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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No information on GLP. Peer reviewed article, methodolgy similar to OECD 414.

Data source

Reference
Reference Type:
publication
Title:
Oral (gavage), in utero and postnatal exposure of Sprague-Dawley rats to low doses of tributyltin chloride. Part I: Toxicology, histopathology and clinical chemistry
Author:
Cooke et al
Year:
2004
Bibliographic source:
Food and Chemical Toxicology, 42, 211-220

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Pregnant Sprague-Dawley rats were purchased from Charles River Canada (St. Constant, QC Canada) and delivered to Health Canada's Animal Resource Division in Ottawa on day 5 of gestation.
All rats were allowed access to standard rat chow (Purina 5001, Agribands Purina Canada Inc, Strathroy, ON, Canada) and water ad libitum. A sample of rat chow was kindly analysed by Dr. R. Dabeka of Health Canada for total tin content by high-resolution ICPMS after digestion with nitric and hydrochloric acids and was determined to be less than 5 ng/g, which represents less than one per cent of the lowest dose of tributyltin used in our studies.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test material dissolved in olive oil at 0, 0.025; 0.25 or 2.5 mg/kg body weight/day until birth and then throughout lactation. The test material solutions were prepared such that the dosing volume was 0.5 mL per 100 g body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Rat pup stomach content samples (0.25 g, previously frozen) were hydrolysed overnight and then extracted with 0.05 % tropolone in ether-hexane. The extracted ionic butyltins were then derivatised to butylethyltin by Grignard reaction in preparation for analysis by gas chromatography (GC)-atomic emission detection (AED).
Details on mating procedure:
No data
Duration of treatment / exposure:
Dams were exposed from day 8 of pregnancy and throughout lactation.
Following weaning at PND 21, pups were selected randomly from within each dose group and gavaged daily with the same dose of the test material that their dams had received.
Frequency of treatment:
Daily
Duration of test:
The effects of exposure to low doses of the test material from day 8 of gestation until adulthood were examined.
Doses / concentrationsopen allclose all
Dose / conc.:
0.025 mg/kg bw/day (actual dose received)
Dose / conc.:
0.25 mg/kg bw/day (actual dose received)
Dose / conc.:
2.5 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Dams: n=16 and 9 per dose group for study 1 and study 2 respectively.
Pups: n=12 and 22 per dose group for study 1 and 2 respectively.
Control animals:
yes
Details on study design:
Rats were implanted with identifying microchips (Biomedic Data Systems Inc., Seaford, DE), randomly assigned to four treatment groups

Examinations

Maternal examinations:
Body weights of pregnant and lactating dams were recorded daily until weaning and food consumption was determined weekly.
Dams were sacrificed post weaning.

Pathology:
Two randomly selected dams from each dose group were also examined microscopically.
Fetal examinations:
Pup weights were recorded on postnatal days 4, 7, 14 and 21 and thereafter, pups were weighed daily and food consumption determined weekly.
In Study 1, pups were sacrificed by exsanguination under isoflurane anaesthesia at 30 days of age (males and females), 60 days of age (females only) and 90 days of age (males only). In study 2, pups were sacrificed at 60 days of age (females only) and 90 days of age (males only). Blood was collected for clinical analyses. Organ weights (adrenal, brain, liver, kidney, spleen and thymus) were recorded at necropsy. Two litters from each dose group from Study 1 were sacrificed and the stomach contents of suckling pups removed at 7 days of age, pooled and stored frozen (-20 °C) until assayed for organotin content.

Recovery experiments:
Samples of the pup's stomach content (0.25 g) were spiked at two levels (58-65 and 231-261 ng g^-1) with a mixture containing BuSnCl3, Bu2SnBr2and Bu3SnCl prior to hydrolysis. The percent recovery of each analyte was calculated by comparing the mean peak area of the recovered butyltin with the mean peak area of the same compound in a blank pup stomach content hydrolysate spiked just prior to derivatisation. Reagent blanks were run concurrently with each set of samples. An Agilent model 6890 GC equipped with a model G2350 AED was used for butyltin determination.

Pathology:
The following tissues were collected for histologic evaluation: thymus, thyroid, heart, lung, liver, kidney, adrenal, spleen, mesenteric and popliteal lymph nodes, ileum and femoral bone marrow. Tissues were fixed in 10 % neutral buffered formalin prior to paraffin embedding. Sections were cut at 5 microns and stained with haematoxylin and eosin.

