Registration Dossier

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The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study Initiation Date: 04 June 2018, Experimental Completion Date: 24 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Pursuant to Articles 10(a)(vi) and/or (vii), 12(1)(d) and 13(4) of the REACH Regulation, a
technical dossier registered at 100 to 1000 tonnes per year shall contain as a minimum the
information specified in Annexes VII to IX of the REACH Regulation.
The basic test design of an extended one-generation reproductive toxicity study (Cohorts 1A
and 1B, without extension of Cohort 1B to include a F2 generation, and without Cohorts 2A,
2B and 3) is a standard information requirement as laid down in column 1 of 8.7.3., Annex
IX of the REACH Regulation if the available repeated dose toxicity studies (e.g. 28-day or
90-day studies, OECD TGs 421 or 422 screening studies) indicate adverse effects on
reproductive organs or tissues or reveal other concerns in relation with reproductive
toxicity. If the conditions described in column 2 of Annex IX are met, the study design
needs to be expanded to include the extension of Cohort 1B, Cohorts 2A/2B, and/or Cohort
3. Adequate information on this endpoint needs to be present in the technical dossier for the
registered substance to meet this information requirement.
a) The information requirement
You have sought to adapt this information requirement according to Section 3 of Annex XI
of the REACH Regulation using the same justification as for the adaptation proposed for the
prenatal developmental toxicity study (Section 2). However, ECHA notes similarly to Section
2, for the prenatal developmental toxicity, that none of the criteria of that adaptation
(3.2(a); 3.2(b); or 3.2(c)) are currently fulfilled.
Therefore, ECHA considers that this adaptation of the information requirement does not
meet the requirements set forth under Section 3 of Annex XI.
ECHA considers that adverse effects on reproductive organs or tissues and other concerns in
relation with reproductive toxicity are observed. More specifically, you provided a screening
study in rats (Waalkens-Berendsen, 2004) performed with the registered substance
according to OECD TG 422. In this study reproductive effects such as increased duration of
gestation, increased implantation loss, an increased number of still born pups and pup
mortality at postnatal day 4, decreased pup weight and an increased number of runts at
postnatal day 1, and a statistically significant increase in the incidence of cysts in the
ovaries of 8 high-dose females were seen, albeit in the high-dose only, in the presence of
maternal toxicity (severe effects in thymus).
Pursuant to Annex IX, Section 8.7.3. an extended one-generation reproductive toxicity
study is thus an information requirement for registration of the registered substance.
ECHA further notes that this OECD 422 study does not provide the information required by
Annex IX, Section 8.7.3., because it does not cover key parameters, exposure duration, life
stages and statistical power of an extended one-generation reproductive toxicity study. The
main missing key aspects/elements are: At least 20 pregnant females per group, an
extensive post-natal evaluation of the F1 generation, and investigation of (developmental)
immunotoxicity.
ECHA concludes that the information available on this endpoint for the registered substance
in the technical dossier does not meet the information requirement. Consequently there is
an information gap and it is necessary to provide information for this endpoint. Thus, an
extended one-generation reproductive toxicity study according to columns 1 and 2 of 8.7.3.,
Annex IX is required. The following refers to the specifications of this required study.
b) The specifications for the required study
Premating exposure duration and dose-level setting
To ensure that the study design adequately addresses the fertility endpoint, the duration of
the premating exposure period and the selection of the highest dose level are key aspects
to be considered. According to ECHA Guidance, the starting point for deciding on the length
of premating exposure period should be ten weeks to cover the full spermatogenesis and
folliculogenesis before the mating, allowing meaningful assessment of the effects on
fertility.
Ten weeks premating exposure duration is required because there is no substance specific
information in the dossier supporting shorter premating exposure duration as advised in the
ECHA Guidance on information requirements and chemical safety assessment R.7a, chapter
R.7.6 (version 4.0, July 2015). The exposure duration is supported also by the lipophilicity
of the substance to ensure that the steady state in parental animals has been reached
before mating.
The highest dose level shall aim to induce some toxicity to allow comparison of effect levels
and effects of reproductive toxicity with those of systemic toxicity. The dose level selection
should be based upon the fertility effects with the other cohorts being tested at the same
dose levels.
It is recommended that results from a range-finding study (or range finding studies) for the
extended one-generation reproductive toxicity study are reported with the main study. This
will support the justifications of the dose level selections and interpretation of the results.
Extension of Cohort 1B
If the column 2 conditions of 8.7.3., Annex IX are met, Cohort 1B must be extended, which
means that the F2 generation is produced by mating the Cohort 1B animals. This extension
provides information also on the sexual function and fertility of the F1 animals.
The use of the registered substance is leading to significant exposure of workers and
consumers because the registered substance has industrial and consumer uses such as in
adhesive, sealants, coatings and paints, thinners, paint removes, textile dyes. Furthermore,
effects indicating endocrine disrupting mode of action such as increase in gestation length
were observed in the OECD TG 422 study (see above). Furthermore, the estimated LogKow
of 9.26 of the registered substance indicates a bioaccumulative potential.
Therefore, ECHA concludes that Cohort 1B must be extended to include mating of the
animals and production of the F2 generation because the uses of the registered substance is
leading to significant exposure of industrial workers and consumers and the internal dose
for the registered substance and or any of its metabolites is estimated to reach a steady
state in the test animals only after an extended exposure based on the high estimated
LogKow of the registered substance and the OECD TG 422 study indicates modes of action
related to endocrine disruption for the registered substance.
Cohort 3
The developmental immunotoxicity Cohort 3 needs to be conducted in case of a particular
concern on (developmental) immunotoxicity as described in column 2 of 8.7.3., Annex IX.
ECHA notes that existing information provided in the dossier on the registered substance in
the OECD TG 422 study shows evidence of immunotoxicity and severe thymus toxicity
(thymus atrophy).
Upon receipt of the draft decision you submitted comments explaining that:
“The registrant agrees to get information concerning reproduction toxicity, on strength of
information lack in a further generation.
However, there exists an OECD 443 (IUCLID section 7.8.3) study on the structural analogue
surrogate Dioctyltin dichloride. This study was not considered as relevant for Dioctyltin
dichloride because of the different gastric metabolism and the different bioavailability of the
substances (chloride vs. oxide) and the fact that the study did not integrate the acute
immune effects in parent and juvenile animals. In subacute, subchronic and development /
reproduction toxicity studies, it is important that for an acute immune suppressive
substance the acute immunotoxicity is considered as a relevant endpoint.
Based on these considerations the lead registrant proposes a tiered approach based on the
outcome of extended OECD 414 (section 2) in progress.
The following justification should be considered:
- Acute immune toxicity is not determined in OECD TG 443, only immune
toxicity, therefore a misinterpretation as repeated exposure toxicity possible.
- If acute immune toxicity is not determined, acute immune toxicity in juvenile
animals may lead to an interpretation as developmental immune toxicity.
- Determination if immune toxicity caused by Dioctyltin oxide follows Habers
Rule.
- If OECD 414 shows a positive teratogenic outcome, performance of OECD 443
may not be appropriate considering animal welfare.
The lead registrant is aware of his responsibility regarding human, environment and nature;
in addition the German “Grundgesetz” includes the fundamental right of animal welfare.
Thus we want to avoid any inappropriate animal experiment without value for human and
environment. Therefore, the proposed tiered approach under consideration of the outcome
of the extended OECD 414 is supports the weight of legally protected rights.”
ECHA acknowledges your comments and the proposed tiered approach for performing the
