procedure, in both the presence and absence of a rat liver metabolic activation system. The presence of the appropriate genetic characteristics was verified for the strains used in this study. The results of the controls were acceptable for all assays.

We conducted the preliminary assay with tester strain TA100 in the presence and absence of metabolic activation containing 4% S-9. The doses used were 10, 50, 100, 500, 1000, and 5000 µg/plate. Toxicity was indicated with revertant counts below the spontaneous background level at the 1000 µg dose and thinning of the background lawn at 5000 µg, both in the presence and absence of metabolic activation. Based on these results, the first assay was conducted at the same dose levels, with all five tester strains, in the presence and absence of 4% S-9. There was no evidence of a dose-related increase in the number of histidine revertants with any tester strain. Toxicity was noted with all tester strains at 5000 µg as background lawn thinning or complete absence of bacterial growth. Some toxicity was also noted at 1000 µg. The second assay was conducted at dose levels of 5, 10, 50, 100, 500, and 1000 µg/plate, and the .percent of S-9 in the metabolic activation mixture was increased to 10% due to the lack of response in the first assay. Again, there was no dose-related increase in the number of histidine revertants with any tester strain. Toxicity was observed at the 1000 µg dose with some strains, indicated by a decrease in the number of revertant colonies below background levels.

Conclusions:

Mixes of methyltin compounds were not mutagenic when tested according to the procedures used in this assay.

Executive summary:

Mixes of Methyltin Compounds were examined for mutagenic activity in the Salmonella/microsome assay using the standard plate incorporation procedure with Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, in both the presence and absence of an Aroclor 1254-induced rat-liver metabolic activation system. No reproducible, dose-related increase in the number of histidine revertant colonies was observed with any tester strain. Therefore, mixes of methyltin compounds were not mutagenic when tested according to the procedures used in this assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance dimethyltin dichloride (DMTC) (EC Number 212-039-2, CAS Number 753-73-1), see attached justification.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 1990 - 25 July 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Percentage of DMTC is only 72%. 28% is MMTC.

Reason / purpose for cross-reference:

other: read across: target

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Tryptophan

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophan auxotroph

Metabolic activation:

with and without

Metabolic activation system:

S-9 liver fraction from male Sprague-Dawley rats given a single 500-mg/kg intraperitoneal injection of Aroclor 1254

Test concentrations with justification for top dose:

10, 50, 100, 500, 1000, and 5000 µg/plate.

Vehicle / solvent:

For test article: Water, deionized
For positive control substances: Dimethylsulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

For test article: Water, deionized For positive control substances: Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: The culture is allowed to sit unshaken for 2 to 4 hours, then gently shaken (100 rpm) for 11 to 14 hours at 37°C.
- Exposure duration: After the top agar has set, the plates are incubated at 37 °C for about 48 hours.
- Expression time (cells in growth medium): The tryptophan-independent revertant colonies are counted following the incubation period; however, if the plates cannot be immediately evaluated, they are refrigerated at 4°C until they can be counted.

NUMBER OF REPLICATIONS: The test article was assayed twice, with three plates per dose level, both with and without a mammalian liver metabolic activation mixture.

NUMBER OF CELLS EVALUATED: 10^8

DETERMINATION OF CYTOTOXICITY
Toxicity is evidenced by several phenomena: a substantial decrease in the number of revertant colonies on the test plates compared with the controls, the clearing or absence of the background lawn growth, or the formation of pinpoint nonrevertant colonies.

Evaluation criteria:

The tryptophan-independent revertant colonies are counted using an automated colony counter. When accurate counts cannot be obtained (e.g., because of precipitation on the plates), the colonies are counted manually using an electric probe colony counter. The actual numbers of revertant colonies observed and the condition of the background lawn growth are presented in the attached tables. No designation or "-7" indicates a normal background lawn. Toxicity is designated by "-5" for background lawn thinning, "-4" for pinpoint colonies, and "-3" for absence of bacterial growth.

CRITERIA FOR INTERPRETATION
Positive - A test material is considered a mutagen when it induces a reproducible, dose-related increase in the number of revertants. This increase should occur for at least three consecutive dose levels.

