Trichlorooctylstannane

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th February - 10th June 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study conducted under GLP. The method of analysis involved derivatization. This method only measures the amount of the alkyltin moiety, MOT, present and does not identify the other ligands attached to the tin. All measured MOT was attributed to the parent substance. Currently there is no analytical method available that can quantify the actual named substance, i.e., the entire organotin compound with its associated chloride ligand.

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 421

Deviations:

no

Principles of method if other than guideline:

Type: other: reproduction/developmental screening study
Method: other: OECD Guide-line 421

GLP compliance:

yes

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

Trichlorooctylstannane

EC Number:

221-435-4

EC Name:

Trichlorooctylstannane

Cas Number:

3091-25-6

Molecular formula:

C8H17Cl3Sn

IUPAC Name:

trichlorooctylstannane

Details on test material:

SOURCE: ORTEP Association Stabilizer Task Force
PURITY: 89.2% Trichlorooctylstannane (monooctyltin trichloride)
ANY OTHER INFORMATION:
Alkyl group distribution % (mass/mass)
Monooctyltin trichloride 85.50
Dioctyltin dichloride 11.07
Trioctyltin chloride 0.50
Tin tetrachloride 0.03
Mono-i-butyltin trichloride 0.42
Monobutyltin trichloride 0.03
Monooctyltin trichloride isomers 0.91
Octyl-i-butyltin dichloride 0.12
Octylbutyltin dichloride 0.05
Monododecyltin trichloride 0.03
Dioctyltin dichloride isomer 0.06
Monohexadecyltin trichloride 1.10
Octylhexadecyltin dichloride 0.17

The alkyl group distribution of trichlorooctylstannane was determined using a Grignard ethylation (ethylmagnesium bromide) followed by GC-analysis with FID detection. The concentration of trichlorooctylstannane in feed was determined by GC-MS analysis of the hexane extracts. The homogeneity and stability of the test substance were determined for each group by a one way ANOVA (sample location and time as grouping factor, respectively).

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Obtained from a colony maintained under SPF conditions at Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: In the 13 weeks study, the animals were 7 weeks old at study initiation, in the satelite group, the animals were 13-14 weeks of age.
- Weight at study initiation: 137.7 - 168.0 g (mean 154.4 g) for males;  110.4 - 135.1 g (mean 122.4 g) for females and 187.1 g to 214..2 g (mean 200.4 g) in the satelite group.
- Housing: Animals were housed under conventional conditions in one room, in macrolon cages, with sterilised wood shavings (Woody Clean 3/4) as bedding material and environmental enrichment (shreds of paper), 2 (dose range finding study) or 5 rats per cage (13-week study), separated by sex. During the premating period females of the satelite groups were housed 3 or 4 per group per cage. During the gestation and lactation period the females were housed individually.
- Diet : RM3 (commercial Rat & Mouse No. 3 Breeding Diet) diet was provided ad libitum as a powder, refreshed once a week, provided in stainless steel cans covered by a perforated steel plate to prevent spillage.
- Drinking water: Tap water was provided ad-libitum in polypropylene bottles.
- Acclimation period: The quarantine and acclimatisation periods between arrival and experimental start date were 5, 13 and 13 days (range finder, definitive test and satelite groups respectively).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): at least 30% not exceeding 70 % other than during room cleaning (max 99.9 %)
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): Artificial light with a sequence of 12 hours light and 12 hours dark.

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The experimental diets were prepared shortly prior to study initiation, and roughly every six weeks thereafter.
- Mixing appropriate amounts with (Type of food): The test material was mixed with RM3 diet with a mechanical mixer.
- Storage temperature of food: <-18 °C freezer until use

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

GC-MS was used to determine the achieved concentration, homogeneous distribution and stability of the test substance in diet samples. The method of analysis involved derivatization. This method only measures the amount of the alkyltin moiety, MOT, present and does not identify the other ligands attached to the tin. All measured MOT was attributed to the parent substance. Currently there is no analytical method available that can quantify the actual named substance, i.e., the entire organotin compound with its associated chloride ligand.