OECD TG 443. However, ECHA notes that unless the outcome of the ongoing OECD 414 is a
classification of the substance as Reproductive toxicity Category 1A or 1B (H360F) and the
available data are adequate to support a robust risk assessment, the PNDT study cannot be
used to waive the EORGTS study. Information from the pre-natal developmental toxicity
study (OECD TG 414) regarding sexual function and fertility is limited to the maintenance of
the pregnancy from implantation up to close to the parturition. It does not provide
information on hazardous properties to the postnatal development including sexual
maturation and histopathological integrity of the reproductive organs at adulthood. Thus,
the information from these studies cannot fulfill the data requirement for this endpoint.
This information does not allow either to assume/conclude that the substance has not
hazardous properties with regard to sexual function and fertility, would you want to use this
study in the context of adaptation possibilities offered by Annex XI. Consequently the draft
decision was not amended.
ECHA concludes that the developmental immunotoxicity cohort 3 needs to be conducted
because there is a particular concern on (developmental) immunotoxicity based on the
results from the above-identified in vivo study on the registered substance.
The study design must be justified in the dossier and, thus, the existence/non-existence of
the conditions/triggers must be documented.
Species and route selection
According to the test method EU B.56/ OECD TG 443, the rat is the preferred species. On
the basis of this default consideration, ECHA considers that testing should be performed in
rats.
ECHA considers that the oral route is the most appropriate route of administration for
substances except gases to focus on the detection of hazardous properties on reproduction
as indicated in ECHA Guidance on information requirements and chemical safety assessment
(version 4.0, July 2015) R.7a, chapter R.7.6.2.3.2. Since the substance to be tested is a
solid, ECHA concludes that testing should be performed by the oral route.
Outcome
Therefore, pursuant to Article 41(1) and (3) of the REACH Regulation, you are requested to
submit the following information derived with the registered substance subject to the
present decision: Extended one-generation reproductive toxicity study (test method EU
B.56/ OECD TG 443), in rats, oral route, according to the following study-design
specifications:
o Ten weeks premating exposure duration for the parental (P0) generation;
o Dose level setting shall aim to induce some toxicity at the highest dose level;
o Cohort 1A (Reproductive toxicity);
o Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B
animals to produce the F2 generation; and
o Cohort 3 (Developmental immunotoxicity).
Notes for your consideration
No triggers for the inclusion of Cohorts 2A and 2B (developmental neurotoxicity) were
identified. However, you may expand the study by including Cohorts 2A and 2B if new
information becomes available after this decision is issued to justify such an inclusion.
Inclusion is justified if the new information shows triggers which are described in column 2
of Section 8.7.3., Annex IX and further elaborated in ECHA Guidance on information
requirements and chemical safety assessment R.7a, chapter R.7.6 (version 4.0, July 2015).
You may also expand the study to address a concern identified during the conduct of the
extended one-generation reproduction toxicity study and also due to other scientific reasons
in order to avoid a conduct of a new study. The justification for the expansion must be
documented. The study design must be justified in the dossier and, thus, the existence/nonexistence
of the conditions/triggers must be documented.
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
yes
Remarks:
see free text
Principles of method if other than guideline:
additional endpoints: Sn in plasma, Sn in milk, bone density
GLP compliance:
yes
Limit test:
no
Justification for study design:
Pursuant to Articles 10(a)(vi) and/or (vii), 12(1)(d) and 13(4) of the REACH Regulation, a
technical dossier registered at 100 to 1000 tonnes per year shall contain as a minimum the
information specified in Annexes VII to IX of the REACH Regulation.
The basic test design of an extended one-generation reproductive toxicity study (Cohorts 1A
and 1B, without extension of Cohort 1B to include a F2 generation, and without Cohorts 2A,
2B and 3) is a standard information requirement as laid down in column 1 of 8.7.3., Annex
IX of the REACH Regulation if the available repeated dose toxicity studies (e.g. 28-day or
90-day studies, OECD TGs 421 or 422 screening studies) indicate adverse effects on
reproductive organs or tissues or reveal other concerns in relation with reproductive
toxicity. If the conditions described in column 2 of Annex IX are met, the study design
needs to be expanded to include the extension of Cohort 1B, Cohorts 2A/2B, and/or Cohort
3. Adequate information on this endpoint needs to be present in the technical dossier for the
registered substance to meet this information requirement.
a) The information requirement
You have sought to adapt this information requirement according to Section 3 of Annex XI
of the REACH Regulation using the same justification as for the adaptation proposed for the
prenatal developmental toxicity study (Section 2). However, ECHA notes similarly to Section
2, for the prenatal developmental toxicity, that none of the criteria of that adaptation
(3.2(a); 3.2(b); or 3.2(c)) are currently fulfilled.
Therefore, ECHA considers that this adaptation of the information requirement does not
meet the requirements set forth under Section 3 of Annex XI.
ECHA considers that adverse effects on reproductive organs or tissues and other concerns in
relation with reproductive toxicity are observed. More specifically, you provided a screening
study in rats (Waalkens-Berendsen, 2004) performed with the registered substance
according to OECD TG 422. In this study reproductive effects such as increased duration of
gestation, increased implantation loss, an increased number of still born pups and pup
mortality at postnatal day 4, decreased pup weight and an increased number of runts at
postnatal day 1, and a statistically significant increase in the incidence of cysts in the
ovaries of 8 high-dose females were seen, albeit in the high-dose only, in the presence of
maternal toxicity (severe effects in thymus).
Pursuant to Annex IX, Section 8.7.3. an extended one-generation reproductive toxicity
study is thus an information requirement for registration of the registered substance.
ECHA further notes that this OECD 422 study does not provide the information required by
Annex IX, Section 8.7.3., because it does not cover key parameters, exposure duration, life
stages and statistical power of an extended one-generation reproductive toxicity study. The
main missing key aspects/elements are: At least 20 pregnant females per group, an
extensive post-natal evaluation of the F1 generation, and investigation of (developmental)
immunotoxicity.
ECHA concludes that the information available on this endpoint for the registered substance
in the technical dossier does not meet the information requirement. Consequently there is
an information gap and it is necessary to provide information for this endpoint. Thus, an
extended one-generation reproductive toxicity study according to columns 1 and 2 of 8.7.3.,
Annex IX is required. The following refers to the specifications of this required study.
b) The specifications for the required study
Premating exposure duration and dose-level setting
To ensure that the study design adequately addresses the fertility endpoint, the duration of
the premating exposure period and the selection of the highest dose level are key aspects
to be considered. According to ECHA Guidance, the starting point for deciding on the length
of premating exposure period should be ten weeks to cover the full spermatogenesis and
folliculogenesis before the mating, allowing meaningful assessment of the effects on
fertility.
Ten weeks premating exposure duration is required because there is no substance specific
information in the dossier supporting shorter premating exposure duration as advised in the
ECHA Guidance on information requirements and chemical safety assessment R.7a, chapter
R.7.6 (version 4.0, July 2015). The exposure duration is supported also by the lipophilicity
of the substance to ensure that the steady state in parental animals has been reached
before mating.
The highest dose level shall aim to induce some toxicity to allow comparison of effect levels
and effects of reproductive toxicity with those of systemic toxicity. The dose level selection
should be based upon the fertility effects with the other cohorts being tested at the same
dose levels.
It is recommended that results from a range-finding study (or range finding studies) for the
extended one-generation reproductive toxicity study are reported with the main study. This
will support the justifications of the dose level selections and interpretation of the results.
Extension of Cohort 1B
