Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 December 2002 - 22 April 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD standards
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
see "principles of method if other than guideline"
Principles of method if other than guideline:
Deviations:
Animals were tail marked prior to allocation
Size of cages were 42.5x26.6x15cm
Weekly food intake measured on day 13 instead of day 14
Blood for hematology was taken from 2 animals instead of 5
Determinations that could not be conducted for individual animals are presented in study appendices

None of the deviations were considered to have influenced the validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetraoctyltin
EC Number:
222-733-7
EC Name:
Tetraoctyltin
Cas Number:
3590-84-9
Molecular formula:
C32H68Sn
IUPAC Name:
tetraoctylstannane
Details on test material:
- Name of test material (as cited in study report): Tetraoctylstannane (TTOT)
- Substance type: Monoconstituent
- Physical state: Colourless liquid
- Analytical purity: 90.79%
- Impurities (identity and concentrations):
Trioctyltin chloride: 8.44%
Dioctyltin dichloride: 0.74%
Monooctyltin trichloride: 0.03%
- Composition of test material, percentage of components: See above
- Purity test date: 11-31 December 2002
- Lot/batch No.: 346272
- Expiration date of the lot/batch: 31 July 2004
- Stability under test conditions: Stable
- Storage condition of test material: At less than -18°C, in the dark

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks old
- Weight at study initiation:
Dose range finding-
Males: 276 to 294 grams
Females: 182 to 192 grams
Main study-
Males: 262 to 268 grams
Females: 179 to 186 grams
- Housing:
Premating period: groups of 4/sex were kept in macrolon type 3 cages (42.5 x 26.6 x 15.0 cm)
Mating period: mating pairs were each placed in macrolon type 3 cages
Post-mating period: mated females place singly in macrolon type 3 cages
- Diet (e.g. ad libitum): Commercial rodent diet (Rat & Mouse No. 3, Breeding Diet RM3) from Special Diets Services, Witham, England. Diet was provided ad libitum as a powder in perforated stainless steel cans. These were refreshed once per week.
- Water (e.g. ad libitum): Tap water suitable for human consumption was provided ad libitum in polypropylene bottles
- Acclimation period: 14 days for dose range finding; 7 days for main study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs light/ 12 hrs dark

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Experimental diets were prepared once shortly before the start of the studies; they were stored in a freezer (less than -18°C) until use. The test substance was weighed in a tray of steel and thereafter the tray was rinsed with RM3 food. The weighed test substance was mixed with approximately 3kg weighed RM3 food in the Stephan cutter for 2 minutes. Thereafter, approximately 3 kg of weighed food was used to rinse the Stephan cutter. Mixing was continued in the Lödige for 2 minutes

DIET PREPARATION
- Rate of preparation of diet (frequency): Once
- Mixing appropriate amounts with (Type of food): RM3
- Storage temperature of food: Less than -18°C in dark
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From each diet sample, 2.0 g was transferred into a 50 mL Greiner tube. An aliquot of the internal standard solution (monoheptyltin trichloride, diheptyltin dichloride, tripropyltin chloride, and tetrapropyltin in methanol) was added. Subsequently, methanol, acetate buffer solution (pH 4.5), 20% aqueous sodiumtetraethylborate (NaBEt4) solution and hexane (with naphthalene as internal standard) were added to each sample and this mixture was shaken and heated to 60°C. During this step, the organotin chlorides were extracted into the hexane layer. Prior to GC-MS analysis, the hexane layer was washed with 2 mol/L HCl in order to remove (most of) the ethylboron compounds that interfere with the GC-MS analysis. The concentration of each test substance in feed was determined by GC-MS analysis of the hexane extracts. Further details about the derivatization, extraction and analysis are below:

