Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the key study (Krul, 2003), a bacterial reverse mutation test was conducted according to OECD Guideline no. 471.

The results the results obtained with the test material in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that the test material was not mutagenic under the conditions employed in this study.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 January 2003 to 17 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
See principles of method if other than guideline
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not applicable
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not applicable
Principles of method if other than guideline:
Deviations from the protocol:
M. van der Kaaden was involved in this study instead of Ms. G.C.D.M. Bruyntjes-Rozier and Ms. M.J.M. van den Wijngaard.
This deviation is considered not to have influenced the integrity and outcome of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine is the target gene for salmonela
Tryptophan is the target gene for E.coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
In total six concentrations were tested: 0.1, 0.3, 0.8, 2.3, 7 and 21 µg/plate.
The actual concentrations of the test material in the test solutions were not determined. Therefore, the concentrations quoted in this report are nominal concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
For TA1535, TA98, TA100 and E.coli WP2 uvrA in the presence of S9-mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
For TA1537 in the presence of S9-mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For TA1535 and TA100 in the absence of S9-mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
For TA1537 in the absence of S9-mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
For TA98 in the absence of S9-mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylnitrosurea
Remarks:
For E.coli WP2 uvrA in the absence of S9-mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: The plates were incubated at ca. 37 °C for 48-72 hours.
- Exposure duration: 48-72 hours.

NUMBER OF REPLICATIONS:
All determinations were made in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is defined as a reduction in the number of revertant colonies andlor a clearing of the background lawn of bacterial growth.

Evaluation criteria:
The mutagenicity study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, and if no more than 5 % of the plates are lost through contamination or other unforeseen events.

A test material is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates is concentration-related increased or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test material is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

In case of an inconclusive first assay, a second independent assay was conducted. The first mutagenicity assay is regarded inconclusive if a positive or equivocal response at only one concentration is observed or if a positive or equivocal responses at several concentrations without a concentration-related increase are observed.

Omission of the second assay under these conditions is acceptable as a single assay does not or hardly results in false negative conclusions.

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested strains.
Statistics:
No statistical analysis was performed.

Both numerical significance and biological relevance are considered together in the evaluation.
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98 & TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: TA 1535, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A dose range finding test to assess the toxicity of the test material to the bacteria was performed with TA98 both in the absence and presence of S9-mix. DMSO was chosen as vehicle. Just before use, the test material was diluted in the vehicle at 50 mg/mL. A transparent colourless solution was obtained. Serial 3-fold dilutions in DMSO were prepared and in total ten dilutions were tested, ranging from 0.3 to 5000 µg/plate.

The test material was diluted in DMSO. A dose range finding test was performed with TA98 in both the absence and the presence of S9-mix with ten different concentrations of the test substance, ranging from 0.3 - 5000 µg/plate. The test material was toxic at concentrations above 7 µg/plate.

In the main bacterial reverse mutation test six different concentrations were tested ranging from 0.1 - 21 µg/plate. Negative controls (solvent) and positive controls were run simultaneously with the test material.

In the absence of S9-mix, the test material was toxic to all Salmonella typhimurium strains at 7 and 21 µg/plate and in the presence of S9-mix to strain TA 1537 at 21 µg/plate, as was evidenced by the presence of pinpoints or a decrease in the mean number of revertant colonies compared to the negative controls.

In both the absence and the presence of S9-mix and in all strains, the test material did not cause a more than two-fold increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control.

The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.

It is concluded that the results obtained with the test material in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that the test material was not mutagenic under the conditions employed in this study.

Conclusions:
It is concluded that the results obtained with the test material in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that the test material was not mutagenic under the conditions employed in this study.
Executive summary:

In a Bacterial reverse mutation test, the results obtained with the test material in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that the test material was not mutagenic under the conditions employed in this study.

In the absence of S9-mix, the test material was toxic to all Salmonella typhimurium strains at 7 and 21 µg/plate and in the presence of S9-mix to strain TA 1537 at 21 µg/plate, as was evidenced by the presence of pinpoints or a decrease in the mean number of revertant colonies compared to the negative controls.