From each diet sample, 2.0 g was transferred into a 50 mL Greiner tube. An aliquot of the internal standard solution (monoheptylin trichloride, diheptylin dichloride, tripopyltin chloride and tetrapropyltin in methanol) was added. Subsequently methanol, acetate buffer solution (pH 4.5), 20 % aqueous NaBEt4 solution (STEB solution) and hexane (with naphtalene as internal standard) were added to each sample, this mixture was agitated and heated to 60 °C. During this step, the organotin chlorides were converted into the corresponding ethylated tetraorganotin derivatives, which were extracted into the hexane layer. Prior to GC-MS analysis, the hexane layer was washed with 2 mol/L HCL to remove the majority of the ethylboron compounds that interfere with the GC-MS analysis of the hexane extracts.
The homogenous distribution, stability and acheived concentration of the test substance in RM3 rat feed was analysed in the batch of diets prepared for the dose range finding study. The homogenous distribution and acheived concentration of the test substance in RM3 rat feed of the high dose-group of the 13-week study (500 mg/kg) was determined in the first batch of diets prepared for the 13 wek study. Validation of the high dose level was performed simultaneously with the homogeneity and content analyses in the first batch of diets prepared for the 13-week study.#
The same diet preparation protocol was used in the dose range finding study and the 13-week studies.
The criteria for homogeneity, stability and content of the test substance in the diet were:
Homogeneity:- For each group a one way analysis of variance was performed using the sample location (1-5) as a grouping factor. An associated F-value with probability p < 0.01 were considered to be significant. The test substance was considered to be homogeneously distributed in the diets if p = 0.01 and/or if the relative standard deviation (RSD) between the sample means was less than or equal to 15%.
Stability:- For each group a one way analysis of variance (Anova) was performed using time as a grouping factor. An associated F-value with probability p < 0.01 was considered to be significant. The test substance was considered to be stable in the diets if p = 0.01 and/or if the mean concentration on the last day was between 80 and 120 % of the mean concentration on the first day (t = 0).
Acheived concentration: For each concentration level, the mean of the concentrations as measured in the study samples used for the assessment of the homogeneity was considered to represent the acheived concentration. The content of the test substance in diet was considered to be "close to intended" if the mean measured concentration was between 80 % and 120 % of the intended concentration. Directly after mixing of each diet for the dose range finding study, samples for the homogeneity/stability experiments were taken in the order; top centre, middle centre, bottom centre, left centre, right centre and labelled as such. Secondly, five samples (of about 50 g each) for examination of the stability were taken from the top centre part of the mixer. All samples wer labelled with the diet codes (TNO study number), the colour-codes, the nominal concentrations of the test substance and the date of preparation. The samples taken for the homogeneity experiments were also used for dose confirmation. In addition, analyses to determine the content (acheived concentration) of the test substance in three batches of diets used in the 13 week study were conducted.
Diet samples for the determination of the content of the diets used in this study were taken immediately after preparation of the diets and stored at ca. -18 °C.

Duration of treatment / exposure:

Diet was available ad libitum throughout the study

Frequency of treatment:

daily

Duration of test:

13 week study

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 100 and 500 mg trichlorooctylstannane/kg diet
Basis:
nominal in diet

No. of animals per sex per dose:

40 females (four dose groups of 10 rats)

Control animals:

yes, concurrent no treatment

Details on study design:

MATING PROCEDURES SATELLITE GROUPS: During the premating period females were housed 3 or 4 per group per cage. Male rats of the 13 week study were mated after a premating period of 10 weeks with female rats. During the mating period one male and one female of the same dose group were caged until copulation occurred or two weeks had elapsed. The day a vaginal smear was detected sperm positive, was considered gestation day 0. During the gestation and lactation periods the females were housed individually. If a male died before or during the mating period before the female was found sperm positive, the female was mated with another, proven male of the same group (i.e. a male which already had a successful copulation, sperm positive smear with another female).

