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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The NOAEL of the test item for develomental toxicity was set to > 25 mg/kg bw, the high dose, because there were no adverse effects on fetal developmentals or incidences for external, soft tissueu or skeletal anomalies

Link to relevant study records
Reference
Endpoint:
reproductive toxicity, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 June 2016 - 10 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD TG 414
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
DBTE > 95 %
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
In-house bred animals (healthy, yound adult animals)
4 Groups 25 pregnant females per group
110 female and 55 males were received. Males were used for cohabitation with females. After cohab
itation, all males, extra mated and non mated females were euthanized under CO2 anesthesia.
Age at initiation of mating: 10 to 12 weeks
Animal Identification: Acclimatisation period: Cage cards and tail marking by marker pen. Treatment
period: cage cards and body marking by tumeric solution.
Animals were housed under standard laboratory conditions, air-conditioned with adequate fresh air su
pply (12-15 Air changes per hour), room temperature 19.8 to 22.6 oC and relative humidity 49 to 68%
, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were
recorded once daily.
Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
Test Item formulation was prepared daily before administration. The required quantity of test item was
weighted into a clean beaker and there by adding little volume of peanut oil (vehicle) in to the beaker
and was mixed well with glass rod and transferred into a measuring cylinder. The beaker was rinsed
with peanut oil and the volume was transferred to measuring cylinder. This procedure was repeated
until to ensure entire quantity of test item formulation was transferred into measuring cylinder. Fin
ally the volume was made up to required quantity with peanut oil to get a desired concentration of
different dose levels
Details on mating procedure:
After minimum five days of acclimatization period, males and females were cohabitated at 1:2 ratio (one male and two females) until evidence of copulation is observed to obtain the required number ofpregnant rats for each group or for two weeks. Every morning, the vaginal smear of each female wasexamined for presence of sperm in the vaginal smear and/or vaginal plug. The day of confirmation of mating was designated as day ‘0’ of gestation. Each day, the body weight of mated rats (day 0 pregnant females) was recorded and arranged in the ascending order of their body weight. These matedfemales were evenly distributed to all the groups based on their body weights so as to maintaincomparable mean body weight for all groups and permanent identification numbers were assigned.
Animals were kept for mating in seven batches to regulate the number of animals sacrificed on a particular day.
Females not mated within 14 days of pairing with the first male were placed with a second provenmale.
After obtaining required number of pregnant females for each group, the extra mated, non-matedfemales and all males were sacrificed without recording any observations
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis for dose concentration verification was was performed for all dose formulations during week 1 and week 4 of dosing period. The dose formulation samples were collected in duplicates (2 x 5 ml ) for each dose formulation including vehicle control and transferred at ambient conditions for dose confirmation analysis at Auriga Research ltd, Unit-III, No 136, 6th Cross, 2nd stage,Yeshwanthpur industrial suburb, Bangalore-560022.

The samples are analyzed for tin content by ICP-OES and the results found to be within the acceptance range of +/- 10 % of the nominal conjcentration.

Results in Appendix 14 of the report
Duration of treatment / exposure:
GD 5 to 19
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1 Vehicle Control
Dose / conc.:
2.5 mg/kg bw/day (nominal)
Remarks:
G2 Low Dose
Dose / conc.:
8.5 mg/kg bw/day (nominal)
Remarks:
G3 Mid Dose
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
G4 High Dose
No. of animals per sex per dose:
25 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
Treatment Group Description Dose (mg/kg bw) Concentration (mg/ml) No of preg. fem.
Vehicle G1 Control 0 0 25
DBTE G2 Low Dose 2.5 0.5 25
DBTE G3 Mid Dose 8.5 1.7 25
DBTE G4 High Dose 25.0 5.0 25
Parental animals: Observations and examinations:

Clinical Signs of Toxicity and Mortality/Morbidity
All animals were observed once daily for clinical signs of toxicity and twice daily for mortality/morbidity.
Body Weight
Individual animal body weight was weighed on Gestation Days (GD) 0, 3, and daily from DG 5 to 20 (day of caesarian section)
Feed Consumption
Individual animal feed intake of mated females was recorded for days 0 to 3, 3 to 5 and daily from day5 to 20 of gestation.
Postmortem examinations (parental animals):
Necropsy
All surviving animals were anaesthetized by exposing to CO2 and subjected to detailed necropsy on
the day (GD 20) of cesarean section. The Thymus gland of each rat was weighed and recorded. The
Thymus gland, ovary and uterus were collected and preserved in 10% neutral buffered formalin so
lution for microscopic examination.
Reproductive indices:
Uteri Observations
On the 20th day of gestation, fetuses were taken out by cesarean section and females were subjecte
d to macroscopic examination. The uteri of non-pregnant females were immersed in 10% ammonium
sulphide and there was no evidence of implantation sites. The weight of the gravid uterus including
cervix was recorded for each pregnant female at hysterectomy. The following counts/observations
were performed for all pregnant animals.
• No. of corpora lutea
• No. of implantations
• No. of live and dead fetuses
• No. of early and late resorptions
Offspring viability indices:
• Sex, number and weight of live fetuses
• External appearance of live fetuses (including oral cavity)
• External anomalies
• Crown-rump length
Live fetuses were killed by keeping them on cool packs and allocated to either skeletal or visceral exa
minations, independent of sex. Approximately one-half of live fetuses from each litter were examine
d for skeletal alterations. The remaining fetuses were examined for soft tissue alterations (visceral
examinations).
Visceral Examination
A detailed soft tissue examination was performed on the fresh fetuses with even numbers using micro
dissection technique (Staples technique) for body and a free-hand serial sectioning technique (Wilson
technique) for head.
After examination, the fetuses along with organs were preserved in a solution of glycerine. Obser
vations of visceral abnormalities and variations were recorded.
Skeletal Examination
The fresh fetuses with odd numbers were skinned and eviscerated, fixed in 95% ethanol, subjected to
preparation of Alcian blue staining for cartilage and Alizarin red S staining for bones and the specim
ens were examined under stereomicroscope for the presence or absence of skeletal malformation
(variations).
After examination, the fetuses were preserved in a solution of glycerine.
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):
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was a treatment related reduction in thymus size observed in the high dose animals at necropsy. Total 14/25 females were noted as having a reduction in the size of thymus in gross examination
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There was a treatment related reduction in thymus size observed in the high dose animals at necropsy. Total 14/25 females were noted as having a reduction in the size of thymus in gross examination
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The treatment related histopathological change of decreased cellularity in the thymic cortex was reported for 15/25 females of the high dose group. The observed decreased cell population in the cortex was described as multifocal to diffuse and severity varied from minimal to marked. The histopathological evaluation of the thymus was extended to the lower dose groups and there was no treatmentrelated changes observed in these animals.
Key result
Dose descriptor:
NOAEL
Effect level:
> 8.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The NOAEL of the test item for develomental toxicity was set to > 25 mg/kg bw, the high dose, because there were no adverse effects on fetal developmentals or incidences for external, soft tissueu or skeletal anomalies
Key result
Reproductive effects observed:
no
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Additional information

All dibutyltin compounds degrade into dibutyltin and the appropriate ligand, and so on this basis, it is possible to read-across between the different dibutyltin compounds to address the toxicity to reproduction endpoints. As no toxicity to reproduction data are available for DBT(2 -EHMA) this endpoint has been addressed by the submission of studies performed on dibutyltin dichloride and using read-across from these studies. In a single-generation reproduction screening study, any influence of dibutyltin chloride on reproduction occurred only at maternally toxic levels; a maternal NOAEL of ca. 0.3 mg/kg bw/day was estimated.

Summary of data:

The study, Waalkens-Berendsen, D. H. (2003), was performed in compliance with GLP and to the OECD guideline 421. Accordingly the study was assigned a reliability score of 1 and considered reliable and adequate for assessment. The study a single-generation reproduction screening study, noted that any influence of dibutyltin chloride on reproduction occurred only at maternally toxic levels; a maternal NOAEL of ca. 0.3 mg/kg bw/day was estimated.


Short description of key information:
The following study has been submitted to address the toxicity to reproduction endpoint:

Waalkens-Berendsen DH (2003). Dibutyldichlorostannane (CAS # 683-18-1): Reproduction/developmental toxicity screening test in rats. Testing laboratory: TNO, Project Organisation, Ecotoxicology, Utrechtseweg 48, P. O. Box 370, 3700 AJ Zeist, The Netherlands. Report no.: V 4906. Owner company: Organotin Environmental Programme (ORTEP) Association, Stabilizer Task Force. Report date: 2003-12-04.