The study was conducted to the OECD Guideline no. 471.

Genetic toxicity in vivo

Description of key information

The test material was examined for its mutagenic potential in a bone marrow micronucleus test in mice according to OECD Guideline no. 474. Under the conditions used in this study, the test material, was cytotoxic to the bone marrow of mice. The positive control goup responded appropriately. The sole statistical difference in the incidences of MPE, specifically in the 300 mg/kg group at the 48 hour time point, was judged incidental because of the lethality seen at this dose level and the unexplained decrease in MPE in the concurrent controls. Thus, the weight of the evidence suggests the test material is not mutagenic in this test, but the data are equivocal.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 January 2003 to 28 April 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
See principles of method if other than guideline
Principles of method if other than guideline:
Deviations from the protocol:
- The dose range finding toxicity test was started on 20 December 2002, instead of 29 October 2002.
- The main micronucleus test was started on 14 January 2003, instead of 26 November 2002.
- During the main micronucleus test, during a short period, the relative humidity in animal room 5.2.10 was slightly higher than 70 %, due to cleaning activities.
- During the main micronucleus test, during a short period, the pressure in animal room 5.2.10 was slightly lower than 5 %, due to cleaning activities.

These deviations did not adversely influence the integrity and/or the validity of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: young adult
- Weight at study initiation: Approx 31-35 g
- Assigned to test groups randomly: Yes, under following basis: animals were allocated by computer randomization to a vehicle control group A, three test groups treated with Tributylchlorostannane (B, C and D) and to a positive control group E.
- Fasting period before study: Administration of the test substance was carried out after a fasting period of 2 hours and 15 minutes.
- Housing: The animals were housed in sterilised Macrolon cages (type I + II), fitted with a grid cover of stainless steel and with a bedding of sterilised softwood chips.
- Diet: With the exception of the fasting period prior to dosing, feed was provided ad libitum from the arrival of the animals until the end of the study.
- Water: With the exception of the fasting period prior to dosing, drinking water was provided ad libitum from the arrival of the animals until the end of the study. The drinking water (tap-water) was given in polypropylene bottles, which were cleaned weekly and filled as needed.
- Acclimation period: During the quarantine and acclimatization period (at least 6 days) the animals were observed daily for overt signs of ill health and anomalies.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: Relative humidity of at least 30 % and not exceeding 70 % other than during room cleaning.
- Air changes (per hr): The animal rooms were ventilated with about 10 air changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was artificial with a sequence of 12 hours light and 12 hours dark.
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On day 0 (prior to dosing), the test material was suspended in corn-oil, at concentrations of 15, 7.5 and 3.75 mg/mL. The orally (by gavage) given dosing volume was 20 mL/kg-bw. Prior to dosing, the animals were weighed. Administration of the test material was carried out after a fasting period of 2 hours and 15 minutes. The dose-levels administered were 300, 150 and 75 mg/kg-bw.
Duration of treatment / exposure:
Single oral dose
Frequency of treatment:
Single oral dose
Post exposure period:
48 hours
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals for doses 75, 150 mg/kg-bw and the positive control group. 10 animals used for the negative control and dose group 300 mg/kg-bw. Three reserve mice were treated with the highest dose-level of 300 mg/kg-bw to replace any mortality in the highest dose-level group.
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Route of administration: Sigma; intraperitoneal injection
- Vehicle: saline
- Doses / concentrations: stock-concentration: 0.075 mg/ml; dosing volume 10 ml/kg-bw; dose level: 0.75 mg/kg-bw
- Other: For safety reasons, the animals of the positive control group (main micronucleus test only) were housed in a laminar down-flow cabinet, just prior to administration and until sacrifice. The animal rooms were ventilated with about 10 air changes per hour and were maintained at a temperature of 22 ± 3 °C and a relative humidity of at least 30 % and not exceeding 70 % other than during room cleaning. Lighting was artificial with a sequence of 12 hours light and 12 hours dark.
Tissues and cell types examined:
Bone marrow cells.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The acute toxicity (LD50) for rats, orally treated with the test material was not known. Therefore, dose levels of 2000 (the limit dose), 750, 375 and 187.5 mg/kg-bw were selected for the dose-range finding acute toxicity test in male and female mice.