CLINICAL OBSERVATIONS AND FREQUENCY:
- Clinical signs: at least once daily (in the morning) and on working days also once in the afternoon.
- Mortality: at least once daily (in the morning) and on working days also at least once in the afternoon.
- Body weight: once during the acclimatization period, once at initiation of the study prior to introduction of feed. The females of the satellite groups were weighed 4 days before gestation, on gestation days 0, 7, 14 and 21 and on postnatal (PN) days 1 and 4. Furthermore, all surviving animals were weighed on the day of necropsy in order to determine their correct organ to body weight ratios.
- Food consumption: measured per cage over weekly periods during the premating period, the gestation periodn and from PN 1-4, by weighing the feeders (in g/animal/day).
- Water consumption: provided ad libitum, the amount consumed was not measured.
- Intake of test substance: the intake of substance per kg/bw/day was calculated from the nominal dietary concentration of the substance, the food consumption and the mean body weight in the period for which the intake of the substance was calculated.
- Mating: during the mating period every consecutive morning vaginal smears were made to ascertain copulation by detection of sperm cells in the smear.
- Parturition and litter evaluation: at the end of the gestation period, females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum, the litters were examined only once daily for dead pups.
- Litter size, sexes and weights: the total litter size and numbers of each sex as well as the number of stillbirths, live and dead pups and grossly malformed pups were evaluated on days 1 and 4 of lactation. The pups were weighed individually and litter weight was calculated for days 1 and 4 of lactation. Mean pup weights were calculated as litter weight/number pups. The number of runts (< 2 sd from the litter mean) were noted and reported.

ORGANS EXAMINED AT NECROPSY:
- Macroscopic: all adult animals were subjected to a complete gross necropsy. Organs weighed included: ovaries, uterus (after counting of the implantation sites), thymus and all gross lesions. Samples of latter organs were preserved for microscopic examination. Necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded. Pups were examined externally for gross abnormalities and killed by hypothermia at < -18°C.
- Microscopic: microscopic examination of the ovaries, uterus and thymus of the control and high dose group (500 mg/kg) was performed. Microscopic examination was extended to the thymus of the females of the 10 and 100 mg/kg groups because of the effects observed in the 500 mg/kg group for this tissue/organ. Furthermore, the reproductive organs of the males of the 10 and 100 mg/kg groups that failed to sire (did not mate or female was not pregnant), and the reproductive organs of females of the 10 and 100 mg/kg groups that were non-mated or non-pregnant, were microscopically examined.

Statistics:

With regard to fertility and reproductive performance, 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
-viability index day 1-4 = (number of pups surviving 4 days/total number  of live pups on day 1) 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

Results and discussion

Effect levels

1

Dose descriptor:

NOEL

Effect level:

100 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Sub-chronic toxicity. Based on the decreased thymic weights and associated histopathological findings. Equivalent to 6.4 mg/kg bw/day for males and 6.8 mg/kg by/day in females.

2

Dose descriptor:

NOEL

Effect level:

100 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive effects: Based on the incidences in several parameters, together considered as post-implantation losses. 6.4 mg/kg bw/day in males and 4.8-7.7 mg/kg by/day in females.

3

Dose descriptor:

NOAEL

Effect level:

10 mg/kg diet

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Maternal toxicity: Based on histopathological changes in the thymus (severe lymphoid depletion and very severe atrophy_/

Observed effects

NOAEL (fertility and developmental effects): 100 mg/kg
NOAEL (maternal toxicity): 10 mg/kg