If the column 2 conditions of 8.7.3., Annex IX are met, Cohort 1B must be extended, which
means that the F2 generation is produced by mating the Cohort 1B animals. This extension
provides information also on the sexual function and fertility of the F1 animals.
The use of the registered substance is leading to significant exposure of workers and
consumers because the registered substance has industrial and consumer uses such as in
adhesive, sealants, coatings and paints, thinners, paint removes, textile dyes. Furthermore,
effects indicating endocrine disrupting mode of action such as increase in gestation length
were observed in the OECD TG 422 study (see above). Furthermore, the estimated LogKow
of 9.26 of the registered substance indicates a bioaccumulative potential.
Therefore, ECHA concludes that Cohort 1B must be extended to include mating of the
animals and production of the F2 generation because the uses of the registered substance is
leading to significant exposure of industrial workers and consumers and the internal dose
for the registered substance and or any of its metabolites is estimated to reach a steady
state in the test animals only after an extended exposure based on the high estimated
LogKow of the registered substance and the OECD TG 422 study indicates modes of action
related to endocrine disruption for the registered substance.
Cohort 3
The developmental immunotoxicity Cohort 3 needs to be conducted in case of a particular
concern on (developmental) immunotoxicity as described in column 2 of 8.7.3., Annex IX.
ECHA notes that existing information provided in the dossier on the registered substance in
the OECD TG 422 study shows evidence of immunotoxicity and severe thymus toxicity
(thymus atrophy).
Upon receipt of the draft decision you submitted comments explaining that:
“The registrant agrees to get information concerning reproduction toxicity, on strength of
information lack in a further generation.
However, there exists an OECD 443 (IUCLID section 7.8.3) study on the structural analogue
surrogate Dioctyltin dichloride. This study was not considered as relevant for Dioctyltin
dichloride because of the different gastric metabolism and the different bioavailability of the
substances (chloride vs. oxide) and the fact that the study did not integrate the acute
immune effects in parent and juvenile animals. In subacute, subchronic and development /
reproduction toxicity studies, it is important that for an acute immune suppressive
substance the acute immunotoxicity is considered as a relevant endpoint.
Based on these considerations the lead registrant proposes a tiered approach based on the
outcome of extended OECD 414 (section 2) in progress.
The following justification should be considered:
- Acute immune toxicity is not determined in OECD TG 443, only immune
toxicity, therefore a misinterpretation as repeated exposure toxicity possible.
- If acute immune toxicity is not determined, acute immune toxicity in juvenile
animals may lead to an interpretation as developmental immune toxicity.
- Determination if immune toxicity caused by Dioctyltin oxide follows Habers
Rule.
- If OECD 414 shows a positive teratogenic outcome, performance of OECD 443
may not be appropriate considering animal welfare.
The lead registrant is aware of his responsibility regarding human, environment and nature;
in addition the German “Grundgesetz” includes the fundamental right of animal welfare.
Thus we want to avoid any inappropriate animal experiment without value for human and
environment. Therefore, the proposed tiered approach under consideration of the outcome
of the extended OECD 414 is supports the weight of legally protected rights.”
ECHA acknowledges your comments and the proposed tiered approach for performing the
OECD TG 443. However, ECHA notes that unless the outcome of the ongoing OECD 414 is a
classification of the substance as Reproductive toxicity Category 1A or 1B (H360F) and the
available data are adequate to support a robust risk assessment, the PNDT study cannot be
used to waive the EORGTS study. Information from the pre-natal developmental toxicity
study (OECD TG 414) regarding sexual function and fertility is limited to the maintenance of
the pregnancy from implantation up to close to the parturition. It does not provide
information on hazardous properties to the postnatal development including sexual
maturation and histopathological integrity of the reproductive organs at adulthood. Thus,
the information from these studies cannot fulfill the data requirement for this endpoint.
This information does not allow either to assume/conclude that the substance has not
hazardous properties with regard to sexual function and fertility, would you want to use this
study in the context of adaptation possibilities offered by Annex XI. Consequently the draft
decision was not amended.
ECHA concludes that the developmental immunotoxicity cohort 3 needs to be conducted
because there is a particular concern on (developmental) immunotoxicity based on the
results from the above-identified in vivo study on the registered substance.
The study design must be justified in the dossier and, thus, the existence/non-existence of
the conditions/triggers must be documented.
Species and route selection
According to the test method EU B.56/ OECD TG 443, the rat is the preferred species. On
the basis of this default consideration, ECHA considers that testing should be performed in
rats.
ECHA considers that the oral route is the most appropriate route of administration for
substances except gases to focus on the detection of hazardous properties on reproduction
as indicated in ECHA Guidance on information requirements and chemical safety assessment
(version 4.0, July 2015) R.7a, chapter R.7.6.2.3.2. Since the substance to be tested is a
solid, ECHA concludes that testing should be performed by the oral route.
Outcome
Therefore, pursuant to Article 41(1) and (3) of the REACH Regulation, you are requested to
submit the following information derived with the registered substance subject to the
present decision: Extended one-generation reproductive toxicity study (test method EU
B.56/ OECD TG 443), in rats, oral route, according to the following study-design
specifications:
o Ten weeks premating exposure duration for the parental (P0) generation;
o Dose level setting shall aim to induce some toxicity at the highest dose level;
o Cohort 1A (Reproductive toxicity);
o Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B
animals to produce the F2 generation; and
o Cohort 3 (Developmental immunotoxicity).
Notes for your consideration
No triggers for the inclusion of Cohorts 2A and 2B (developmental neurotoxicity) were
identified. However, you may expand the study by including Cohorts 2A and 2B if new
information becomes available after this decision is issued to justify such an inclusion.
Inclusion is justified if the new information shows triggers which are described in column 2
of Section 8.7.3., Annex IX and further elaborated in ECHA Guidance on information
requirements and chemical safety assessment R.7a, chapter R.7.6 (version 4.0, July 2015).
You may also expand the study to address a concern identified during the conduct of the
extended one-generation reproduction toxicity study and also due to other scientific reasons
in order to avoid a conduct of a new study. The justification for the expansion must be
documented. The study design must be justified in the dossier and, thus, the existence/nonexistence
of the conditions/triggers must be documented.
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
other: in rodent diet
Analytical verification of doses or concentrations:
yes
Dose / conc.:
0 mg/kg diet
Dose / conc.:
5 mg/kg diet
Dose / conc.:
25 mg/kg diet
Dose / conc.:
200 mg/kg diet
Control animals:
yes, plain diet
Oestrous cyclicity (parental animals):
No test article-related changes were noted
Sperm parameters (parental animals):
No test article-related changes were noted
Clinical signs:
no effects observed
Description (incidence and severity):
No test article-related clinical observations were noted.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test article-related effects on body weight gain were observed for males and females provided with 200 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test article-related effects on food consumption were observed for females provided with 200 ppm.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematology assessments showed marginally higher neutrophil levels for males and
females provided with 200 ppm, compared with control, at the end of the in-life
phase. Percentage leucocytes and monocytes were also increased on LD 22 for
females provided with 200 ppm, compared with controls. Mean cell volume, mean
cell hemoglobin, and reticulocytes were increased on LD 22 for females provided
with 200 ppm, and lower mean red cell distribution width was observed.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
a) Bones
No test article-related effect on femur length or bone area, bone mineral content, and
bone mineral density was observed following exposure at all dose levels, compared
with controls