Negative - A test material is considered a nonmutagen when no dose-related increase in the number of revertants is observed in at least two independent experiments.

Inconclusive - When a test material cannot be identified clearly as a mutagen or nonmutagen, the results are classified as inconclusive.

Statistics:

The results are a tabulation of the number of colonies appearing on the plates. Mean and standard deviation values were determined for the number of revertants at each dose level.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The E. coil WP2 (uvrA)/microsome assay was used to evaluate the ability of mixes of methyltin compounds to induce genetic damage in E. coli strain WP2 (uvrA). The assays were performed using the standard plate incorporation procedure, in both the presence and absence of a rat-liver metabolic activation system. The presence of the appropriate genetic characteristics was verified for the strain used in this study. The results of the controls were acceptable for all assays.

The first assay was conducted in the presence and absence of metabolic activation containing 4% S-9. The doses used were 10, 50, 100, 500, 1000, and 5000 µg/plate. There was no evidence of a dose-related increase in the number of tryptophan revertants. Slightly low revertant counts at the 5000 µg dose may indicate toxicity. Based on these results, the second assay was conducted at the same dose levels, but the percent of S-9 in the metabolic activation mixture was increased to 10% due to the lack of response in the first assay. Note that the portion of the second assay with 10% S-9 was repeated on a separate day due to unacceptable 2-anthramine control values. There was no dose-related increase in the number of tryptophan revertants, and low revertant counts were observed at the 5000 µg dose level, confirming toxicity at this dose.

Conclusions:

In conclusion, mixes of methyltin compounds were not detected as being mutagenic when tested according to these procedures.

Executive summary:

Mixes of methyltin compounds were examined for mutagenic activity in the the Escherichia coli WP2 (uvrA)/microsome plate incorporation assay performed using the standard plate incorporation procedure with Escherichia coli strain WP2 uvrA, in both the presence and absence of an Aroclor 1254-induced rat-liver metabolic activation system. A reproducible, dose-related increase in the number of tryptophan revertant colonies was not observed. Therefore, mixes of methyltin compounds was not mutagenic when tested according to the procedures used in this assay.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance dimethyltin dichloride (DMTC) (EC Number 212-039-2, CAS Number 753-73-1), see attached justification.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Read-across to a mixture of DMTC (Dimethyltin dichloride CAS No. 753 -73 -1) and MMTC (Monomethyltin chloride CAS No. 99-16-8)

- In vivo mammalian cell study (Hamilton 1993)

Under the conditions of the test, the test material was not a genotoxic agent in the livers of male F-344 rats after oral treatment.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5th April - 14th June 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Reason / purpose for cross-reference:

other: read across: target

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, 401 South New Hope Road, Raleigh, North Carolina, 27610, USA
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: 161.9-199.6 (range-finding study); 195.8-228.3 g (first definitive UDS assay); 175.1-208.2 g (second definitive UDS assay)
- Assigned to test groups randomly: yes
- Fasting period before study: NDA
- Housing: no more than 3 per cage in polycarbonate cages containing hardwood-chip bedding.
- Diet (e.g. ad libitum): Purina Certified Rodent Chow No. 5002 ad libitum.
- Water (e.g. ad libitum): Deionized, UV-exposed tap water was provided ad libitum
- Acclimation period: ca. 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 58 - 78
- Humidity (%): 32 to 89
- Photoperiod (hrs dark / hrs light): 12/12

The temperature and humidity in the rooms housing the animals was not within the recommended range (64.4-78.8°F, and 40-70% humidity). The temperature of the room housing the rats during the range-finding assays fluctuated between 58 and 79°F. This circumstance did not affect the outcome of the study because the dose levels for the second definitive UDS assay, which provided the UDS data in this report, were based on the pattern of animal deaths that occurred in the first definitive UDS assay, not the range-finding assays. The temperature and humidity in the room housing the rats used during the first and second definitive assays was approximately 64.5 to 71°F with 40 to 55% humidity, and 67 to 73°F with 56 to 69% humidity, respectively. It is not believed that these conditions affected the outcome of the study.