The dose levels and the choice of sex, selected for the main micronucleus test, were based on the clinical signs observed during the performance of the dose-range finding acute toxicity test as described below. For both sexes, severe toxicity was observed in this dose-range finding acute toxicity test at the three highest dose levels of 2000, 750 and 375 mg/kg-bw. These results obtained were discussed with the sponsor. Thereafter, it was decided to perform the main micronucleus test with dose levels of 300, 150 and 75 mg/kg-bw and with male mice only.

Dose-range finding acute toxicity test:
A dose-range finding acute toxicity test was performed in order to determine the dose level(s) and sex to be used in the main micronucleus test. Dose levels of 2000 (the limit dose), 750, 375 and 187.5 mg/kg-bw were selected for the dose-range finding acute toxicity test in male and female mice.
After a fasting period of ca. 2 hours and 45 minutes, two males and two females were treated once (by gavage) with each of the four dose-levels (2000, 750, 375 and 187.5 mg/kg-bw) of the test material. Observations with respect to all signs of reaction to treatment were recorded 1 hour, 4 hours, 1 day and 2 days post treatment. The animals were coded by an earmark (V-mark in the ears). The females were allocated odd numbers and the males even numbers. Body weights were recorded prior to dosing (day 0) and on day 3. Sex differences could not be demonstrated in the dose-range finding acute toxicity test.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Signs of reactions to treatment were recorded from 1- 4 hours after treatment and daily thereafter.

At the sacrifice time of 24 hours after dosing, 5 mice treated with the vehicle control, 15 mice treated with the test material (5 mice per dose-level) and 5 mice treated with the positive control substance mitomycin C, were killed by cervical dislocation. At the sacrifice time of 48 hours after dosing, 5 mice treated with the vehicle control, together with 5 mice treated with the highest dose-level of the test material, were killed by cervical dislocation.


DETAILS OF SLIDE PREPARATION:
From each mouse, the bone marrow cells of both femur were immediately collected into foetal calf serum and processed into glassdrawn smears. Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grunwald Giemsa solution. The other smear was stored as a reserve slide.

The slides were randomly coded by a person not involved in the scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear.
The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.

Due to the unexplained reduction in MPE in the 48 hour control group, the sponsor requested that the slides of the 48 hours vehicle control animals be recounted to verify the results of the first count.

Evaluation criteria:
The study is considered valid if the positive controls give a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls are within the historical range.

A response is considered to be positive if the mean number of MPE/2000 PE is statistically significantly higher, when compared to the mean number of the vehicle controls.

A test material is considered to cause chromosomal damage and/or damage to the mitotic apparatus, if a clear dose-related increase in the mean numbers of MPE/2000 PE is observed, when compared to the mean number of the vehicle controls, and/or if a single positive dose level is observed.

A test material is considered to be negative in the micronucleus test if it produces no positive response at any of the dose-levels and time points analysed.

The test material or its metabolites are considered to have reached the general circulation and thereby the bone marrow, if the test material statistically reduce the mean number of PE/E or causes systemic toxicity.

Both statistical significance and biological relevance are considered together in the evaluation.
Statistics:
1) At time point 24 hours after administration, data on MPE and PE were subjected to a One Way Anova with factor group (A,B,C and D). If the Anova yielded a significant effect (p<0.05), it was followed by pooled error variance t-tests or, if variances were not homogenous, separate variance t-tests. These t-tests were applied to the negative control group A versus treatment groups B, C and D. In addition, the positive control group E and the negative control group A were compared using pooled error variance t-tests or, if variances were not homogenous, separate t-tests.

2) At time point 48 hours after administration, for treatment groups A and D, data on MPE and PE were subjected to pooled error variance t-tests or, if variances were not homogenous, separate variance t-tests.