b) Milk
Milk samples obtained from lactating females from all groups, including controls,
showed tin levels to be lower than the lower limit of quantitation (LLOQ).
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Systemic effects in the F0 generation included changes in thymus following
administration of 200 ppm for both sexes and following administration of 25 ppm to
females. The kidney changes noted for F0 females were considered not adverse;
therefore, a no observed adverse effect level (NOAEL) was established as 25 ppm for
systemic toxicity for males and 5 ppm for females for the F0 generation.
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg diet
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg diet
System:
immune system
Organ:
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test article-related clinical observations were noted for animals provided with
200 ppm, and consisted of isolated instances of high stepping gait noted for one male
during the weekly detailed observations. For females during gestation and lactation,
tail elevation was recorded for two females
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For 200 ppm males, lower mean body weights and reduced food consumption were
observed from the initiation of the F1 phase, and remained lower until the end of the
phase, with an initial reduction in body weight gain observed during the first week of
the F1 phase compared with controls.
Lower mean body weight and food consumption were also observed during the
first five weeks of the F1 phase for females maternally exposed and provided with
200 ppm, compared with controls. Lower mean body weight gain was also noted
during the first week. Mean body weights were similar to controls during early and
mid-gestation. However, by GD 20, marginally lower mean body weights and body
weight gains were observed, with values still marginally lower during the
first four days of the lactation phase, with a corresponding reduction in food
consumption, compared with controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
For 200 ppm males, lower mean body weights and reduced food consumption were
observed from the initiation of the F1 phase, and remained lower until the end of the
phase, with an initial reduction in body weight gain observed during the first week of
the F1 phase compared with controls.
Lower mean body weight and food consumption were also observed during the
first five weeks of the F1 phase for females maternally exposed and provided with
200 ppm, compared with controls. Lower mean body weight gain was also noted
during the first week. Mean body weights were similar to controls during early and
mid-gestation. However, by GD 20, marginally lower mean body weights and body
weight gains were observed, with values still marginally lower during the
first four days of the lactation phase, with a corresponding reduction in food
consumption, compared with controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males maternally exposed and provided with 200 ppm showed a reduction in
hemoglobin levels and corresponding increases in reticulocytes (counts and %),
compared with controls
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females maternally exposed and provided with 200 ppm showed an
increase in alanine aminotransferase activity, compared with controls. Elevated mean
cholesterol levels, with a reduction in albumin levels, were also observed for males,
compared with controls.
Females maternally exposed and provided with 200 ppm also showed a reduction in
total protein and an increase in blood urea, compared with controls.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Description (incidence and severity):
No notable immunotoxicology findings for immunophenotyping and T-Cell
Dependent Antibody Response assessments were noted.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Lower mean thymus weights (absolute, and relative to body weight and brain weight)
were recorded for animals provided with 200 ppm, compared with controls. Although
no macroscopic findings were noted in the thymus, increased thymic atrophy was
recorded for animals maternally exposed and provided with 200 ppm, compared with
controls.
Increased liver weight ratios were recorded for males and increased heart weight
ratios were recorded for both sexes maternally exposed and provided with 200 ppm,
although no macroscopic or microscopic correlates were observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The percentage post-implantation losses were twice as much in the 200 ppm group,
compared with controls. Litter size was also lower in this group on PND 1, although
sex ratio was unaffected.
Four F2 pups were recorded as missing (presumed cannibalized) and one dead pup
was recorded in the 200 ppm litters, compared to no dead/missing pups in control
litters. This was considered test article-related, which resulted in the lower PND 4
viability index for this group, and a smaller litter size on PND 4.
Test article-related clinical observations were noted for the 200 ppm litters. Blackened
/ dead tissue on the tail apex was recorded for three pups from two separate 200 ppm
litters. Red and thin appearance was also noted for four pups from one litter. These
observations were not observed for control litters. No further test article-related
clinical observations were noted in the 200 ppm F2 pups. No test article-related
clinical observations were noted in the 5 or 25 ppm F2 generation pups.
Marginally lower mean body weights (adjusted for litter size) were observed for
males and females from 200 ppm litters; these were considered not to have
represented an adverse effect on body weight.
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
other: post-implamentation lost, litter size
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg diet
System:
immune system
Organ:
blood
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test article-related clinical observations were noted for animals provided with
200 ppm, and consisted of isolated instances of high stepping gait noted for one male
during the weekly detailed observations. For females during gestation and lactation,
tail elevation was recorded for two females.
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For 200 ppm males, lower mean body weights and reduced food consumption were
observed from the initiation of the F1 phase, and remained lower until the end of the
phase, with an initial reduction in body weight gain observed during the first week of
the F1 phase compared with controls.
Lower mean body weight and food consumption were also observed during the
first five weeks of the F1 phase for females maternally exposed and provided with
200 ppm, compared with controls. Lower mean body weight gain was also noted
during the first week. Mean body weights were similar to controls during early and
mid-gestation. However, by GD 20, marginally lower mean body weights and body
weight gains were observed, with values still marginally lower during the
first four days of the lactation phase, with a corresponding reduction in food
consumption, compared with controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
For 200 ppm males, lower mean body weights and reduced food consumption were
observed from the initiation of the F1 phase, and remained lower until the end of the
phase, with an initial reduction in body weight gain observed during the first week of
the F1 phase compared with controls.
Lower mean body weight and food consumption were also observed during the
first five weeks of the F1 phase for females maternally exposed and provided with
200 ppm, compared with controls. Lower mean body weight gain was also noted
during the first week. Mean body weights were similar to controls during early and
mid-gestation. However, by GD 20, marginally lower mean body weights and body
weight gains were observed, with values still marginally lower during the
first four days of the lactation phase, with a corresponding reduction in food
consumption, compared with controls.
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males maternally exposed and provided with 200 ppm showed a reduction in
hemoglobin levels and corresponding increases in reticulocytes (counts and %),
compared with controls.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females maternally exposed and provided with 200 ppm showed an
increase in alanine aminotransferase activity, compared with controls. Elevated mean
cholesterol levels, with a reduction in albumin levels, were also observed for males,
compared with controls.
Females maternally exposed and provided with 200 ppm also showed a reduction in
total protein and an increase in blood urea, compared with controls.
Urinalysis findings:
not examined
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
Sexual maturity was delayed for males and females maternally and directly exposed
to 200 ppm, compared with controls. This is a secundary effect based on lower food
consumtion and delayed body weight increase
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Lower mean thymus weights (absolute, and relative to body weight and brain weight)
were recorded for animals provided with 200 ppm, compared with controls. Although
no macroscopic findings were noted in the thymus, increased thymic atrophy was
recorded for animals maternally exposed and provided with 200 ppm, compared with
controls.
Increased liver weight ratios were recorded for males and increased heart weight
ratios were recorded for both sexes maternally exposed and provided with 200 ppm,
although no macroscopic or microscopic correlates were observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Lower mean thymus weights (absolute, and relative to body weight and brain weight)
were recorded for animals provided with 200 ppm, compared with controls. Although
no macroscopic findings were noted in the thymus, increased thymic atrophy was
recorded for animals maternally exposed and provided with 200 ppm, compared with
controls.
Increased liver weight ratios were recorded for males and increased heart weight
ratios were recorded for both sexes maternally exposed and provided with 200 ppm,
although no macroscopic or microscopic correlates were observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Increased liver weight ratios were recorded for males and increased heart weight
ratios were recorded for both sexes maternally exposed and provided with 200 ppm,
although no macroscopic or microscopic correlates were observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
no effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental immunotoxicity
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
25 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg diet
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
Oral (dietary) administration of control article (vehicle) or 5, 25 or 200 ppm
(equivalent to a nominal dose level of 0, 0.4, 1.8, or 11.8 mg/kg/day) Dioctyltin Oxide
to Han Wistar rats for up to two consecutive generations resulted in test article-related
effects at all dose levels.
Systemic effects in the F0 generation included changes in thymus following
administration of 200 ppm for both sexes and following administration of 25 ppm to
females. The kidney changes noted for F0 females were considered not adverse;
therefore, a no observed adverse effect level (NOAEL) was established as 25 ppm for
systemic toxicity for males and 5 ppm for females for the F0 generation.
Marginally lower mating and fertility indices were noted in the F1 generation
following administration of 200 ppm, however, this was considered nonadverse,
therefore the no observed effect level (NOEL) for reproductive toxicity is 200 ppm.
Immunotoxicity effects in the F1 generation consisted of a reduction of TDAR
coupled with slight reductions in circulating blood T lymphocyte numbers at all dose
levels, compared with controls, with the effect more prominent for males. In the
absence of any associated changes in hematology parameters, these findings were
considered nonadverse. As such, the NOAEL is 200 ppm for developmental
immunotoxicity.
Based on lower body weights, delayed sexual maturation which resulted as a
secondary effect to the lower mean body weights, in the F1 generation, and smaller
litter sizes and pup mortality in the F2 generation following administration of
200 ppm, the NOEL for offspring growth and development is established as 25 ppm.
No evidence of any endocrine disturbance was noted in this study.
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 November 2003 to 8 March 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted in accordance with the standardised testing guideline OECD 422 and in accordance with GLP with no deviations thought to affect the quality of the presented data. The study was reported to a high standard, sufficient to assess the reliability of the data presented.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10-11 weeks
- Weight at study initiation: The weight variation of the animals for each sex did not exceed 20 %.
- Diet: ad libitum
- Water: tap water ad libitum in polypropylene bottles

