Dimethyltin dichloride

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

Type of genotoxicity: 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Deviations from the Protocol:
Extra animals were dosed in the 16 hour high dose groups of both definitive UDS assays to avoid loss of data because of animal death. This circumstance did not affect the outcome of the study.

Circumstances That May Have Affected the Outcome of the Study:
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.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Mixture of methyltin chloride compounds
- Substance type: crystalline
- Physical state: solid
- Storage condition of test material: ≤25ºC

Test animals

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

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

Administration / exposure

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)

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

Remarks:

Doses / Concentrations:
600 and800 mg/kg
Basis:
actual ingested
Second range finding study

Doses / concentrations 3

Remarks:

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

Doses / concentrations 4

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):

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

Examinations

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".

Results and discussion

Test results

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.

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
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.