The humidity of the room housing the animals dropped as low as 32% and sharp spikes reaching higher than 70% occurred while the animal room was being washed down. It is not believed that these circumstances affected the outcome of the study.

IN-LIFE DATES: From: 26th April 1993 To: 14th June 1993

Route of administration:

oral: gavage

Vehicle:

Water

Details on exposure:

The route of dosing and the method of test article administration were chosen to maximize exposure of the liver to the test article and controls. All test article dosing solutions were prepared within 1 hour before dosing.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Once

Post exposure period:

7 days (range finding study)
2 or 16 hours (UDS assays)

Remarks:

Doses / Concentrations:
25, 50, 100, 200 and 400 mg/kg
Basis:
actual ingested
First range finding study

Remarks:

Doses / Concentrations:
600 and 800 mg/kg
Basis:
actual ingested
Second range finding study

Remarks:

Doses / Concentrations:
90, 175 and 350 mg/kg
Basis:
actual ingested
First definitive UDS assay

Remarks:

Doses / Concentrations:
50, 110 and 225 mg/kg
Basis:
actual ingested
Second definitive UDS assay

No. of animals per sex per dose:

First range finding assay: 3 males at each dose
Second range finding assay: 3 males at each dose
First definitive UDS assay (dosed 2 hours prior to sacrifice): 3 males at each dose, except negative control with 0
First definitive UDS assay (dosed 16 hours prior to sacrifice): 3 males at each dose, except highest dosage group with 4 and positive control with 0
Second definitive UDS assay (dosed 2 hours prior to sacrifice): 3 males at each dose, except negative control with 0
Second definitive UDS assay (dosed 16 hours prior to sacrifice): 3 males at each dose, except highest dosage group with 4 and positive control with 0

Control animals:

yes

Positive control(s):

Positive control: Dimethylnitrosamine
- Route of administration: oral
- Doses / concentrations: 10 mg/kg bw

Tissues and cell types examined:

Primary cell cultures were obtained from livers of treated male rats. Livers were perfused in situ with a collagenase solution. Isolated hepatocytes were combed out of the perfused livers, and cell concentrations were calculated from a hemocytometer count. Viable cells per millilitre were determined by the trypan blue exclusion method, and approximately 2.5-5.0 x 10^5 cells were inoculated into six-well culture dishes (containing coverslips) in Williams medium E (WE, pH 6.8) supplemented with 2 mM l-glutamine, 50 µg/ml gentamicin sulfate, and 10% fetal bovine serum. After 1.5 to 2.0 hours of incubation in a humidified atmosphere at 37°C, 5% CO2, the cultures were washed to remove nonviable cells (those not attached to the coverslips). All washes and subsequent culturing were performed in serum-free medium. Cultures were incubated in WE containing 10 µCi/ml 3H-methylthymidine (specific activity, approximately 80 Ci/mmol) for 4 hours at 37°C, 5% CO2, followed by 14 to 18 hours in WE containing 0.25 mmol unlabeled thymidine.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
Death data from both range-finding assays were analysed together to estimate the LD50 at 443 mg/kg bw methyltin chlorides. The LD calculated by an SRI generated program on the VAX-3100 (LD50) that uses a point estimate of the linear interpolation of the log doses. The dose levels of methyltin chloride in the first definitive in vivo-in vitro hepatocyte DNA repair (UDS) assay were set at 90, 175 and 350 mg/kg bw on the basis of the results of the dose range-finding assays (approximately 20, 40 and 80% of the estimated LD50).

TREATMENT AND SAMPLING TIMES
Animals were dosed once, either 2 or 16 hours prior to sacrifice.

DETAILS OF SLIDE PREPARATION
The cultures were washed with culture medium, swelled in 1% sodium citrate, fixed in 1:3 glacial acetic acid:ethanol, and washed with deionized water. The coverslips were mounted on slides, dipped in Kodak NTB-2 emulsion, and exposed at -20°C for approximately 7 days before development. Cells were stained with 1% methyl green pyronin Y.