Column: fused silica HP5 MS, 30 m, 0.25 mm ID, 0.25 µm film
Pre-column: fused silica HP5 Ms, 2.5 m, 0.25 mm, 0.25 µm film
Column temperature: after 3 min at 45°C at a rate of 5°C/min to 80°C; then at a rate of 15°C/min to 260°C; 15 min at 260°C
Carrier: helium; 1.5 mL/min constant flow
Injection volume: 1 µL
Injection temperature: start at 60°C, then at a rate of 14.5°C/s to 300°C; 5 min at 300°C
Injection method: splitless
Ionisation: electron impact, 70 eV
Mass range: 60-600 amu
Mass fragments used:
TTOT, m/z= 459; 347; 235
TTPT, m/z= 249

Results & Conclusions:

The test substance TTOT was considered to be homogenously distributed in all diets.
The test substance TTOT was considered to be stable in the diets upon storage at room temperature for 7 days and upon storage at less than -18°C for 55 days.
The content of the test substance TTOT was considered to be close to intended for all diets. Only the 500 mg/kg dose level of the dose-range finding study did not meet the criteria (relative difference from intended concentration was -24%), but when the low recovery of 76% obtained during the validation of TTOT at this dose level is taken into account, the achieved concentration seems to be close to the intended concentration.
In general, the analytical results of TTOT in study samples seem to be influenced by varying efficiencies of TTOT from the diet, resulting from sticking of TTOT to the diet.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until pregnancy occurred
- Proof of pregnancy: sperm in vaginal smear, designated day 0 of pregnancy

Sperm positive females that were not pregnant were killed 24 days after copulation
Duration of treatment / exposure:
Dose-range finding study: 3 January 2003
Main study:
Males: 28 days- 28 February 2003 to 28 March 2003
Females: 38 to 48 days -28 February 2003 to 7-17 April 2003
Frequency of treatment:
ad libitum in diet.
Duration of test:
Dose-range finding study: 3 January 2003
Main study:
Males: 28 days- 28 February 2003 to 28 March 2003
Females: 38 to 48 days -28 February 2003 to 7-17 April 2003
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1,500 & 7,500 mg/kg in diet
Basis:
nominal in diet
No. of animals per sex per dose:
Dose-range finding study: Four males and four females per treatment level (five total including control)
Main study: 12 males and 12 females per treatment level (four total including control)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The doses of the range finding study were selected by the sponsor. Dosing for the main study was based on the results of the range finding test.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily (morning hours) for both dose-range finding and main studies

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were checked daily for moribundity, abnormalities, reaction to treatment, ill health

BODY WEIGHT: Yes
- Time schedule for examinations:
Dose-range finding study: on day -2 ( for randomization purposes), day 0, 3, 7, 10 and 14 (day of sacrifice)
Main study:
Pre-mating period: on day -2 ( for randomization purposes), day 0, 7, and 13
Mating period: males weighed weekly until sacrifice; females weighed on day 0, 7, 13
Post-mating period: females weighed on day 0, 7, 14, and 21 (during presumed gestation), and on day 1 and 4 of lactation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the pre-mating period
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia
- Animals fasted: Yes
- How many animals: 5 rats/sex/group
- Parameters checked: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of pre-mating period
- Animals fasted: Yes
- How many animals: 5 rats/sex/group
- Parameters checked: fasting glucose, alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase activity, gamma glutamyl transferase activity, total protein, albumin, ratio albumin to globulin, urea, creatine, total bilirubin, total cholesterol, triglycerides, phospholipids, calcium, sodium, potassium, chloride, inorganic phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to test initiation; weekly during exposure
- Dose groups that were examined: All
- Battery of functions tested: Functional Observation Battery (FOB)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Uterus and ovaries weight: Yes
- Number of lost implantation sites
- Number of lost implantations
Fetal examinations:
A necropsy was performed on stillborn pups and pups that died during the study; macroscopic abnormalities were recorded. Pups were examined externally for gross abnormalities and killed by hypothermia at <-18 °C on PN 4 or shortly thereafter.
Statistics:
Clinical findings were evaluated by Fisher's exact probability test
Body/organ weight, food consumption were evaluated by one-way ANOVA followed by Dunnett's multiple comparison test
Mated/pregnant females evaluated with Fisher's exact probability test
Implantation sites, live and dead pups were evaluated by Kruskal-Wallis nonparametric ANOVA with Mann-Whitney U-test.
Blood counts, chemistry values, organ weights evaluated by one-way ANOVA followed by Dunnett's multiple comparison tests
Reticulocytes and relative differential white blood cell counts evaluated by Kruskal-Wallis non-parametic ANOVA followed by Mann-Whitney U-tests.
Continuous FOB parameters assessed via Kruskal-Wallis ANOVA; categorical FOB parameters were evaluated by Pearson chi-square analysis
Indices:
Fertility and reproductive performance:
For each mating the following data are presented for each group:
- number of females placed with males
- number of males mated with females
- number of successful copulations (= number of females mated)
- number of males that became sires
- number of pregnant females as demonstrated by the presence of implantation sites observed at autopsy.
- number of females surviving delivery
- number of females with liveborn and (all) stillborn pups
- number of pups delivered (live- and stillborn)
- number of live pups at day n
- number of pups lost
- number of litters lost entirely
- number of male pups at day n
- number of implantation sites
- number of lost implantations
- litter size