This study has been allocated a Klimisch score of 2 on the basis that it was performed to the appropriate guideline under GLP and the test material was a read-across substance for DBT(2-EHMA) .

Effects on developmental toxicity

Description of key information
The following studies have been submitted to address the developmental toxicity/teratogenicity endpoint:
Ema M & Harazono A (2000). Adverse effects of dibutyltin dichloride on initiation and maintenance of rat pregnancy.
Ema et al (1991). Teratogenicity of di-n-butyltin dichloride in rats.
Ema et al (1992). Susceptible period for the teratogenicity of di-n-butyltin dichloride in rats.
Ema et al (1995). Comparative Developmental Toxicity of Butyltin Trichloride, Dibutyltin Dichloride and Tributyltin Chloride in Rats.
Ema et al (1996). Comparative Developmental Toxicity of Di-, Tri- and Tetrabutyltin Compounds after Administration during Late Organogenesis in Rats
Noda T et al (1993). Teratogenic effects of various di-n-butyltins with different anions and butyl(3-hydroxybutyl) tin dilaurate in rats
Osterburg I (1993). Dibutyltin dichloride oral (gavage) teratogenicity study in the rat
Waalkens-Berendsen DH (2003) Dibutyldichlorostannane (CAS # 683-18-1): Reproduction/developmental toxicity screening test in rats
Osterburg (1993) has been allocated a Klimisch score of 2 as the study was conducted to recognised guidelines and GLP using dibutyltin dichloride as the test material to read-across to DBT(2-EHMA)
All other references have been allocated a Klimisch score of 4.
Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
5 mg/kg bw/day
Additional information

All dibutyltin compounds degrade into dibutyltin and the appropriate ligand (in gastric conditions, dibutyltin dichloride), on this basis, it is possible to read-across between the different dibutyltin compounds to address in vivo toxicity endpoints. As no adequate developmental toxicity studies are available on DBT(2 -EHMA), this endpoint has been addressed by the submission of studies performed on dibutyltin dichloride and using a read-across approach, with the available data on dibutyltin oxide as a supporting study. In several studies of development and teratogenicity, dibutyltin chloride as well as several other organotins were repeatably and reliably associated with a syndrome of malformations of the oroglossal region. Malformations appear to be limited to dose levels also associated with maternal toxicity; however, it is not clear how relevant maternal toxicity may be to the syndrome of malformations reported.

SUMMARY OF AVAILABLE DATA

Osterburg. I (1993) was performed in compliance with GLP and conducted according to the guideline OECD 414. The study was accordingly assigned a reliability score of 2 and considered adequate for assessment of the endpoint. The study was performed in Wistar rats, dosed via the oral route (gavage). The test material was determined to have a NOAEL of 1.0 mg/kg bw/day for maternal toxicity and 5.0 mg/kg bw/day for teratogenicity.

 

Seven studies were provided as supporting information and are briefly summarised below.

 

Reference: Ema M & Harazono A (2000)

Reliability and rationale for score: 2 (reliable with restrictions) Study meets generally accepted scientific standards, is well documented, and acceptable for assessment. Partial organogenetic period exposures.

Results: The study was conducted to evaluate the adverse effects of dibutyltin dichloride (DBTCl) on initiation and maintenance of pregnancy after maternal exposure during early pregnancy in rats. The NOAEL for maternal toxicity was determined to be <3.8 mg/kg/day and for teratogenicity was 15.2 mg/kg/day.

 

 

Reference: Ema et al (1991)

Reliability and rationale for score: 2 (reliable with restrictions) Study was conducted using less than the recommended number of animals (20). Purity of test material not reported. Doses were not adjusted for body weight reductions. Body weights were reduced in the two highest treatment groups; therefore, these animals may have received higher doses than reported. The number of fetuses examined for internal malformations was less than the EPA-recommended number. The two highest dose levels tested, 7.5 and 10 mg/kg, were generally lethal, killing 42% and 75% of the dams, respectively.

Results: Pregnant rats were given di-n-butyltin dichloride (DBT) by gastric intubation at a dose of 0, 2.5, 5.0, 7.5or 10.0 mg/kg on days 7-15 of pregnancy.The NOAEL for maternal toxicity was determined to be 5.0 mg/kg/day and for teratogenicity was 2.5 mg/kg/day.