All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
(Equivocal)
Toxicity:
yes
Remarks:
300 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Severe clinical signs were observed in the dose-range finding acute toxicity test. These severe clinical signs were observed in both male and female mice. The results of the dose-range finding acute toxicity test were reported to the sponsor. Thereafter, it was decided to perform the main micronucleus test with the dose levels of 300, 150 and 75 mg/kg-bw and with male mice only.

All results tables can be seen in the attached illustration.

Clinical signs in the main micronucleus test:

A-group (corn-oil): no clinical signs were observed.

B-group (75 mg/kg-bw): no clinical signs were observed.

C-group (150 mg/kg-bw): C44 showed piloerection. C46 showed blepharospasm.

D-group (300 mg/kg-bw): D62, D64, D66 and D80 showed sluggishness, blepharospasm and piloerection. D68, D70 and D74 showed sluggishness and blepharospasm. D72 died before sacrifice time and was replaced by reserve mouse D94. D76 died before sacrifice time and was replaced by reserve mouse D96 D78 died before sacrifice time and could not be replaced because one of the reserve animals died after treatment.

Micronucleated Polychromatic Erythrocytes (MPE):

In this test the incidences of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in treatment groups B (75 mg/kg-bw) and C (150 mg/kg-bw) and D (300 mg/kg-bw) were 2.2, 2.2, and 4.0, respectively. The incidence of MPE per 2000 PE in the positive control group (E), at the 24 hour time point, was 45.0.

At the 48 hour time point there was an unexplained reduction in the incidence of MPE in the negative controls compared to the 24 hour time point. The slides were re-counted and both results are presented. At 48 hours the 4.0 incidence of MPE per 2000 PE in treatment group D was the same as the incidence at 24 hours. Also in the 300 mg/kg-bw group (D), 3 animals died prior to scheduled sacrifice at 48 hours and only 2 were able to be replaced with reserve animals leaving n=4 for this group.

Polychromatic Erythrocytes (PE) in the mice of the main micronucleus test:

At 24 and 48 hours after treatment, in the vehicle controls, the number of PE per 200 E were approximately equal. At 48 hours after treatment, the number of PE per 200 E were reduced in treatment group D (300 mg/kg-bw) relative to controls. This is a biologically relevant effect and indicates that the test substance reached the bone marrow and induced cytotoxicity to the bone marrow cells.

Statistical analysis of the main micronucleus test results:

At 24 hours after treatment, the two-way ANOVA indicated there were no statistically significant differences in the incidences of MPE and PE in any treatment group compared to the negative controls. At 24 hours, the incidence of MPE per 2000 PE in the positive control group was statistically significantly greater than (p<0.001) that in the negative controls. The latter result demonstrates the sensitivity of the test system and the validity of the assessments.

At 48 hours after treatment, the two-way ANOVA indicated a statistically significant difference between treatment group D (300 mg/kg-bw) and the negative control group for MPE (p<0.05).

At 48 hours after treatment, the ANOVA indicated a statistically significant difference between treatment group D (300 mg/kg-bw) and the negative control group for MPE (p<0.05).

This difference was judged to be due to the incidental drop in the control incidence of MPE rather than an increase in the incidence of MPE in the treated group. Two observations support this judgement: 1) the incidence data for PE per 200 E in controls at the 24 and 48 hour time points are essentially the same, and 2) the maximal number of MPE per PE among the mice in the 300 mg/kg-bw dose group is no higher than the highest incidence in mice from the vehicle controls. Thus, the observations suggest that the statistical difference, while real, is not indicative of an underlying increase in the effect of Tributylchlorostannane on MPE in this case. At 48 hours after treatment, the two-way ANOVA indicated there was a statistically significant decrease in the number of PE in treatment group D (300 mg/kg-bw) (**p<0.01), when compared to the negative control group A (corn-oil). This indicated that the test material reached the bone marrow and was cytotoxic to bone marrow cells.

