2-ethylhexyl 10-ethyl-4,4-dioctyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study conducted under GLP. Justification for read-across : This study was initially considered to be relevant and adequate read-across data for the Dioctyltin bis (EHMA) (CAS N0 15571-58-1) as DOT(2-EHMA) was demonstrated to be readily hydrolysed to Dichlorodioctyltilstanane (CAS no.3542-36-7) under physiological conditions (see section 7.1.1). Thus DOTC(Dichlorodioctylstannane) was considered to be an appropriate anchor compound and surrogate for the mammalian toxicology endpoints of repeated dose, in vivo genetic toxicity, reproduction and developmental effects, when it is dosed via the oral route of administration. Read-across to the substance DOTC is no longer considered as wholly appropriate based on the results of the recent Hydrolysis study, as reported by Naßhan, H, 2014 (see section 7.1.1) which indicate the substance DOTCl is the only metabolite of DOTE which is formed in a simulated mammalian gastric environment; no dioctyltindichloride was formed under the conditions of the study.

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 421- reproduction/ developmental screening study

Deviations:

yes

Remarks:

None that affected the validity of the study.

GLP compliance:

yes

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

Dichlorodioctylstannane (dioctyltin dichloride) 94% purity

IUPAC Name:

Dichlorodioctylstannane (dioctyltin dichloride) 94% purity

Constituent 2

Reference substance name:

Dichlorodioctylstannane

EC Number:

222-583-2

EC Name:

Dichlorodioctylstannane

Cas Number:

3542-36-7

Molecular formula:

C16H34Cl2Sn

IUPAC Name:

dichloro(dioctyl)stannane

Details on test material:

SOURCE: ORTEP Association Stabilizer Task Force
PURITY: 94.1% Dichlorodioctylstannane (dioctyltin dichloride)

ANY OTHER INFORMATION:
Alkyl group distribution % (m/m)
Monooctyltin trichloride 2.25
Dioctyltin dichloride 94.09
Trioctyltin chloride 0.70
Tin tetrachloride 0.05
Mono-i-butyltin trichloride 0.01
Octyl-i-butyltin dichloride 0.78
Octylbutyltin dichloride 0.16
Octylhexyltin dichloride 0.03
Dioctyltin dichloride isomers 0.64
Octyldodecyltin dichloride 0.03
Monohexadecyltin trichloride 0.03
Octylhexadecyltin dichloride 1.19
Tetraoctyltin 0.04

The alkyl group distribution of the test substance was determined using a Grignard ethylation (ethylmagnesium bromide), followed by GC analysis with FID detection. The concentration of dichlorooctylstannane in feed was determined by GC-MS analysis of the hexane extracts. The homogeneity and stability of the test substance was 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 ORGANISMS:
- Species: Albino rats
- Strain: Wistar outbred (Crl:(WI)WU BR)
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 15-16 weeks old
- Weight at study initiation:  185.5 - 212.8 g (mean 197.4 g) for females

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

- Type of exposure: via the diet (rodent diet: Rat & Mouse No.3 breeding diet RM3)
- Post exposure period: none
- Vehicle: not applicable
- Concentration in vehicle: not applicable
- Total volume applied: not applicable
- Doses: 0, 10, 100, 300 mg dichlorooctylstannane/kg diet
- Particle size: not applicable
- Type or preparation of particles: not applicable

The test substance intake of the female animals of the 10, 100 and 300 mg/kg groups were, respectively: Premating period days 0-7: 0.6, 5.8 and 13.5 mg/kg bw/day days 7-14: 0.7, 5.9 and 16.4 mg/kg bw/day Gestation period GD 0-7: 0.7, 6.2 and 16.6 mg/kg bw/d.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

Duration of exposure: daily for 2 consecutive weeks during the premating period, daily during gestation (up to 26 days after study initiation) and up to euthanasia at or shortly after postnatal day (PN) 4.

Frequency of treatment:

daily

Duration of test:

Approximately 18 weeks for the males and 6 weeks for females of the satellite groups

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 100, 300 mg dichlorooctylstannane/kg diet
Basis:
nominal in diet

No. of animals per sex per dose:

Number of animals: 44 females (four dose groups of 10 rats/sex)

Control animals:

yes, concurrent no treatment

Details on study design:

Duration of test: 6-8 weeks

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  (see section 5.4 Repeated Dose Toxicity) were mated after a premating  period of 10 weeks with the 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 and once weekly thereafter. The females of the satellite groups were weighed 4 days before gestation, on gestation days 0, 7, 14 and 21 and on postnatal 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 premating and gestation period 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 is 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 as well.