Any other information on results incl. tables

NOAEL (fertility and developmental effects): Based on reproductive and  developmental effects observed after mating of female animals of the 500  mg/kg group, the mid dose level of 100 mg substance/kg diet (equivalent  to 6.4 mg MOTC/kg bw/day in males and 4.8-7.7 mg MOTC/kg bw/day for  females) can be considered as a NOAEL for fertility and developmental  effects.
NOAEL (maternal toxicity): Based on the observed histopathological  changes in the thymus (severe lymphoid depletion) of the female animals  of the 100 and 500 mg/kg groups, the low dose of 10 mg substance/kg diet  (equivalent to 0.5-0.7 mg MOTC/kg bw/day for females) can be considered  as a NOAEL for maternal toxicity.
TEST SUBSTANCE INTAKE:
The test substance intake of the female animals of the 10, 100 and 500  mg/kg groups was respectively:
Premating period
days 0-7: 0.6, 6.1 and 29.1 mg MOTC/kg bw/day
days 7-14: 0.6, 6.1 and 28.3 mg MOTC/kg bw/day
Gestation period
GD 0-7: 0.7, 6.9 and 31.7 mg MOTC/kg bw/day
GD 7-14: 0.7, 6.8 and 32.6 mg MOTC/kg bw/day
GD 14-21: 0.5, 4.8 and 22.3 mg MOTC/kg bw/day
Lactation period
PN 1-4: 0.7, 7.7 and 33.0 mg MOTC/kg bw/day
Analysis of the test subtance in diet samples revealed that the test  substance dose was close to the nominal level for all diets.  Mean  measured concentrations of trichlorooctylstannane in the test diets  ranged from 101-117% of nominal concentrations.
Homogeneity: The test substance was considered to be homogeneously  distributed in all diets.
Stability: The test substance was considered to be stable in the diets  upon storage at room temperature for 7 days and upon storage at < -18°C  for 6 weeks.
MATERNAL TOXIC EFFECTS:
- Mortality and day of death: One animal was found dead on PN 2 (i.e. 39  days after the start of exposure).
- Pre-coital time: comparable among the control and treated groups
- Mating index: 100% for all groups
- Number pregnant per dose level: 9, 9, 7 and 7 for the control, 10, 100  and 500 mg/kg groups, respectively.
- Female fecundity index: comparable among the control and treated groups
- Female fertility index: comparable among the control and the treated  groups
- Male fertility index: comparable among the control and the treated  groups.
- Number aborting: No data available.
- Number of resorptions: No data available.
- Number of implantations: 11-15 (control group), 10-14 (10 mg/kg), 9-13  (100 mg/kg), 10-14 (500 mg/kg).
- Gestation index: 100, 100, 100 and 71% in the control, 10, 100 and 500  mg/kg groups, respectively.
- Live birth index: 100, 99, 97 and 74% in the control, 10, 100 and 500  mg/kg groups, respectively.
- Number of females with live born pups: 9, 9, 7 and 5 in the control,  10, 100 and 500 mg/kg groups, respectively.
- Number of females with stillborn pups: 0, 1, 1 and 3 for the control,  10, 100 and 500 mg/kg groups, respectively.
- Number of females with all stillborn pups: Only in the 500 mg/kg group;  2 females with all still born pups were observed.
- Post implantation loss: 6.4, 6.3, 14.5 and 41.3% for the control, 10,  100 and 500 mg/kg groups, respectively.
- Number of corpora lutea: No data available.
- Duration of Pregnancy: 21-23 days
- Clinical signs: No treatment related clinical signs were observed  during the premating, mating, gestation and lactation periods.
- Body weight (gain): During the premating, gestation and lactation  periods, mean body weights and body weight changes of the animals were  similar among the groups.
- Food consumption: During the premating, gestation and lactation  periods, mean food consumption of the animals were similar among the  groups.
- Description, severity, time of onset and duration of clinical signs: No  data available.
- Hematological findings incidence and severity: No data available.
- Clinical biochemistry findings incidence and severity: No data  available.
- Gross pathology incidence and severity: No treatment-related changes  were observed, including in the female animal that died on PN 2.
- Organ weight (changes): Mean absolute uterus weight was statistically  significantly increased in the females of the 10 mg/kg group when  compared to the control group.  This difference was considered to be a  chance finding.  No other differences were observed in absolute and  relative uterus and ovary weights.  Mean absolute and relative thymus  weights of the females of the 100 and 500 mg/kg groups were decreased by  24-26% and 31-35%, respectively.  However, these decreases were not  statistically significant.
- Histopathology incidence and severity: Only reproductive organs and  thymi were examined microscopically.  A cause of death for the female  which died on PN 2 could not be established.
Examination of the thymus revealed moderate to severe lymphoid depletion  in 8/9 females of the 500 mg/kg group (evaluation of the thymus of the  female which died on PN 2 was hampered by autolytic changes).  Lymphoid  depletion was characterized by a decrease in the size of the thymic  lobules which can be ascribed to extensive loss of cortical and medullary  small lymphocytes.  Consequently, the distinction between the cortical  and medullary areas was blurred.  In severe cases the cortex was very  small, or even partially absent.  The remaining lymphoid cells visible in  the cortical areas were predominantly lymphoblasts.  Lymphoblastic cells  and reticuloepithelial cells had increased in number or these cells were  more pronounced due to the disappearance of small lymphocytes and  collapse of thymic stroma.  In three females of the 100 mg/kg group and  in one female of the 500 mg/kg group, only remnants of thymic tissue were  seen, denoted as 'very severe atrophy'.  This thymic tissue was  characterized by very small lobules, with complete loss of the normal  architecture, consisting predominantly of reticuloepithelial cells/stroma  and only a few lymphoblasts and lymphocytes.  The statistically  significant decrease in incidence of pregnancy/lactation involution seen  in the thymus of animals of the 500 mg/kg group, is most probably due to  the presence of overt treatment related lymphoid depletion, which  obscured the lactation-related involution.  Pregnancy/lactation  involution, characterized by a decreased size of the thymic lobules  exhibiting normal architecture, is a common observation in pregnant or  lactating animals.
FETAL DATA:
- Litter size: The mean number of pups delivered per litter amounted to  11.6, 11.4, 10.3 and 8.9 for the control, 10, 100 and 500 mg/kg groups,  respectively.  The number of pups delivered was statistically  significantly decreased in the 500 mg/kg group.
- Litter weight: Mean pup weights and pup weight changes were comparable  among the control and treated groups.
- Pup mortality: 0, 1.0, 2.8 and 26% for the control, 10, 100 and 500  mg/kg groups, respectively (PN 1); 1, 7.8, 0 and 20% for the control, 10,  100 and 500 mg/kg groups, respectively (PN 4). 
- Number viable: The viability index (PN 1-4) was 99, 92, 100 and 80% in  the control, 10, 100 and 500 mg/kg groups, respectively.
- Number live pups per litter: 11.6, 11.3, 10.0 and 9.2 for the control,  10, 100 and 500 mg/kg groups, respectively (PN 1); 11.4, 10.4, 10.0 and  9.3 mg/kg for the control, 10, 100 and 500 mg/kg groups, respectively (PN  4)
- Sex ratio: No difference was observed in the sex ratio between the  groups.
- Postnatal growth: No data available; screening study.
- Postnatal survival: No data available; screening study.
- Grossly visible abnormalities: The incidence of runts in the 10 mg/kg  group was statistically significantly increased on PN 4; this observation  was only significant on pup basis.  No statistically significant  difference in the number of runts was observed between the treated groups  and the control group.  For these reasons, the effect in the 10 mg/kg  group is considered to be a chance finding.  Macroscopic observation of  the stillborn pups revealed no abnormalities of the pups.  Four stillborn  pups of the 500 mg/kg group were (partly) cannibalized; as far as  possible no abnormalities were observed in these pups.
- External abnormalities: No data available; screening study.
- Soft tissue abnormalities: No data available; screening study.
- Skeletal abnormalities: No data available; screening study.