Conclusions:
Under the conditions used in this study, the test material, was cytotoxic to the bone marrow of mice. The positive control goup responded appropriately. The sole statistical difference in the incidences of MPE, specifically in the 300 mg/kg group at the 48 hour time point, was judged incidental because of the lethality seen at this dose level and the unexplained decrease in MPE in the concurrent controls. Thus, the weight of the evidence suggests the test material is not mutagenic in this test, but the data are equivocal.
Executive summary:

The test material was examined for its mutagenic potential in a bone marrow micronucleus test in mice. The study consisted of a dose-range finding acute toxicity test carried out with male and female mice followed by a main micronucleus test with male mice.

Results of the dose range finding acute toxicity test indicated that there were no differences in the response which could be attributed to the sex of the animals. Severe toxicity was observed in both sexes at dose levels of 2000, 750, and 375 mg/kg-bw. Male mice were chosen for the main micronucleus test and doses of 300, 150, and 75 mg/kg-bw were adopted.

For the main micronucleus test, animals were treated once by gavage with three graded dose levels of the test material. The high dose group (D), consisted of 10 males, and each animal received a dose of 300 mg/kg-bw. The mid dose group (C), consisted of 5 males, and each animal received a dose of 150 mg/kg-bw. The low dose group (B), consisted of 5 males, and each animal received a dose of 75 mg/kg-bw. The vehicle control group A consisted of 10 males, and each animal was dosed in a similar way with the corn-oil vehicle only. A positive control group consisted of 5 males, and each animal was given a single intraperitoneal dose of mitomycin C at 0.75 mg/kg-bw. At 24 hours after treatment, 5 animals of each dose-level of the test material, 5 negative control animals and 5 positive control animals, were euthanised. At 48 hours after treatment, the remaining 5 animals of group D, the high dose group and the remaining 5 negative control animals, were euthanised. From both femurs of each animal, the bone marrow cells were collected in foetal calf serum and processed into smears for microscopic examination. At both 24 and 48 hours after treatment, the number of polychromatic erythrocytes (PE) per 200 erythrocytes (E) and the number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) were counted for each mouse. The group mean numbers of MPE per 2000 PE and the group mean numbers of PE per 200 E for each group were reported. The historical vehicle control and historical positive control data for MPE per 1000 PE were also presented.

At 24 and 48 hours after treatment in the vehicle controls, the number of PE per 200 E were approximately equal. At 48 hours after treatment, the number of PE per 200 E were reduced in treatment group D (300 mg/kg-bw) relative to controls. This is a biologically relevant effect and indicated that the test material reached the bone marrow and induced cytotoxicity to the bone marrow cells. At 24 hours after treatment, there were no statistically significant differences in the incidences of MPE or PE in any treatment group compared to the negative controls. At 24 and 48 hours, the incidence of MPE per 2000 PE in the positive control group was increased (P<0.001) relative to the negative controls. The latter results demonstrates the sensitivity of the test system and the validity of the assessments.

At the 48 hour time point there was an unexplained reduction in the incidence of MPE per 2000 PE in the negative controls compared to the 24 hour time point. This was confirmed in a recount of the slides. The reduced incidence of MPE per 2000 PE in the high dose group (D) at 48 hours was unchanged compared to the incidence at 24 hours. Also in the 300 mg/kg-bw group (D), 3 animals died prior to scheduled sacrifice at 48 hours and only 2 were able to be replaced with reserve animals leaving n=4 for this group. 5.At 48 hours after treatment, the ANOVA indicated a statistically significant difference between treatment group D (300 mg/kg-bw) and the negative control group for MPE (p<0.05). This difference was judged to be due to the incidental drop in the control incidence of MPE rather than an increase in the incidence of MPE in the treated group. Two observations support this judgement: 1) the incidence data for PE per 200 E in controls at the 24 and 48 hour time points are essentially the same, and 2) the maximal number of MPE per PE among the mice in the 300 mg/kg-bw dose group is no higher than the highest incidence in mice from the vehicle controls. Thus, the observations suggest that the statistical difference, while real, is not indicative of an underlying increase in the effect of Tributylchlorostannane on MPE in this case.