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 (300 mg/kg) groups was performed. Microscopic examination was extended to the thymus of the females of the low (10 mg/kg) and mid dose (100 mg/kg) groups because of the effects observed in the 300 mg/kg group for this tissue/organ. Furthermore, the reproductive organs of the males of the low (10 mg/kg) and mid dose (100 mg/kg) groups that failed to sire (did not mate or female was not pregnant) and the reproductive organs of females of the low (10 mg/kg) and mid dose (100 mg/kg) groups that were non-mated or non-pregnant were microscopically examined.

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

Statistics:

- Test substance analysis: Homogeneity: one way analysis of variance (Anova) using the sample location (1-5) as grouping factor. 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: one way analysis of variance (Anova) using time as grouping factor. 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).
Achieved concentration: for each concentration level, the mean of the concentrations, as measured in the diet samples used for the assessment of
the homogeneity, was considered to represent the achieved concentration. The content of the test substance in the diet was considered to be
'close to intended' if the mean measured concentration was between 80% and 120% of the intended concentration.

- Body weight: one way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests.
- Food consumption and food efficiency: one way analysis of variance (Anova) followed by L.S.D. tests.
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females and females with live pups. Number of implantation sites, live and dead pups were evaluated by Kruskal-Wallis non-parametric Anova followed by the Mann-Whitney U-test.
- The litter was used as the statistical unit for calculation of fetal values.
- Histopathological changes: Fisher's exact probability test.

All tests were two-sided. Probability values of p<0.05 were considered significant.

Results and discussion

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

reproduction toxicity

Effect level:

0.5 - 0.7 mg/kg bw/day

Sex:

female

Basis for effect level:

other: Based on reproductive and developmental effects: animals showing only implantations at necropsy, animals delivering only dead pups, decreases in gestation, live birth and viability indices and increases in post-implantation loss and number of runts

2

Dose descriptor:

LOAEC

Remarks:

general toxicity

Effect level:

0.5 - 0.7 mg/kg bw/day

Sex:

female

Basis for effect level:

other: decreases in absolute and relative thymus weights associated with treatment related lymphoid depletion at 10, 100 and 300 mg/kg/day groups

Observed effects

TEST SUBSTANCE INTAKE:
The test substance intake of the female animals of the 10, 100 and 300 mg/kg groups were, respectively:
Premating period
days 0-7: 0.6, 5.8 and 13.5 mg/kg bw/day
days 7-14: 0.7, 5.9 and 16.4 mg/kg bw/day
Gestation period
GD 0-7: 0.7, 6.2 and 16.6 mg/kg bw/day
GD 7-14: 0.7, 6.2 and 17.0 mg/kg bw/day
GD 14-21: 0.5, 4.2 and 11.0 mg/kg bw/day
Lactation period
PN 1-4: 0.7, 5.0 and 8.4 mg/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 dichlorooctylstannane ranged from 94-114% of the nominal concentration.
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 24 hours, but not for 4 and 7 days, and
upon storage at < -18°C for 6 weeks.

For the toxic effects observed, see Table 1 below.

Any other information on results incl. tables

Table 1: Toxic effects observed

MATERNAL TOXIC EFFECTS:
-Mortality and day of death:	No mortality occurred.
- Pre-coital time:	Comparable among the control and treated groups.
- Mating index:	90-100% for all groups
- Number pregnant per dose level:	7, 8, 7 and 8 for the control, 10, 100  and 300 mg/kg groups, respectively.
- Female fecundity index:	Comparable among the control and treated groups.
- Female fertility index: .	Comparable among the control and treated groups
- Male fertility index: .	Comparable among the control and treated groups
- Number aborting:	No data available.
- Number of resorptions:	No data available.
- Number of implantations:	11-14 (control group), 11-16 (10 mg/kg), 10-13  (100 mg/kg), 8-13 (300 mg/kg)
- Gestation index:	86, 100, 71 and 50% in the control, 10, 100 and 300  mg/kg groups, respectively.
- Live birth index:	99, 95, 53 and 60% in the control, 10, 100 and 300  mg/kg groups, respectively.
- Number of females with live born pups:	6, 8, 5 and  4 in the control,  10, 100 and 300 mg/kg groups, respectively.
- Number of females with stillborn pups:	1, 1, 4 and 3 for the control,  10, 100 and 300 mg/kg groups, respectively.
- Number of females with all stillborn pups:	0, 0, 2 and 1 in the  control, 10, 100 and 300 mg/kg groups, respectively.
- Post implantation loss:	22.3, 21.0, 49.2 and 70.0% for the control, 10,  100 and 300 mg/kg groups, respectively.
- Number of corpora lutea:	No data available.
- Duration of Pregnancy:	21-23 days
- Clinical signs:	No clinical signs were observed during the premating  period. During the gestation period, piloerection was observed in one  animal of the control group and one animal of the 300 mg/kg group.  In  addition, blepharospasm was observed in the same animal of the 300 mg/kg  group.  During lactation, piloerection was observed in one animal of the  100 mg/kg group and in two animals of the 300 mg/kg group.  Blepharospasm  was observed in one animal of the 300 mg/kg group.  Two animals of the  100 mg/kg group and one animal of the 300 mg/kg group were considered to  be thin.  In addition, two animals of the 100 mg/kg group showed a pale appearance.  Some animals were sparsely haired during gestation and/or  lactation.  However, this finding is considered normal for this strain.
- Body weight (gain):	During the premating period no significant  differences in mean body weight were observed. Mean body weight change  was statistically significantly reduced in the 100 and 300 mg/kg groups during the first week of the premating period (4.2 -6.2 and 8.4 -17.0 mg/kg/day, respectively). During the gestation  period, mean body weight was statistically significantly reduced from GD  7-21 in the 300 mg/kg group (from 30 to 60 % over the gestation period).   Body weight change was statistically significantly reduced during the entire gestation period. During the  lactation period (day 1 -4), the mean body weight was statistically significantly reduced in the 100 and 300 mg/kg groups (5.08 and 3.88 g vs. 11.52 in controls).