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 30-70 5
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

DOSE-RANGE FINDING TEST IN-LIFE DATES: From: 12 November 2003 To: 26 November 2003
MAIN TEST IN-LIFE DATES: From: 14 January 2004 To: 8 March 2004
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): The experimental diets were prepared once shortly before the study
- Mixing appropriate amounts with (Type of food): The test substance was weighed and placed in a small grinder. The tray was rinsed with food which was then also added to the grinder and mixed for 2 x 30 seconds. This mixture was moved to a Stephan cutter and the grinder was rinsed with food and moved to the cutter. Approximately 3 kg weighed food was mixed into the cutter for 2 x 2 minutes and moved. This was then moved to the Lödige cutter. The Stephan cutter was rinsed with approximately 3 kg of food and that was also moved to the Lödige cutter. Mixing was continued in the Lödige cutter for 2 minutes with the total amount of food.
- Storage temperature of food: <-18 °C
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until pregnancy occurred
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The feed was checked for homogenous distribution, stability and concentration of the test substance for all the doses in the dose-range finding study. The same dose preparation for the dose-range finding study was also used for the main test. The homogenous distribution and achieved concentration of the low dose in the main test was also analysed.
Directly after preparation of the diet for the dose-range finding study, samples for homogeneity and stability were taken. Five samples were taken (approximately 50 g each) to examine the homogeneity of the dose from the top centre, middle centre, bottom centre, left centre and right centre of the mixer. Secondly samples (around 50 g) were taken from the top centre part of the mixer to measure the stability. The samples taken for measurement of the homogeneity were also used for dose confirmation. In addition the content (achieved concentration) of the test substance in the batch of diet used in the main study. Diet samples were taken for analysis immediately after preparation and stored at – 18 °C.
Samples of the 0, 25, 75, 200 and 500 mg/kg diets from the dose-range finding study and doses of 0, 25 and 250 mg/kg from the main study as well as all related calibration samples were derivatised.
A calculated amount of internal standard solution (MHT, DHT and TTPT in methanol) was added to 2.0 g of diet in a 50 mL Corning tube. 10 mL of 100 % acetic acid was then added and the Corning tube was closed and shaken for 60 minutes (250 rpm). 10 mL of acetate buffer solution (pH 4.5) was added along with 10 mL methanol. 2.0 mL of 20 % (m/V) aqueous STEB solutions was added, followed by 10 mL hexane (containing naphthalene, approximately 0.1 mg/L). The tube was shaken for 15 minutes (250 rpm) then placed in an oven at 60 °C for 15 minutes. After phase separation, the hexane layer (approximately 3 mL) was removed and washed with 3 mL of 2 mol/L HCl (30 minutes shaking at 250 rpm). The hexane top layer was diluted with hexane: hexane extracts from sample with dose levels of 0, 25 and 75 mg/kg were diluted five times, the higher doses were diluted fifty times. The resulting solutions were transferred into an amber coloured glass vial and anaylysed using GC-MS.
The procedure for the samples from the main study at doses of 0 and 5 mg/kg followed the same derivatisation procedure, except the initial quantity of the diet was 5.0 g not 2.0 g
The concentration of organotin compounds in the extracts was determined using GC-MS. For calculation of the amount of DOTO in the samples, the peak area of DHT was used as an internal standard. Quantitiation was achieved using the calibration graphs constructed from the calibration solutions.
The following conditions were used:
- Column: Fused silica HP5 MS, 30 m, 0.25 mm ID, 0.25 µm film
- Precolumn: fused silica HP5 MS, 2.5 m, 0.25 mm ID, 0.25 µm film
- Column temperature: after 3 minutes 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: DOT m/z = 375*; 263; 151, DHT 347*; 249; 179
Mass fragments marked with an asterisk were used for quantitation
Duration of treatment / exposure:
28 days (treated food was available ad libitum)
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg diet
Remarks:
Range finding study
Dose / conc.:
25 mg/kg diet
Remarks:
Range finding study
Dose / conc.:
75 mg/kg diet
Remarks:
Range finding study
Dose / conc.:
200 mg/kg diet
Remarks:
Range finding study
Dose / conc.:
500 mg/kg diet
Remarks:
Range finding study
Dose / conc.:
0 mg/kg diet
Remarks:
Main study
Dose / conc.:
5 mg/kg diet
Remarks:
Main study
Dose / conc.:
25 mg/kg diet
Remarks:
Main study
Dose / conc.:
250 mg/kg diet
Remarks:
Main study
No. of animals per sex per dose:
22 males and 22 females in the 14-day dose range finding study (5 groups of 4 male and 4 female rats)
52 males and 52 females in the main study (4 groups of 12 male and 12 female rats)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses were selected on the results of the range finding test
- Rationale for animal assignment: Randomised
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Every morning throughout the study, and a second observation in the afternoon of working days.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the first exposure and then once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female rats were taken on day -2 (randomisation) and on days 0 (first day of dosing), 7 and 13 of the premating period.
Males were weighed weekly during the mating period until sacrifice. Females were weighed during mating (day 0, 7 and 13) and mated females were weighed on day 0, 7, 14 and 21 during presumed gestation and on day 1 and 4 of lactation. All animals were weighed at sacrifice.