Clinical chemistry:
Clotted blood, collected via the abdominal aorta with serum separator tubes, was centrifuged at 700xg for 20 min to prepare serum. A Beckman Synchron CX5 clinical system (Beckman Instruments Canada, Inc., Missisauga, ON, Canada) and Beckman reagent kits were used to measure clinical endpoints. The following endpoints were measured in sera from control and treated rats: glucose (non-fasting), blood urea nitorgen (BUN), creatinine, total protein, albumin, total bilirubin, calcium, sodium, potassium, chloride, amylase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase, creatine kinase, alkaline phosphatase, γ-glutainyltransferase (GGT), cholesterol, triglycerides, phosphate, uric acid, magnesium, thyroxine (T4), thyroxine uptake, osmolality and albumin/globulin ratio.
Statistics:
Dams body weights until weaning and pups body weights from weaning until sacrifice were analysed to obtain the orthogonal polynomial regression estimates for intercept (mean body weight across the study period), p1 (average slope across the study period), p2 (curvature across the period of the study) and p3 (sigmoidal change in curvature) [general linear model (GLM) procedure, SAS Institute Inc., Carey NC]. Significant treatment effects on the intercept, p1, p2 and p3 values were determined using ANOVA followed by Tukey's Studentised Range test.
For all other statistical comparisons, SigmaStat programme version 1 for Windows was used: dam body weights were analysed in two phases, prior to and post parturition. Food consumption data were analysed using two-way ANOVA and significant differences between treatments were detected by Tukey's test. Ratios of weekly food consumption to weekly body weight gain were arcsine transformed prior to comparison using two-way ANOVA and significant differences between treatments were detected by Tukey's test. Litter size, survival to weaning, sex ratio and organ weights were analysed by one way ANOVA followed by Tukey's test to identify differences between treatments and clinical chemistry data were analysed using Pearson Product Moment analysis for trends and also by one-way ANOVA, significant differences between treatments being detected by Tukey's test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Kidney: all 8 dams selected for histopathology exhibited mild multifocal chronic interstitial nephritis.
Heart: one dam dosed at 0.25 mg/kg/day had chronic mild epicarditis with acute focal myocardial necrosis and the other dam, also dosed at 0.25 mg/kg/day, had chronic mild epicarditis with focal atrial fibrosis.
Lung: both dams dosed at 0.25 mg/kg/day had similar changes: pyogranulomatous pleuritis with plant material (feed) and bacteria in the reaction. One dam dosed at 2.5 mg/kg/day, showed mild chronic inflammatory infiltrate in the pleura with slight fibrosis without feed and bacteria present, confirming the diagnosis of pleuritis made during gross examination.
No lesions were detected in thyroid, liver, adrenal, and colon.

Maternal developmental toxicity

Other effects:
no effects observed
Description (incidence and severity):
No effects on dams' body weights, food consumption, litter size, sex ratio or survival of pups to weaning.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
2.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No effects on dams' body weights, food consumption, litter size, sex ratio or survival of pups to weaning.

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
In male pups dosed at 2.5 mg/kg/day, reduced serum thyroxine levels were evident, indicating that the thyroid is a target for test material toxicity. No histopathological lesions were seen in the liver but elevated serum alanine aminotransferase, gamma-glutamyl transferase and amylase indicated hepatotoxicity. Significant decreases in liver weights in female pups exposed to 0.025 mg/kg/day were observed at PND 60. Decreases in spleen and thymus weights also pointed towards toxic effects of the test material on the immune system. The 0.025 mg/kg/day dose should have been a no affect dose and yet this dose caused significant effects on growth profiles, decreased liver weights and elevated serum GGT levels in females.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
< 0.025 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Dams body weights, food consumption, litter size, sex ratio and pup survival:

There were no effects of the test material exposure on the bodyweights or food consumption of the dams (Fig. 1). All dams gave birth at the expected time (day 22-23 of gestation). There were no significant differences in litter sizes, sex ratio or pup survival to weaning (Table 1).

Pup's stomach contents:

Recoveries of test material at the two spiking levels ranged from 89-93 %, indicating a consistent, nearly quantitative extraction of the butyltins from the pup stomach contents.

In the first study, the growth rates of all test material treated pups were significantly lower compared with vehicle controls

Orthogonal polynomial regression analysis revealed that the effect of the test material was to decrease both the mean body weights (intercept) and the average slopes of the growth profiles. In the second study, female pups dosed with the test material at 0.25 mg/kg/day, exhibited a greater average slope compared with controls but the other test material doses were without effect. All test material doses in the second study significantly affected the curvature of the male pups growth profiles. There were no significant effects in sigmoidal change in curvature in either study.

The average daily food consumption of female pups post-weaning was not affected by the test material (P>0.05) but male pups exposed to 0.25 mg/kg/day consumed a small but statistically significantly greater quantity (P<0.05). When the ratio of weekly food consumption to weekly body weight gain was determined, it was observed that for the female pups, those dosed with 0.25 mg/kg/day, the ratio was significantly lower compared with controls (group means: control, 3.62 vs 3.31 in 0.25 mg/kg/day) (2-way ANOVA of arcsine transformed ratios; P<0.05), indicating a more efficient conversion of food to body mass. However, for the male pups, a dose related increase in the ratio was observed (group means: control, 3.46; 0.025 mg/kg/day, 3.51; 0.25 mg/kg/day, 3.58; 2.5 mg/kg/day 3.67) which, for the 2.5 mg/kg dose group, was significantly higher compared with the controls (2-way ANOVA of arcsine transformed ratios; P <0.05), indicating a less efficient conversion of food to body mass.