Applicant's summary and conclusion

Conclusions:

Based on the incidences in several parameters which together are considered post-impantation losses at the 500 mg/kg dose, the mid-dose level of 100 mg Trichlorooctylstannane/kg (equivalent to 6.4 mg/kg bw day in males and 4.8 - 7.7 mg/kg bw day for females) is judged to be the NOAEL for reproductive effects.
Based on the observed histopathological changes in the thymus (severe lymhoid depletion and very severe atrophy) of the 100 and 500 mg/kg female animals of the satellite groups, 10 mg Trichlorooctylstannane/ kg diet (equivalent to 0.5 - 0.7 mg/kg bw day) can be considered as a NOAEL for maternal toxicity.

Executive summary:

Based on the incidences in several parameters which together are considered post-impantation losses at the 500 mg/kg dose, the mid-dose level of 100 mg Trichlorooctylstannane/kg (equivalent to 6.4 mg/kg bw day in males and 4.8 - 7.7 mg/kg bw day for females) is judged to be the NOAEL for reproductive effects.

Based on the observed histopathological changes in the thymus (severe lymhoid depletion and very severe atrophy) of the 100 and 500 mg/kg female animals of the satellite groups, 10 mg Trichlorooctylstannane/ kg diet (equivalent to 0.5 - 0.7 mg/kg bw day) can be considered as a NOAEL for maternal toxicity.