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, also calculated as g/animal/day

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the premating period
- Anaesthetic used for blood collection: Yes, CO2/02 anaesthesia
- Anit-coagulant: K2-EDTA
- Animals fasted: Yes, overnight
- How many animals: 5 rats/sex/group
- Parameters checked:
Haemoglobin
Packed cell volume
red blood cell count
reticulocytes
Total white blood cell count
Prothrombin time
Thrombocyte count
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the premating period
- Anaesthetic used for blood collection: Yes, CO2/02 anaesthesia
- Animals fasted: Yes, overnight
- How many animals: 5 rats/sex/group
- Parameters checked:
Fasting glucose
Alkaline phosphatase activity (ALP)
Aspartate aminotransferace activity (ASAT)
Alanine aminotransferace activity (ALAT)
Gamma glutamyl transferase activity (GGT)
Total protein
Albumin
Ratio albumin to globulin
Urea
Creatinine
Bilirubin (total)
Cholesterol (total)
Triglycerides
Phospholipids
Calcium (Ca)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Inorganic phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Arena testing was performed prior to the first exposure and then once weekly until the end of dosing and in females until the end of lactation. Males and females selected for the functional observation battery test (FOB) and spontaneous activity measurements were excluded from the final arena testing.
At the end of the study, FOB test and spontaneous motor activity measurements were performed on day 27 for males and on post natal day for for females.
- Dose groups that were examined: All animals were subject to the arena testing. For the other two tests, 5 animals per sex were randomly selected from each dose group.
- Battery of functions tested:
Autonomic: lacrimation, salivation, pupil response to light, palpebral closure, piloerection, defecation and urination
Neuromuscular: gait, mobility, forelimb and hindlimb gripstrength, landing foot splay, righting reflex
Sensorimotor: response to tail pinch, click, tough and approach of a visual object
Convulsive: clonic and tonic movements
Excitability: ease of removal, handling reactivity, arousal and vocalisations
Activity: rearing and motor activity
Physiological: body temperature
Oestrous cyclicity (parental animals):
-
Sperm parameters (parental animals):
-
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in the offspring:
The total litter size and numbers of each sex as well as the number of stillbirths, livebirths and grossly malformed pups and pups showing abnormalities were recorded on PN 1 and PN 4. The pups were weighed individually on PN 1 and PN 4. Mean pup weights were calculated as litter weight/number pups. The number of runts (pup weight less than mean pup weight of the control group minus 2 standard deviations) was calculated.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Males were sacrificed after 28 days of exposure
- Female animals: Sperm positive females that were not pregnant were killed 25 days after copulation, mothers with litters were killed on post natal day 4.

GROSS PATHOLOGY: Yes
The following were taken from all animals:
Ovaries
Uterus
Testes
Epididymides
Seminal vesicles
Prostate
Organs and tissues showing macroscopic abnormalities
The following were taken from 5 animals/sex/group:
Adrenals
Axillary lymph node
Bone marrow (femur)
Brain
Caecum
Coagulation glands
Colon
Duodenum
Eyes
Heart
Jejunum
Lungs
Kidneys
Liver
Mammary gland (females only)
Mesentric lymph node
Parathyroids
Peyer's patches
Pituitary
Rectum
Sciatic nerve
Spinal cord
Spleen
Stomach
Thymus
Thyroids
Trachea
Urinary bladder

The following organs were weighed:
Adrenals
Brain
Heart
Kidneys
Liver
Spleen
Thymus

HISTOPATHOLOGY: Yes Microscopic examination was performed on the collected organs of all rats in the control and high-dose group.
The liver and ovaria of females and the thymus of the male and female rats in the low and mid-dose groups were also evaluated
The following tissues, though collected were not subject to histopathological examination:
Coagulation glands
Mammary gland (females only)
In addition, reproductive organs of males that failed to sire (mated female which was not pregnant) and females that were non-mated or non-pregnant of the mid and low dose groups were microscopically examined.
Postmortem examinations (offspring):
SACRIFICE
- The offspring were sacrificed at post natal day 4 by hypothermia at <-18 °C.
Pups were examined externally for gross abnormalities.