The following parameters were calculated:
- pre-coital time = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- mating index= (number of females mated/number of females placed with males) x 100
- male fertility index = (number of males that became sires/number of males placed with females) x 100
- female fertility index = (number of pregnant females/number of females placed with males) x 100
- female fecundity index = (number of pregnant females/number of females mated) x 100
- gestation index = (number of females with live pups/number of females pregnant) x 100
- live birth index = (number of pups born alive/number of pups born) x 100
- pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x 100
- number of lost implantations = number of implantations sites - number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100
Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY:
No mortalities were observed.
No clinical signs were observed in the males from the start of the study until sacrifice.
During the pre-mating period, female C569 of the mid-dose group was sparsely haired and in female D585 of the high-dose group a kinked tail was observed. Animal D585 showed a kinked tail until sacrifice. Exopthalmus was observed after orbital puncture in female C565 of the mid-dose group and female D575 of the high-dose group. This observation lasted until GD 0 for animal D575 and GD 3 for animal C565. Thereafter, panophthalmitis was observed in both animals until sacrifice. During the gestation period, animals A509 (control group, GD 20), C553 and C569 (mid-dose group, GD 1 and GD 20, respectively) were sparsely haired. During the lactation period, the following animals were sparsely - haired: animal A509 (control, PN 0-5) and C551 and 553 (mid-dose, PN 0-4).


BODY WEIGHT AND WEIGHT GAIN:
During the pre-mating, gestation and lactation periods, the mean body weight and body weight change of the female animals of the tetraoctylstannane-treated groups was comparable to the control group.


FOOD CONSUMPTION AND COMPOUND INTAKE:
During the pre-mating period, mean food consumption expressed as g/animal/day of the female animals of the low-, mid-, and high-dose group was statistically significantly decreased from days 0-7 and expressed as g/kg body weight/day in the mid- and high-dose group from days 0-7.


ORGAN WEIGHTS: The absolute (0.049 high-dose versus 0.121 g control) and relative (0.236 high-dose versus 0.599 g/kg body weight control) thymus weight of the female animals of the high-dose group was statistically significantly decreased.

GROSS PATHOLOGY:
Female animals with a litter were sacrificed on PN 4 or 5. The absolute (0.049 high-dose versus 0.121 g control) and relative (0.236 high-dose versus 0.599 g/kg body weight control) thymus weight of the female animals of the high-dose group was statistically significantly decreased.
No other effects on organ weights (absolute and relative) were observed.