 

 

Reference: Ema et al (1992)

Reliability and rationale for score: 2 (reliable with restrictions) Study meets generally accepted scientific standards, is well documented, and acceptable for assessment. Purity of test material not reported. Partial organogenetic period exposures.

Results: Pregnant rats were given di-n-butyltin dichloride (DBT) by gastric intubation at a dose of 20 mg/kg on days 7-9,10-12 or 13-15 of pregnancy or at a dose of 20 or 40 mg/kg on day 6, 7, 8 or 9 of pregnancy. It could be concluded that, following maternal exposure to DBT in rats, developing offspring are not susceptible to teratogenic effects of DBT on day 6 and that day 7 is the earliest susceptible period, day 8 is the highest susceptible period and day 9 is no longer a susceptible period for teratogenesis of DBT.

 

 

Reference: Ema et al (1995)

Reliability and rationale for score: 2 (reliable with restrictions). Study meets generally accepted scientific standards, is well documented, and acceptable for assessment. Purity of test material not reported. Partial organogenetic period exposures.

Results: Butyltin trichloride (BT), dibutyltindichloride (DBT)and tributyltin chloride (TBT) were compared for their developmental toxicity including teratogenic potential following administration during the susceptible period for the teratogenesis of DBT. Pregnant rats were given DBT at a dose of 10 or 15 mg/kg by gastric intubation on days 7 and 8 of pregnancy. Treatment with DBT resulted in a significantly lower maternal weight gain, lower fetal weight and higher postimplantation embryo lethality. A significantly and markedly increased incidence of fetuses with malformations, such as exencephaly, cleft jaw, cleft lip, ankyloglossia, club foot, deformity of the vertebral column in the cervical and thoracic regions and of the ribs and ano- ormicrophthalmia, was observed in both groups treated with DBT. It could be concluded that BT, DBT and TBT are different in the susceptibility and spectrum of developmental toxicity.

 

 

Reference: Ema et al (1996)

Reliability and rationale for score: 2 (reliable with restrictions) Study meets generally accepted scientific standards, is well documented, and acceptable for assessment. Purity of test material not reported. Partial organogenetic period exposures.

Results: Dibutyltin dichloride (DBT), tributyltin chloride (TrBT) and tetrabutyltin (TeBT) were compared fortheir developmental toxicity and teratogenic potential following administration during the susceptibleperiod for teratogenesis of TrBT.

Pregnant rats were given DBT at a dose of 165 or 330 µmol/kg on days 13-15 of pregnancy. The findings of the study suggest that DBT has no teratogenic effect when administered during late organogenesis at doses that induced overt maternal toxicity.It could be concluded that there is a difference in the manifestation and degree of developmental toxicity between DBT, TrBT and TeBT.

 

 

Reference: Noda T et al (1993)

Reliability and rationale for score: 2 (reliable with restrictions) The number of animals and dose groups used in this study were less than the recommended amounts in OECD Guideline 414. Study meets generally accepted scientific standards, is well documented, and acceptable for assessment. Partial organogenetic period exposures.

Results: In the oral (gavage) teratogenicity study in the rat the test material was determined, not to be toxic maternally, but was teratogenic to developing fetuses. The NOAEL is therefore 24.3 mg/kg for maternal toxicity and <24.3 mg/kg for teratogenicity.

 

 

Reference: Waalkens-Berendsen DH (2003)

Reliability and rationale for score: 2 (reliable with restriction) The study was performed in compliance with GLP and in accordance with the guideline OECD 421.

Results:In the Reproduction/developmental toxicity screening test in rats (TNO study number: V 4906) the test material was determined to have a NOAEL for general toxicity established on the low-dose level of 5 mg/kg diet and the NOAEL for reproductive toxicity was established at the mid-dose level of 30 mg/kg diet. No mortalities were observed. No clinical signs were observed in the male and female animals from the start of the study until sacrifice. Examination of the thymus revealed severe to very severe lymphoid depletion in 12/12 high-dose females, and moderate to severe lymphoid depletion in 6/12 (pregnant) mid-dose females.Three females showed late resorptions (autolytic fetuses) in the uterus during necropsy.

Justification for classification or non-classification

The substance is classified with Repro. Cat. 2; R60 -61 according to Directive 67/548/EEC. According to Regulation (EC) no 1272/2008 the test substance would be classified as a Repr. 1B with Hazard statement: H360FD: May damage fertility or the unborn child and should be accompanied with the signal word 'Danger'.