GROSS NECROPSY
- Pups that died during the study were necropsied and macroscopic abnormalities were recorded.
Statistics:
Clinical findings, histopathological changes, the number of mated and pregnant females and females with live pups were evaluated using Fisher’s exact probability test.
Number of implantation sites, live and dead pups were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann Whitney U-test.
Bodyweight, bodyweight gain, organ weights, food consumption, red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values and organ weights were assessed by one-way ANOVA followed by Dunnett’s multiple comparison tests.
Reticulocytes and relative differential white blood cell counts were assessed using Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-tests.
The results of the functional observations were measured on different scales. Continuous measurements were analysed by one-way analysis of variance at each time point, if found to be statistically significant, a post-hoc group comparison was performed. Rank order data were analysed by Kruskal-Wallis analysis of variance at each test time point, followed by planned multiple comparisons between dose groups were a significant results occurred. Categorical data were assessed using Pearson chi-square analysis.
Motor activity data were assessed by one-way analysis of variance at each time point with a post-hoc group comparison performed on significant results.
All tests were two sided and the level of probability p<0.05 was considered as significant. Effects of treatment on habituation were analysed by repeated measures of analysis variance in five 6 minute time blocks. Statistical evaluations on pup variables were considered on a litter basis. Additional evaluations on a pup basis were performed to identify any specific dose-related effect that may have occurred.
Reproductive indices:
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
Offspring viability indices:
Live birth index = (number of pups born alive/number of pups born) x 100
Pup mortality day n = (Number of dead 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 implantation sites – number of pups born alive
Pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lyutea] x 100
Post-implantation loss = [(number of implantation sites – number of pups born alive)/number of implantation sites] x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the 25 mg/kg diet grou there were adverse finding in the thymus
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY
One female in the high dose group was found dead on gestation day 24; this animal was pregnant and 11 dead foetuses were found in the uterus.
One male animal in the high dose group showed exopthalmus from week 2 and complete degeneration of the eye from week 3 onwards. This was observed after orbital punction. No other clinical signs were noted in the males.
The only finding during the gestation period was a sparsely haired animal in the 250 mg/kg group. During the lactation period, sparsely haired animals were noted in the control (n = 1), 5 mg/kg (n = 1) and in the 250 mg/kg group (n = 1). No other findings were noted in the female animals.

BODY WEIGHT AND WEIGHT GAIN
Mean bodyweights of the treated male animals was comparable to the controls. The bodyweight change of the male animals of the high-dose group was significantly decreased from days 13-21 (body weight gain 69% lower than controls).
Mean bodyweights of the dams of the high-dose group was statistically significantly decreased on gestation day 21 (8.5% lower than controls) and post-natal day 1 (9.3% lower than controls). Mean bodyweight changes of the dams of the high-dose group were more markedly affected during pregnancy, as the overall mean weight gain for the control animals accounted to 74.31 g, whereas that in the high-dose animals it was only 51.43 g (approx. 31% lower); durig gestation day 14 to 21, the reduction in mean weight gain in the high-dose animals attained statistical significance, compared to controls, and was approximately 52% lower. Bodyweights and bodyweight change of the dams of all the treated groups were comparable to the controls group at all other times of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Mean food consumption (g/kg/day) of male animals of the 250 mg/kg diet group was decreased by 7.5%, compared to controls, from day 7-13, and the difference attained statistical significance. No other treatment related effects were observed in the male animals.
During the gestation period and lactation period (gestation days 7-14 and 14-21 and post-natal days 1-4), food consumption (expressed as g/animal/day and g/kg bodyweight/day) of the dams in the high-dose group was significantly decreased. Considering the entire pregnancy period, high-dose animals ate approx. 13% less food than controls (in terms of g/kg/day), and approx. 24% less food than controls during the 4-day lactation period. No other treatment-related effects were observed.

Test substance intake was as follows:
- Males (listed as low, mid and high dose groups; 5, 25 and 250 mg/kg diet respectively)
premating days 0-7: 0.4, 1.7 and 17.4 mg/kg bodyweight/day
premating days 7-13: 0.3, 1.6 and 15.4 mg/kg bodyweight/day
post-mating days 21-28: 0.3, 1.5 and 14.5 mg/kg bodyweight/day

- Females (listed as low, mid and high dose groups; 5, 25 and 250 mg/kg diet respectively)
premating days 0-7: 0.4, 1.7 and 17.4 mg/kg bodyweight/day
premating days 7-13: 0.3, 1.6 and 15.4 mg/kg bodyweight/day
gestation days 0-7: 0.4, 2.0 and 17.4 mg/kg bodyweight/day
gestation days 7-14: 0.4, 1.9 and 16.7 mg/kg bodyweight/day
gestation days 14-21: 0.3, 1.4 and 11.2 mg/kg bodyweight/day
Post-natal days 1-4: 0.5, 2.4 and 17.4 mg/kg bodyweight/day

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The number of pregnant females and the number of males that became sires were: 11, 12, 11 and 10 for the control, low, mid and high dose groups respectively. The number of females with liveborn pups was 11, 11, 11 and 8 for the control, low, mid and high dose respectively.
The mating index was 100% in all groups. The female fecundity index, female fertility index and male fertility index were comparable among the all groups and ranged from 83-100%. The gestation index was comparable 100% in the control, low- and mid-dose groups, and 80% in the high-dose group.
The duration of gestation was statistically significantly increased in the 250 mg/kg diet (high-dose) group, although all animals gave birth between day 21 and 22. One female was found dead in the high dose group on gestation day 24. The animal was pregnant and 11 dead foetuses were discovered in the uterus. No difference on the duration of gestation was observed amongst the control and the low- and mid-dose groups.
Stillborn pups were observed in the high dose in three litters, and 1 female with all stillborn pups was observed.
Pre-implantation loss was 8.4. 2.2. 12.3 and 6.2% for the control, low-, mid- and high-dose groups and was considered comparable.
The statistically increased number of implantation sites in the 5 mg/kg diet (low-dose) group was not considered to be treatment related.
Pos-implantation loss was 9.8, 11.6, 7.5 and 38.7% for the control, low-, mid- and high-dose groups respectively. The post-implantation loss recorded in the high-dose group was statistically significantly increased.

HAEMATOLOGY
All treated groups were found to be comparable to controls.

CLINICAL CHEMISTRY
Statistically significantly increased alkaline phosphatase levels (U/L) were found in the high-dose males. Bilirubin (µmol/L) was found to be statistically significantly increased in the high-dose females. These findings were considered to be treatment related. Other effects such as the statistically significant increase in chloride in the 25 mg/kg diet male rats and the significant decrease in calcium in the 5 mg/kg diet females were not considered to be related to treatment. No other changes were observed.

NEUROBEHAVIOUR
No treatment-related effects were observed.

ORGAN WEIGHTS
The absolute thymus weight in the males of the 250 and 25 mg/kg diet groups were found to be significantly decreased (-23 and -80%, respectively, compared to controls). Relative thymus weight was found to be significantly decreased in the high dose male rats (-77% compared to controls).
The absolute and relative weight of the female animals of the high dose group was significantly decreased (-69 and -66%, respectively, compared to controls). In the mid-dose group the relative thymus weight was also statistically significantly decreased (-36% compared to controls).
In the female animals of the high-dose group, the relative kidney and liver weights were statistically significantly increased (+14 and +22%, respectively, compared to controls).
No other effects were observed in either male or female animals.

GROSS PATHOLOGY
At necropsy, a decrease in thymic size was seen in all animals in the 250 mg/kg diet groups, 11 females in the 25 mg/kg diet group, 7 females in the 5 mg/kg diet group and 5 animals in the control group.
Examination of the female that was found dead revealed hydrothorax, haemorrhagic lungs, dilation of the vena cava and haemorrhagic discharge in the vagina, these were considered to be indicative of problems during parturition.