- Food consumption:	During the premating period, mean food consumption (g/animal/day and g/kg bw/day) of the female animals of the 100 and 300  mg/kg groups was statistically significantly decreased.   During the  gestation period, food consumption (g/animal/day) of the females of the 100 mg/kg group was statistically significantly decreased from GD 7-14. Mean food consumption of the 300 mg/kg group was statistically  significantly decreased (g/animal/day) during the entire gestation period  and as g/kg bw/day from GD 0-14.  During the lactation period, food  consumption of the female animals of the 300 mg/kg group was  statistically significantly decreased.
- 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
-Organ weight changes:	Absolute and relative uterus and ovary weight  were similar among all groups.  The absolute and relative thymus weights  were statistically significantly decreased in the 100 mg/kg (62 and 67%)  and 300 mg/kg (31 and 38%) groups relative to the control group.
-Histopathology incidence and severity:	Microscopic examination revealed  moderate to very severe lymphoid depletion in the thymus, which was  considered related to treatment.  Lymphoid depletion was characterized by  a decrease in the size of the thymic lobuleswhich can be ascribed to  extensive loss of cortical and medullary small lymphocytes.   Consequently, the distinction between the cortical and medullary area was  blurred.  Lymphoid depletion was observed in 5/10 of the 10 mg/kg groupand in all animals of the 100 and 300 mg/kg groups.   Surprisingly, one  control animal also had very severe lymphoid depletion in the thymus. However, this was most probably associated with the fact this animal was  physiologically disturbed,as was demonstrated by 12 resorptions in the  uterus and an abnormal kidney (gross change: flabby and yellow patches).    Some animals of the 10 mg/kg group showed thymic involution as a result  of pregnancy/lactation.  This picture was similar to the thymic  pregnancy/lactation involution in control animals and was characterized  by a decreased size of thymiclobules exhibiting normal architecture. This phenomenon is a common observation in pregnant or lactating animals.   However, the lymphoid depletion in the animals of the 10 mg/kg group was  similar to the thymic change in the animals of the 100 and 300 mg/kg  groups. Therefore, lymphoid depletion in animals of the 10, 100 and 300  mg/kg groups was considered related to treatment with  dichlorooctylstannane

FETAL DATA:
- Litter size:	The mean number of pups delivered per litter amounted to  11.7, 11.0, 10.3 and 8.6 for the control, 10, 100 and 300 mg/kg groups,  respectively.
- Litter weight:	Mean pup weights and pup weight changes were similar in  the 10 and 100 mg/kg groups when compared to the control group.  Pup  weight of the 300 mg/kg group (PN 1, 3 litters and PN 4, 1 litter) was  reduced
- Pup mortality:	1.4, 4.5, 47 and 40% in the control, 10, 100 and 300  mg/kg groups, respectively (PN 1); 5.8, 8.3, 26 and 88% in the control,  10, 100 and 300 mg/kg, respectively (PN 4)
- Number viable pups:	The viability index (PN 1-4) was 94, 92, 74 and 12% in   the control, 10, 100 and 300 mg/kg groups,respectively.
- Number live pups per litter:	11.5, 10.5, 7.6 and 6.5 for the control,   10, 100 and 300 mg/kg groups, respectively (PN 1); 10.8, 11.0, 9.3 and  3.0 for the control, 10, 100 and 300 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:	On PN 1 and 4, the number of runts was  statistically significantly increased in the 100 and 300 mg/kg groups.  In addition, the number of cold pups was increased in the 300 mg/kg group on PN 1.  Macroscopic observations in the stillborn pups revealed no treatment related abnormalities of the 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:

DOTC has reproductive toxic effects, resulting in a NOAEL of 0.5-0.7 mg/kg bw/d. The LOAEL for general toxicity, based on histological changes in the thymus (lymphoid depletion), is 0.5-0.7 mg/kg bw/d.