Organ weights:

Organ weights of pups (Table 2) demonstrated that the test material had no effect on brain or kidney weights. Liver weights were unaffected in female pups at 30 days of age but were significantly reduced by the test material at 60 days of age in the 0.025 and 2.5 mg/kg/day dose groups (P<0.05). The weight reductions reached 20 % and were significant on both wet weight and liver to brain weight ratio bases. The liver of male pups was not affected at 30 days of age (P>0.05) but at 90 days of age, liver weights were reduced in a dose dependent manner with the highest dose of the test material significantly reducing liver weights by 15 %. Adrenal weights were unaffected by the test material with the exception that at 30 days of age, female pups exposed to 2.5 mg/kg/day had significantly heavier adrenals compared with controls (135 %). In all groups, spleen weights tended to decrease with increasing test material dose but was statistically significant for 60 day old females exposed to 2.5 mg/kg/day (20 % decrease in weight, P <0.05) and 30 day old males exposed to 0.25 mg/kg/day (20 % decrease in weight, P<0.05). Similarly, thymus weights tended to decrease with increasing test material dose, the reductions being statistically significant for females at day 60 (0.25 and 2.5 mg/kg/day) and male pups at day 30 (2.5 mg/kg/day).

Pathology:

- Gross pathology

Dams: Two dams from each dose group in study 1 were selected randomly for histopathology. Both of the dams that had been dosed at 0.25 mg/kg/day and 1 dam dosed at 2.5 mg/kg/day had fibrinopurulent pleuritis, which likely resulted from esophageal perforation from gavaging. The other dams appeared normal.

Pups: Six males and six females from each dose group and at each time point from study 1 were selected randomly for histopathological examination. These pups were not related to the dams used for histopathology. Gross lesions were uncommon. Five pups had unilateral right-sided hydronephrosis (2 males and 2 females dosed at 2.5 mg/kg/day. One female dosed at 0.25 mg/ kg/day and 1 female dosed at 0.025 mg/kg/day had unilateral left-sided hydronephrosis).

- Histopathology

Pups: Chronic mild interstitial nephritis was common in kidneys from controls and treated rats. Mild focal myocarditis was seen occasionally in hearts from controls and treated groups. However there was no evidence of any particular association of this lesion with treatment. Thyroids from the 90 day old males dosed at 2.5 mg/kg/day, showed frequent apoptosis within the follicular epithelial lining of the thyroid (4/6 pups) as compared to controls (1/6 pups) and the other doses of the test material(0/6 pups) (Chi squared, P<0.05). However, when the number of apoptotic sites were quantified for each rat in the control and 2.5 mg/kg/day dose group, using a graticulated microscope, the extent of the apoptosis was not statistically significant (z-test, P>0.05). No lesions were detected in liver, adrenal, and colon.

Serum clinical chemistry:

Clinical chemistry changes in pups exposed to the test material are summarised in Table 3. At PND 30, serum Mg levels tended to increase in female pups with increasing test material dose, while male pups had elevated serum triglycerides and reduced thyroxine uptake. In female pups at PND 60 there were a number of significant clinical changes. The kidney function markers BUN (blood urea nitrogen) and creatinine were significantly depressed, as were serum triglycerides, amylase, magnesium and potassium. Serumγ-glutamyl transferase levels were significantly elevated. In male pups at PND 90, serum alanine aminotransferase (ALT) and uric acid were significantly elevated, while thyroxine and amylase levels were depressed.

Applicant's summary and conclusion

Conclusions:
No effects on dams' body weights, food consumption, litter size, sex ratio or survival of pups to weaning. Parameters of the growth parameters of the pups were affected.
Executive summary:

The effects of exposure to low doses of the test material from day 8 of gestation until adulthood were examined. Pregnant rats were gavaged daily with 0, 0.025, 0.25 or 2.5 mg/kg body weight from day 8 of gestation until weaning. Stomach contents of suckling pups contained undetectable levels of TBT and dibutyltin (DBT) levels were detectable only in the highest test material dose used, indicating negligible lactational transfer to pups. Post weaning, pups were gavaged daily with the same dose of the test material administered to their mothers and sacrificed on post-natal days (PND) 30 (males and females), 60 (females) and 90 (males). The test material had no effects on dams body weights, food consumption, litter size, sex ratio or survival of pups to weaning. However, all doses of the test material significantly affected parameters of the growth profile of the pups (mean body weights, average slope, curvature) and the ratio of weekly food consumption to weekly body weight gain indicated enhanced food conversion to body mass in females but a decreased conversion in males. Liver, spleen and thymus weights were also affected by the test material.

In male pups dosed at 2.5 mg/kg/day, reduced serum thyroxine levels were evident, indicating that the thyroid is a target for the test material toxicity. No histopathological lesions were seen in the liver but elevated serum alanine aminotransferase, gamma-glutamyl transferase and amylase indicated hepatotoxicity. Significant decreases in liver weights in female pups exposed to 0.025 mg/kg/day the test material were observed at PND 60. Decreases in spleen and thymus weights also pointed towards toxic effects of the test material on the immune system. The 0.025 mg/kg/day dose should have been a no affect dose and yet this dose caused significant effects on growth profiles, decreased liver weights and elevated serum GGT levels in females.