Female animals with a litter were sacrificed on PN 4 or 5; non-pregnant animals were sacrificed on GD 24. At necropsy an increased incidence of accumulation of fluid (hydrometra), was seen in the uteri of three non-pregnant low-dose females. Two of these animals showed swelling of the cervix as well. Since hydrometra of the uterus is part of common background pathology and a dose-response relationship was absent, no significance was attached to this finding. The other macroscopic observations are common findings in rats of this strain and age and occurred only incidentally.
The decreased size of the thymus was not reported as a gross lesion as decreases in thymus weights were considered to reflect the diminished size of the thymus in individual animals more accurately.


HISTOPATHOLOGY (NON-NEOPLASTIC):
Examination of the thymus revealed very severe lymphoid depletion in 5/5 high-dose females. Lymphoid depletion was characterized by a decrease in the size of thymic lobules because of an extensive loss of cortical and medullary small lymphocytes.


HISTOPATHOLOGY (NEOPLASTIC):
In the mesenteric lymph nodes of 3/5 high-dose females and 1/5 mid-dose female, the presence of clusters of swollen yellowish macrophages in the medullary cords, and occasionally in the paracortical areas, was conspicuous (denoted as very slight to slight macrophage accumulations). The medullary cords had been enlarged by these macrophage accumulations.


OTHER FINDINGS: REPRODUCTIVE EFFECTS:
In each group 12 females were placed with males and all females were mated. Pre-coital time was comparable for the control, low-, mid-dose and high dose groups; 1 female of the control and 2 females of the mid-dose group mated in the period day 8-14. The number of pregnant females and the number of males that became sires amounted to 9, 5, 10 and 11 for the control, low-, mid- and high-dose groups, respectively. The number of females with live-born pups was 8, 5, 9 and 10 for the control, low-, mid-and high-dose groups, respectively.
The mating index was 100% in all groups. The female fecundity index, female fertility index and male fertility index were comparable among the control, mid- and high-dose groups and ranged from 75-92%. In the low-dose group these indices were relatively low (42%). The gestation index was comparable in all groups and in the range of 89-100%. No difference in the duration of gestation was observed amongst the control and the tetraoctylstannne-treated groups. Stillborn pups were observed in 1, 0, 1, and 2 litters of the control, low-, mid- and high-dose group, respectively. Only in the control group 1 female with all stillborn pups was observed.
One female of the mid-dose group (C565) showed only 2 implantation sites in the uterus at necropsy.
At necropsy on GD 24 one female of the high-dose group (D587) showed an autolytic foetus and placenta in the left uterus horn with possibly an abdominal hernia and a live large pup in the right uterus horn; 4 implantation sites were observed. The abdominal hernia might have been due to the autolytic status of the foetus. No other indications of developmental toxicity were observed.
Post-implantation loss was 23.6, 9.6, 18.4 and 18.0% for the control, low-, mid- and high-dose groups, respectively.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
7 500 mg/kg diet
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 500 mg/kg diet
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter size and sex:
The number of pups delivered per litter was comparable in all groups and amounted to 9.1, 10.8, 10.9 and 10.3 for the control, low-, mid- and high-dose groups, respectively. The number of liveborn pups amounted to 81, 54, 97 and 101 for the control, low-, mid-and high-dose groups, respectively. The number of stillborn pups was 1, 0, 1 and 2 for the control, low-, mid- and high-dose groups, respectively. Pup mortality on PN 4 was
comparable in all groups and amounted to 4, 0, 2, 5 (incidences 4.9, 0, 2.1 and 5.0 %) in the control, low-, mid- and high-dose groups, respectively. In the control, low-, mid-and high-dose groups 1, 0, 0 and 0 litters, respectively were lost entirely between PN 0-4. The number of live pups per litter was comparable between the groups on PN 1 (10.1, 10.8, 10.8, 10.1 for the control, low-, mid- and high-dose groups, respectively) and PN 4 (9.6, 10.8, 10.6, 9.6. for the control, low-, mid- and high-dose groups, respectively).
No difference was observed in the sex ratio between the groups.

Litter weight:
No effect on pup weight and pup weight change was observed in the tetraoctylstannane-treated groups when compared to the control group.