METHOD OF ANALYSIS
Measurement of UDS:
Quantitative autoradiographic grain counting was accomplished by use of an ARTEK Model 880 or 980 colony counter interfaced with a Zeiss Universal microscope via an ARTEK TV camera Data were fed directly into a VAX 3100 computer via an ARTEK BCD-RS232 Omni-Interface using an SRI-generated data collection program (ARTEK). Thirty morphologically unaltered cells on a randomly selected area of the slide were counted. The highest count from two nuclear-sized areas over the most heavily labelled cytoplasmic areas adjacent to the nucleus was subtracted from the nuclear count to give the net grains/nucleus (NG). The percentage of cells in repair (% IR) indicates the extent of the response throughout the liver (cells in repair are those showing at least 5 NG). For each dose, a minimum of 3 slides were scored for each animal The data were summarised by SRI-generated programs on the VAX 3100 and the average NG and % IR were calculated for each dose group.

Evaluation criteria:

CRITERIA FOR A VALID ASSAY
Slide Evaluation: Slides were evaluated under a light microscope after the autoradiographic procedures. At that point, the groups were evaluated for UDS and unscorable groups (if any) were determined.

Unscorable Slide Criteria: Unscorable slides may result for any of the following reasons: (1) animal death after dose with test material, (2) poor or no cell attachment, or (3) pyknotic cells or other obvious morphologic changes.

DATA ANALYSIS AND INTERPRETATION
Criteria for a Valid Assay: The UDS data generated were considered acceptable if the vehicle-control data were within historical ranges (-1 mean NG, ≤10% IR) and if positive controls had significant elevations in NG and % IR.

Statistics:

When results did not clearly establish a positive or negative response, the presence of a dose response, the frequency distribution of cellular responses, increases in the % IR, the nuclear and cytoplasmic grain counts, and the reproducibility of data among animals were all considered. The following decision tree was used for classifying the response of the test material:

1. If there was no absolute increase in the nuclear counts of test groups over vehicle controls, the test material was considered "negative."
2. If there was no absolute increase in the nuclear counts and all dose groups had less than 0 NG but the % IR of individual dose groups was elevated, the net grains of cells in repair (NGIR, the average NO value of all cells with ≥5 NG) was evaluated.
a. If the NGIR was ≤10, the test material was considered "negative."
b. If the NGIR was ≥10, this value suggests that a small subpopulation of cells may have been preferentially affected by the test material but that most cells did not respond; therefore, the test article was considered "equivocal."
3. If there were increases in nuclear counts, NG and % IR for individual animals in one or more dose groups (dose-related or nondose-related) but the response was not observed in all animals in the dose group, the results of the test were considered "inconclusive."
4. If other unusual biological effects occurred that confounded the interpretation of the data to a point where a clear determination could not be rendered, the results of the test were considered "inconclusive".

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

One rat from the positive control group yielded unscorable slides because of poor cell attachment and pyknotic nuclei and nuclei not associated with cytoplasm.

Additional information on results:

RANGE-FINDING ASSAYS
In the first range-finding assay one rat in the 400 mg/kg dose group died on the second day after dosing. Clinical signs of rats dosed with methyltin chloride included rough fur, diarrhea, weakness, humped back, difficulty breathing, bloody nose, lacklustre eyes, and blood around eyes. The two surviving rats in the high-dose group had extreme weight loss (rats weighed 52 and 66% of their Day 0 weight when they were sacrificed on Day 7). All of the rats in the second range-finding assay died before their scheduled sacrifice. Clinical signs of rats dosed with 600 or 800 mg/kg bw methyltin chloride included rough fur, weakness, diarrhea, lacklustre eyes, humped back, hypoactive, and blood around eyes. The LD50 of methyltin chloride was estimated at approximately 443 mg/kg bw by summarising results of both range-finding assays together.