Executive summary:

A sub-chronic (13 week) oral toxicity study in rats (OECD Test guideline 408), including a satellite group for a reproduction/developmental screening study (OECD Test guideline 421), was carried out with Dichlorodioctylstannane (DOTC). The satellite study used four groups of 10 female rats (reproduction/developmental screening study). The control group was kept on untreated diet and the three test groups received experimental diets containing 10, 100 and 300mg/kg (ppm) of the test substance. The dose levels used in both studies were selected based on the results of a preceding dose range finding study. In the satellite study, female rats were fed their respective diets beginning two weeks prior to the mating period (with males of the subchronic study), and continued through mating, gestation, and up to PN 4 or shortly thereafter. Male rats from the main study were mated after a premating period of 10 weeks with female rats of the satellite groups, which were fed the same dose of test diet. Clinical observations, growth, food consumption, food conversion efficiency, organ weights and gross examination at necropsy, microscopic examination of various organs and tissues and assessment of various reproductive and developmental parameters were used as criteria for detecting the effects of treatment.

The test substance intake for the females ranged from 0.5-0.7, 4.2-6.2 and 8.4-17.0 mg Dichlorodioctylstannane/kg body weight for the 10, 100 and 300 mg/kg groups, respectively.

The following general treatment related effects were observed on the dams:

Mean body weight change in the 100 mg/kg group was only statistically significantly decreased during the first week of the study.

Mean body weight and body weight change of the 300 mg/kg group were statistically significantly reduced during the entire study; a level of significance was achieved during several periods (from 30 to 60% during the gestation period).

In the 100 mg/kg group food consumption was statistically significantly decreased during the premating period and from GD 7-14.

Food consumption of females given 300 mg/kg group was statistically significantly reduced during the entire study; a level of significance was achieved during most periods.

The following treatment related effects were observed on fertility and developmental parameters:

In the 10 mg/kg/day, no treatment-related effects on reproduction and development of the pups were observed.

In the 300 mg/kg group, the following effects were indicative of a reproductive and developmental effect of Dichlorodioctylstannane in the diet :

• Three animals of the 300 mg/kg group showed only implantations at necropsy, one dam delivered only dead pups, the gestation index was only 50% vs.86% in the control group. The live birth index in the 300 mg/kg group was 60% vs. 99% in the control group. The viability index PN 1-4 in the 300 mg/kg group was 12% vs. 94% in the control group. Post-implantation loss of the 300 mg/kg group was 70% vs. 22.3% in the control group. The number of runts on PN 1 and 4 was increased.

In the 100 mg/kg group, the following effects were indicative of a reproductive and developmental effect of Dichlorodioctylstannane in the diet:

• Two dams delivered only dead pups, the gestation index was only 71% vs.86% in the control group. The live birth index in the 100 mg/kg group was 53% vs. 99% in the control group. The viability index PN 1-4 in the 100 mg/kg group was 74% vs. 94% in the control group. Post-implantation loss of the 100 mg/kg group was 49.2% vs. 22.3% in the control group. The number of runts was increased on PN 1 and 4.

Mean absolute and relative thymus weights of the 100 and 300 mg/kg group were decreased by 38-33% and 69-62%, respectively.

At microscopic examination, treatment-related lymphoid depletion was observed in the 10, 100 and 300 mg/kg animais.

Conclusion

Based on reproductive and developmental effects (particularly severe post-implantation losses and fetal losses) observed after mating of 100 and 300 mg/kg female of the satellite groups with male animals of the main study, the low dose level of 10 mg Dichlorodioctylstannane/kg diet (equivalent to 0.7 mg/ kg body weight/day in males and 0.5-0.7 mg/kg body weight for females) can be considered as a NOAEL for fertility and developmental effects.

Based on the treatment related histological changes in the thymus (lymphoid depletion) of the 10 mg/kg female animais of the satellite groups, 10 mg Dichlorodioctylstannane/ kg diet (equivalent to 0.5-0.7 mg/kg body weight/day) was considered to be a LOAEL for maternal toxicity.