Pup observations:
Although a slight increase in the number of runts (pup weight less than mean pup weight of the control group minus 2 standard deviations) was observed in the high-dose group on PN 4, there was no statistically significantly difference observed between the tetraoctylstannane-treated groups and the control group. The findings were normal for pups of this age and are considered not to be related to treatment.

Pathology of pups:
Macroscopic observation of the stillborn pups revealed one partly cannibalized pup in the control group and 2 autolytic pups in the high-dose group; in these pups no abnormalities were observed. In the stillborn pup of the mid-dose group, no abnormalities were observed.

Effect levels (fetuses)

Remarks on result:
other: not specified

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the observed effects in the high-dose animals, decrease in thymus (female animals), the No Observed Advers Effect Level (NOAEL) for general toxicity is established at the mid-dose level (1,500 mg/kg diet which is equivalent to 80-141 mg/kg body weight/day for the female animals). As no significant reproductive toxicity was established at the high dose level (7,500 mg/kg diet which is equivalent to 426-624 mg/kg body weight in the female animals).
Executive summary:

The objective of this study was to provide data on the possible reproductive and developmental effects of tetraoctylstannane [CAS # 3590-84-9] after oral administration via the diet to Wistar rats of both sexes. The study was combined with a repeated dose toxicity study and preceded by a dose-range finding study. In the dose-range finding study, rats were fed diets containing 0, 100, 500, 2,000 and 10,000 mg tetraoctylstannane/kg diet for 14 days. In the main study, rats were fed diets containing 0, 500, 1,500 and 7,500 mg tetraoctylstannane/kg diet for up to 33 days (males) or during 2 weeks pre-mating, mating, gestation and up to day 4 or 5 of lactation (females).

No treatment-related mortalities or clinical signs were observed.

In male animals tested after 4 weeks of treatment and in female animals tested on postnatal day (PN) 4, no changes indicative of neurotoxic potential of the test substance were observed in the neurobehavioural observations and motor activity assessment.

Mean body weight and body weight change in male and female animals of the tetraoctylstannane-treated groups were similar amongst the groups.

The test substance intake of the male animals during the study ranged from 29-33, 86-99 and 445-480 mg/kg body weight/day for the low-, mid- and high-dose group, respectively.

The test substance intake of the female animals during the pre-mating, gestation and lactation period ranged from 25-42, 80-141 and 426-624 mg/kg body weight/day for the low-, mid- and high-dose group, respectively.

In each group 12 females were placed with males and all females were mated. The number of pregnant animals was comparable in the control, mid- and high-dose groups and amounted to 9, 10 and 11 respectively. In the low-dose group a rather low number of 5 pregnant females was observed. At necropsy, three of the non-pregnant females of this group showed hydrometra of the uterus, which was confirmed upon microscopic examination by luminal dilation. In the absence of similar findings in the mid- and high-dose groups this finding is considered to be incidental.

No statistically significant effects were observed on the female fecundity, male and female fertility, gestation index, pre-coital time, number of females with live-born pups, sex ratio, number of females with stillborn pups, number of females with only stillborn pups, post-implantation loss, number of pups delivered, number of live-born pups, number of live pups/litter, number of stillborn pups, pup mortality PN 4, pup weight PN 1 and 4 and number of runts.

At the end of the pre-mating period, no treatment-related findings were observed in plasma of male and female animals on haematology and clinical chemistry. weight of the male animals of this group was decreased (approx. 32%), but not statistically significantly. In the females of the high-dose group, the absolute and relative thymus weights were statistically significantly decreased by approx. 60%.

No other effects on organ weights (absolute or relative) were observed.

As no significant reproductive effects were observed in the tetraoctylstannane-treated groups, the NOAEL for reproductive toxicity was established at the high-dose level (7,500 mg/kg diet which is equivalent to 445-480 mg/kg body weight in the male animals and 426-624 mg/kg body weight in the female animals).