UDS ASSAY
Dose levels for the first definitive in vivo-in vitro hepatocyte DNA repair (UDS) assay were set at 90, 175 and 350 mg/kg bw (approximately 20, 40 and 80% of the LD50). Three rats from the 16 hour 350 mg/kg methyltin chloride dose group were found dead on the morning after dosing. All rats from the 16 hour 175 mg/kg dose-group had rough fur on the morning after dosing. One rat from the 2 hour 90 mg/kg dose-group and two rats from the 2 hour 350 mg/kg dose-group had diarrhea. The surviving rat from the 16 hour 350 mg/kg dose-group had rough fur, humped back, diarrhea, labored breathing, and was hypoactive. The first definitive UDS assay was terminated after the first eight test animals produced insufficient viable cells for evaluation of UDS probably because of a technical error in the preparation of the cell culture medium.

A second definitive UDS assay was conducted using a new batch of cell culture medium. The LD50 was adjusted downward to approximately 278 mg/kg bw methyltin chloride based on the pattern of death observed in the first definitive assay. Dose levels for the second UDS assay were set at 50, 110 and 225 mg/kg bw (approximately 20, 40, and 80% of the adjusted LD50). One rat in the 16 hour 110 mg/kg dose-group was found the morning after dosing. The remaining rats in the 16 hour 110 and 225 mg/kg dose-groups had rough fur at the time of sacrifice. One rat from the 16 hour 225 mg/kg dose-group had diarrhea, and another rat from the high-dose group had a sore on its right eye at the time of sacrifice. Three rats from the 2 hour 225 mg/kg dose-group had rough fur and diarrhea at the time of sacrifice. Only three of the five rats dosed in the 16 hour high-dose group were evaluated for UDS. One rat from the positive control group yielded inscorable slides because of poor cell attachment and pyknotic nuclei and nuclei not associated with cytoplasm. Slides obtained from rats treated with the negative control 16 hour before sacrifice or 50, 110 or 225 mg/kg bw methyltin chlorides either 2 or 16 hour before sacrifice yielded slides with ≤6.1 mean net grains per nucleus (NG) and ≤2 percentage of cells in repair (% ER). In contrast, rats treated with 10 mg/kg bw DMN 2 hours before sacrifice yielded slides with 28.4 NG and 93% IR. These results indicate that the test material did not induce unscheduled DNA synthesis under the conditions of this study.

Table 1: Summary of Definitive UDS Assay

Treatment	Dose (mg/kg)	Time (hr)	n	NG (mean±S.E.)	% IR (mean±S.E.)	NGIR (mean±S.E.)
Control/water	---	16	3	-6.8 ± 0.7	1 ± 0	8.0 ± 1.6
Methyltin chloride	50	2	3	-7.4 ± 1.0	2 ± 1	6.6 ± 0.8
	50	16	3	-9.0 ± 0.4	0 ± 0	--- ± ---
	110	2	3	-7.7 ± 0.5	2 ± 1	7.2 ± 0.8
	110	16	2	-6.5 ± 0.4	1 ±1	6.4 ± 0.0
	225	2	3	-8.9 ± 0.2	0 ± 0	--- ± ---
	225	16	3	-6.1 ± 0.4	1 ± 0	9.1 ± 2.7
Dimethylnitrosamine	10	2	2	28.4 ± 6.0	93 ± 2	30.8 ± 5.9

Standard errors (S.E.) represent variation among animals

n = Number of animals

NG = Net grains/nucleus

% IR = Percentage of cells in repair (with at least 5 NG)

NGIR = Average net grains/nucleus of cells in repair

Conclusions:

Under the conditions of the test, methyltin chloride compound is not a genotoxic agent in the livers of male F-344 rats after oral treatment.

Executive summary:

The purpose of this study was to evaluate the ability of a mixtures of methyltin chloride compounds (methyltin chloride) to induce unscheduled DNA synthesis (UDS) in male Fischer-344 (F-344) rat hepatocytes after a single oral administration 16 or 2 hours before collection of liver specimens.