Under the conditions used in this study, the test material was cytotoxic to the bone marrow of mice. The positive control group responded appropriately. The sole statistical difference in the incidences of MPE, specifically in the 300 mg/kg-bw group at the 48 hour time point, was judged incidental because of the lethality seen at this dose level and the unexplained decrease in MPE in the concurrent controls. Thus, the weight of the evidence suggests the test material is not mutagenic in this test, but the data are equivocal.

Additional information

In vitro:

In the key study (Krul, 2003), a bacterial reverse mutation test was conducted according to OECD Guideline no. 471.

The reliability rating for this study is 1, according to the criteria of Klimisch, 1997 as the study was conducted to recognised guideline and GLP. This study is considered the most relevant and reliable study for this endpoint as all of the supporting information comes from literature papers or other less reliable sources, therefore this study will be used for classification purposes for this endpoint. The results the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that the test material was not mutagenic under the conditions employed in this study.

- For the ATSDR (2005) report a reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as this information is from a secondary source. (Reference: Hamasaki et al, 1993). The test material was negative without metabolic activation.

- In the Hamasaki et al (1992) paper, genotoxicity of the test material was studied with the SOS chromotest. The test material was not shown to produce DNA damage by the SOS chromotest. A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as the purity of test material was not reported and it was only conducted without metabolic activation, however it meets generally accepted scientific standards with acceptable restrictions.

- In the Hamasaki et al (1992) paper, genotoxicity of the test material was studied with the Bacillus subtilis recombination assay test. Tri-n-butyltin chloride showed DNA-damaging potency at a low dose, 10µg/disk. Tri-n-butyltin chloride could not be tested at more than 1000µg/disk, because of its antibacterial activity. Tri-n-butyltin chloride showed enhanced genotoxicity with increasing dose. A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as the purity of test material was not reported and it was only conducted without metabolic activation, however it meets generally accepted scientific standards with acceptable restrictions.

- In the Hamasaki et al (1993) paper, a bacterial reverse mutation test was conducted. By the mutagenicity assay with S. typhimuruim TA100, n-butyltin trichloride was found to be mutagenic without activation. A reliability rating of 3 was assigned to this study, according to the criteria of Klimisch, 1997 as although the study meets generally accepted scientific standards it lacks information on plate counts and numbers of revertant colonies, multiple S. typhimurium strains were not tested, nor did the authors evaluate the mutagenicity potential of the tested substance in the presence of a metabolic activation system, these deviations are considered to have possibly affected the outcome of the study.

- In the Sasaki (1992) paper, the potentiating effects of the test material on chromosome aberrations were studied in Chinese hamster CHO K1 cells. The test material did not show any clastogenic activity under the experimental conditions without rat liver S9. Post-treatment with organotins, however, increased the number of breakage-type (but not exchange-type) chromatid aberrations induced by MMC, an S-phase-dependent clastogen. Enhancement of the induction of chromosome aberrations by MMC was observed when cells were treated with the test material during the G2 phase. These results suggest that organotin G2 effect causes potentiating effects. A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as the purity of test material was not reported and there is no information on GLP, however it meets generally accepted scientific standards with acceptable restrictions.

- For the ATSDR (2005) report a reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as this information is from a secondary source. (Reference: Jensen et al (1991b)). The test material was found to be positive for spindle inhibition of the Chinese hamster cells.

In vivo:

In the key study (de Vogel, 2003), a micronucleus assay was conducted according to OECD Guideline no. 474. The reliability rating for this study is 1, according to the criteria of Klimisch, 1997 as the study was conducted to recognised guideline and GLP. The test material is not mutagenic in this test, but the data are equivocal.

Justification for classification or non-classification

Although data suggests that the test material is not mutagenic, the results for the in vivo study were equivocal and so mutagenicity can't be ruled out. Based on information for similar substances and the industry standard that all dibutyltins are considered to have the potential to be mutagenic, for precautionary measures, the test material is considered to be classified as Muta. Cat. 2; H341: Suspected of causing genetic defects, according to Regulation (EC) no 1272/2008.