A dose range-finding assay was conducted to determine dose levels for the definitive UDS assay. Male rats approximately 8 weeks old (162-200 g) were dosed at 25, 50, 100, 200 or 400 mg/kg bw with a single oral dose on day 0 and were observed daily for morbidity or other clinical signs. No animals died within the first 24 hours so two more dose groups were added to the range-finding assay on day 1 (600 and 800 mg/kg bw) to ensure that animal death would be observed in the range-finding assay. On day 7, the surviving test animals were sacrificed and their internal organs were examined. One rat from the 400 mg/kg dose group and three rats each from the 600 and 800 mg/kg dose groups died before their scheduled sacrifices. The LD50 was estimated to be approximately 443 mg/kg bw.

Dose levels for the definitive UDS assay were set at 90, 175 and 350 mg/kg bw (approximately 20, 40 and 80% of the estimated LD. Male rats approximately 9 weeks of age (196-228 g) were dosed 2 or 16 hours before sacrifice with test material, vehicle control or the positive control (dimethylnitrosamine given 2 hours before sacrifice). All dose groups contained three rats except the 16 hour 350 mg/kg dose-group, which contained four rats. One extra rat was dosed to avoid loss of data because of animal death. Three rats from the 16 hour 350 mg/kg dose group were found dead on the morning after dosing. This experiment was terminated after the first eight test animals yielded insufficient viable hepatocytes to evaluate for UDS probably because of a technical error in the preparation of the cell culture medium. A second UDS assay was conducted using new cell culture medium.

Another LD50 was estimated to be approximately 280 mg/kg bw based on the pattern of death observed in the first UDS assay. Dose levels for the second UDS assay were set at 50, 110 and 225 mg/kg bw (approximately 20, 40 and 80% of the LD50). A second definitive UDS assay was conducted as described above using the lower dose levels. Male rats approximately 9 weeks of age (175-208 g) were given a single oral dose of the test or control articles 16 or 2 hours before sacrifice. All dose groups contained three animals except the 16 hour high-dose group, which contained five animals Two extra animals were dosed to avoid loss of data because of animal death. One rat in the 110 mg/kg 16 hour dose group was found dead the following morning and could not be evaluated for UDS.

Primary cultures of liver cells on coverslips were obtained from these rats and labelled in vitro with 3H-methylthymidine for 14 to 18 hours. These culture coverslips were coated with Kodak NTB-2 emulsion, exposed, developed, and stained, and the autoradiographic grain counts were quantitated. The net grains/nucleus and percentage of cells in repair were determined. The test article was considered positive if the mean net grain count (NG) was greater than zero and the percentage of cells in repair (IR) for that group was greater than 20%.

Hepatocytes from male rats given up to 225 mg/kg mixtures of methyltin chloride or the negative control 16 or 2 hours before evaluation of UPS yielded NG between -9.0 and -6.1 and ≤2% ER. In contrast, positive control rats given DMN two hours before liver collection yielded 28.4 NG and 93% ER.

Liver cells from male rats were monitored in vitro for unscheduled DNA synthesis (UDS) after these rats were given oral doses of methyltin chloride (50, 110 or 225 mg/kg) 16 or 2 hours before collection of liver specimens. Results indicate that methyltin chloride is not genotoxic in male F-344 rat hepatocytes.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance dimethyltin dichloride (DMTC) (EC Number 212-039-2, CAS Number 753-73-1), see attached justification.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

other: One rat from the positive control group yielded unscorable slides because of poor cell attachment and pyknotic nuclei and nuclei not associated with cytoplasm.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Read-across to a mixture of DMTC (Dimethyltin dichloride CAS No. 753 -73 -1) and MMTC (Monomethyltin chloride CAS No. 99-16-8)

- In vitro gene mutation study in bacteria

(Holmes 1990a)

The test material was examined for mutagenic activity in the Salmonella/microsome assay using the standard plate incorporation procedure with Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, in both the presence and absence of an Aroclor 1254-induced rat-liver metabolic activation system. No reproducible, dose-related increase in the number of histidine revertant colonies was observed with any tester strain. Therefore, the test material was not considered to be mutagenic when tested according to the procedures used in this assay.

(Holmes 1990b)

The test material was examined for mutagenic activity in the the Escherichia coli WP2 (uvrA)/microsome plate incorporation assay performed using the standard plate incorporation procedure with Escherichia coli strain WP2 uvrA, in both the presence and absence of an Aroclor 1254-induced rat-liver metabolic activation system. A reproducible, dose-related increase in the number of tryptophan revertant colonies was not observed. Therefore, the test material was not considered to be mutagenic when tested according to the procedures used in this assay.

- In vitro chromosome aberration study in mammalian cells (Blachman 1990)

The ability of the test material to induce chromosomal aberrations (CA) in cultured human peripheral lymphocytes in the presence and the absence of rat liver S-9 metabolic activation was determined in a study performed in line with OECD Guideline 473.

Dose range-finding trials were conducted in the absence and presence of metabolic activation. In the absence of metabolic activation, complete cytotoxicity (no metaphase cells seen) was noted at concentrations of 40 µg/mL and above. No suppression of the mitotic index (MI) or retardation of cell cycle was noted at 8 µg/mL. In the presence of metabolic activation, complete toxicity (no metaphase cells seen) was noted at doses of 200 µg/mL and above. A slight suppression of the MI was seen at 40 µg/mL. No retardation of the cell cycle was noted in the absence of metabolic activation.

In the aberration assay in the absence of metabolic activation, chromosomes were examined from cultured human peripheral lymphocytes at concentrations of 8 and 16 µg/mL with a 16 to 18 hour harvest and at a concentration of 32 µg/mL with a 28 to 30 hour harvest. In the aberration assay in the presence of metabolic activation, chromosomes were examined from cultured human peripheral lymphocytes exposed to test material at concentrations of 20 and 40 µg/mL with a 16 to 18 hour harvest and at a concentration of 80 µg/mL with a 28 to 30 hour harvest.

No significant increase in either the number of cells with structural chromosomal damage or the frequency of structural chromosome abnormalities per cell was noted in the absence of metabolic activation. A significant increase in both the number of cells with structural chromosomal damage and the frequency of structural chromosome abnormalities per cell was noted after exposure to doses of 40 and 80 µg/mL in the presence of metabolic activation.

Therefore, under the conditions of the study it was concluded that the test material induced chromosomal aberrations in cultured human peripheral lymphocytes in the presence but not in the absence of metabolic activation.

- In vitro gene mutation study in mammalian cells (Bakke 1990)

The purpose of this study was to evaluate the test material for toxicity and mutagenic activity at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese Hamster Ovary cells. Mutant (HGPRT-) cells were identified by their resistance to 6-thioguanine.

The test material was evaluated in experiments in the presence and absence of an exogenous metabolic activation system (S9). The cells were treated for 4 hours with culture medium containing the test sample at concentrations ranging from 49 to 120 µg/mL without S9 and at 31.4 to 150 µg/mL with S9. In these experiments, the test material reproducibly reduced the average cloning efficiency to less than 20% that of the solvent controls at concentrations above 96 µg/mL without S9 and at 76.8 µg/mL with S9 activation.

The criteria for a positive response was a threefold or greater increase in the frequency of 6-thioguanine-resistant colonies, compared with the solvent control cultures, in treated cultures with an average initial survival rate of at least 20%. The test material did not induce a concentration-dependent increase in mutant frequency either without or with S9 and was evaluated as negative under both activation conditions.

- In vitro study (Dopp et al., 2007)

The genetic toxicity potential of dimethyltin dichloride was examined by by looking at the effects on CHO-9 cells using the following methods; cytokinesis blocked micronucleus assay (CBMN), nuclear division index (NDI), chromosome aberrations (CA) and sister chromatid exchanges (SCE). No guideline was followed.

Analysis of chromosome aberrations and sister chromatid exchanges in CHO-9 cells following treatment with different concentrations (10 µM–1 mM) for 1 hour indicated that DMT induced a significant in percentage of chromosomal aberrations and sister chromatid exchanges at a cytotoxic concentration of 1mM.

- In vivo mammalian cell study: DNA damage and/or repair (Hamilton 1993)

The purpose of this study was to evaluate the ability of the test material to induce unscheduled DNA synthesis (UDS) in male Fischer-344 (F-344) rat hepatocytes after a single oral administration 16 or 2 hours before collection of liver specimens.

A dose range-finding assay was conducted to determine dose levels for the definitive UDS assay. Male rats approximately 8 weeks old (162-200 g) were dosed at 25, 50, 100, 200 or 400 mg/kg bw with a single oral dose on day 0 and were observed daily for morbidity or other clinical signs. No animals died within the first 24 hours so two more dose groups were added to the range-finding assay on day 1 (600 and 800 mg/kg bw) to ensure that animal death would be observed in the range-finding assay. On day 7, the surviving test animals were sacrificed and their internal organs were examined. One rat from the 400 mg/kg dose group and three rats each from the 600 and 800 mg/kg dose groups died before their scheduled sacrifices. The LD50 was estimated to be approximately 443 mg/kg bw.

Dose levels for the definitive UDS assay were set at 90, 175 and 350 mg/kg bw (approximately 20, 40 and 80% of the estimated LD. Male rats approximately 9 weeks of age (196-228 g) were dosed 2 or 16 hours before sacrifice with test material, vehicle control or the positive control (dimethylnitrosamine given 2 hours before sacrifice). All dose groups contained three rats except the 16 hour 350 mg/kg dose-group, which contained four rats. One extra rat was dosed to avoid loss of data because of animal death. Three rats from the 16 hour 350 mg/kg dose group were found dead on the morning after dosing. This experiment was terminated after the first eight test animals yielded insufficient viable hepatocytes to evaluate for UDS probably because of a technical error in the preparation of the cell culture medium. A second UDS assay was conducted using new cell culture medium.

Another LD50 was estimated to be approximately 280 mg/kg bw based on the pattern of death observed in the first UDS assay. Dose levels for the second UDS assay were set at 50, 110 and 225 mg/kg bw (approximately 20, 40 and 80% of the LD50). A second definitive UDS assay was conducted as described above using the lower dose levels. Male rats approximately 9 weeks of age (175-208 g) were given a single oral dose of the test or control articles 16 or 2 hours before sacrifice. All dose groups contained three animals except the 16 hour high-dose group, which contained five animals Two extra animals were dosed to avoid loss of data because of animal death. One rat in the 110 mg/kg 16 hour dose group was found dead the following morning and could not be evaluated for UDS.

Primary cultures of liver cells on coverslips were obtained from these rats and labelled in vitro with 3H-methylthymidine for 14 to 18 hours. These culture coverslips were coated with Kodak NTB-2 emulsion, exposed, developed, and stained, and the autoradiographic grain counts were quantitated. The net grains/nucleus and percentage of cells in repair were determined. The test material was considered positive if the mean net grain count (NG) was greater than zero and the percentage of cells in repair (IR) for that group was greater than 20%.

Hepatocytes from male rats given up to 225 mg/kg test material or the negative control 16 or 2 hours before evaluation of UPS yielded NG between -9.0 and -6.1 and ≤ 2% ER. In contrast, positive control rats given DMN two hours before liver collection yielded 28.4 NG and 93% ER.

Liver cells from male rats were monitored in vitro for unscheduled DNA synthesis (UDS) after these rats were given oral doses of test material (50, 110 or 225 mg/kg) 16 or 2 hours before collection of liver specimens. Results indicate that the test material is not genotoxic in male F-344 rat hepatocytes.

- In vivo Micronucleus Assay (SRI International, 1991)

The genetic toxicity of dimethyltin dichloride was examined in Swiss Webster mice dosed at 100, 200, 400 mg/kg bw in water, using gavage. 15 mice per dosage group were used. A micronucleus assay was then performed with counts at 24, 48 and 72 hours.

No induction of micronuclei was observed in male or female Swiss Webster mice upon gastric intubation of dimethyltin dichloride.

Justification for classification or non-classification

In accordance with the criteria outlined in Regulation (EC) No. 1272/2008 (CLP), the substance does not meet the criteria for classification for genotoxicity.