HISTOPATHOLOGY
Microscopic evaluation of the thymus revealed moderate to very severe lymphoid depletion in all animals (both sexes) of the 250 mg/kg diet group and in all females of the 25 mg/kg diet group. Lymphoid depletion was characterised by a decrease in the thymic lobules due to an extensive loss of cortical and medullary small lymphocytes. The distinction between the cortical and medullary areas was unclear. In the more extreme effects observed, the cortex was very small, or absent. The remaining lymphoid cells visible in the cortical areas were mainly lymphoblasts. Lymphoblastic cells and reticuloepithelial cells had increased, and/or higher numbers of these cells were visible due to the disappearance of small lymphocytes and the collapse of the thymic stroma. In 3 high-dose females, lymphoid depletion was accompanied by lymphoid depletion in the PALS (periateriolar lymphocyte sheath areas) in the spleen. The macroscopically observed thymi in 5 control and 7 low dose females exhibited no microscopic abnormalities. In the thymi of the 2 control and 2 low dose females pregnancy/lactation involution was observed. The thymic lobules were decreased in size but exhibited normal structure with the histological appearance of age-involution. Increased glycomeric vacuolation, viz moderate versus very slight was seen in the liver of 4 high dose females and was considered to be a potential cause of the increased weight.
Examination of the reproductive organs revealed a statistically significant increased in the incidence of cysts in the ovaries of 8 high-dose females.
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
> 0.3 - < 0.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Decreased thymus weight
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
> 0.3 - < 0.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Decreased thymus weight and microscopic and macroscopic changes in the thymus
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg diet
System:
immune system
Organ:
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
The number of pups delivered per litter was comparable in all groups, 9.8, 10.8, 9.6 and 8.0 in the control, low-, mid- and high-dose groups respectively. The number of liveborn pups was 108, 129, 104 and 64 in the control, low-, mid- and high-dose groups and was considered to be statistically significantly decreased in the high dose group.
The number of stillborn pups in the control, low- and mid-dose groups were 0, in the high-dose group a statistically significant increase was noted, with 8 stillborns recorded (6 of these from the same litter, 1 each in two additional litters).
Pup mortality on post-natal day 4 (PN 4) was comparable in all groups except the high dose groups in which there was a statistically significant increase. 1, 2, 2 and 22 mortalities (incidences 0.9, 1.6, 1.9 and 34.0%) were recorded in the control, low-, mid- and high-dose groups respectively.
Only in the high-dose group, 3 litters were lost entirely between post-natal day 0 and 4.
The number of live pups per litter on PN 1 (9.8, 10.8, 9.6 and 8.0) and PN 4 (9.7, 10.6, 9.4 and 7.0), was not found to be statistically significantly different even though the number of live pups was decreased in the high-dose group on PN1 and 4.
No difference was observed in the sex ratio.

CLINICAL SIGNS (OFFSPRING)
On post-natal day 1, the number of runts was statistically significantly increased in the 250 mg/kg diet group. No other treatment related abnormalities were recorded.

BODY WEIGHT (OFFSPRING)
On post-natal day 1 and 4, a statistically significant decrease in pup bodyweight in the high-dose group was recorded (day 1 mean value for males+females in the high dose group was 4.54 g, compared to 5.08 in the control group). The pup weight change (PN 1-4) was significantly decreased in the male pups of the high-dose group (mean body weight gain of 1.38 g, compared to 2.56 g in the controls). No effects were noted in the other treatment groups.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic evaluation of the stillborn pups revealed 3 partially cannibalized pups and 3 autolytic pups in the high-dose group; the latter pups had no abnormalities. In addition 2 stillborn pup with no abnormalities in the high-dose groups were examined.
Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
250 mg/kg diet
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no pimary effects on reporduction
Key result
Critical effects observed:
no
Reproductive effects observed:
not specified

Table 1: Test material concentration in experimental diets

Nominal concentration (mg/kg)

Mean Nominal Measured Concentration (mg/kg)

Percent of Nominal

0 (#1)

<0.05

NA

0 (#2)

<0.05

NA

0 (#3)

<0.05

NA

5 (#1)

4.52

90

5 (#2)

4.93

99

5 (#3)

4.40

88

25 (#1)

24.9

100

25 (#2)

26.5

106

25 (#3)

26.3

105

250 (#1)

247

99

250 (#2)

240

96

250 (#3)

244

98

#3: repeated analysis of batch no. 2

 

Table 2: Summary of relevant treatment related findings

Parameter

Dose levels

5 mg/kg diet

25 mg/kg diet

250 mg/kg diet

Bodyweight: GD 21, PN 1 (females only)

Decreased

Bodyweight change: GD 14-21 (females only)

Decreased

Food consumption: PM 7-13 (males) GD 7-14, 14-21 and PN 1-4 (females only)

Decreased

Bilirubin (females only)

Increased

Alkaline phosphatase (males only)

Increased

Relative liver weight (females only)

Increased

Relative kidney weight (females only)

Increased

Absolute and/or relative thymus weight

Decreased

Decreased

Thymus: lymphoid depletion (males only)

Increased

Thymus: lymphoid depletion (females only)

Increased

Increased

Ovary: cysts (females only)

Increased

Liver: glycogenic vacuolation (females only)

Increased

Duration of gestation

Increased

 

Table 3: Reproduction and litter data (high dose group)

Parameter

High dose group

Control group

Mean duration of gestation (days)

21.7*

21

Number of females with liveborn pups

8

11

Gestation index (%)

80

100

* Statistically significantly different.

 

Conclusions:
Based on the effects noted in the thymus in both male and female rats in the 25 mg/kg diet groups, the NOAEL for general toxicity was concluded to be the lowest group tested, 5 mg/kg diet which was equivalent to 0.3-0.4 mg/kg bw/day for male animals and 0.3-0.5 mg/kg bw/day for female animals.
A NOAEL for reproductive toxicity was not considered necessary because the reproductive effects observed were non-specific and considered to be related to maternal toxicity.
Executive summary:

The toxicological effects of the test substance and possible effects on reproduction was assessed in a repeated dose toxicity and reproductive and developmental screening study in rats. The study was performed in accordance with GLP and to the standardised guideline OECD 422.

Based on the effects noted in the thymus in both male and female rats in the 25 mg/kg diet groups, the NOAEL was concluded to be the lowest group tested, 5 mg/kg diet which was equivalent to 0.3-0.4 mg/kg bw/day for male animals and 0.3-0.5 mg/kg bw/day for female animals.

A NOAEL for reproductive toxicity was not considered necessary because the reproductive effects observed were non-specific and considered to be related to maternal toxicity.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
11.8 mg/kg bw/day
Study duration:
chronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
11.8 mg/kg bw/day
Species:
rat
Additional information

The key study Barraclough, Dioctyltin Oxide: Oral (Dietary) Extended One-Generation Reproductive Toxicity Study in the Rat (OECD 443), was performed in accordance with GLP and to the standardised guideline OECD 443. The study was performed to a high standard and the reporting was to a sufficient level to assess the quality of the data presented. The study was therefore awarded a reliability score of 1 in accordance with the criteria for assessing data quality as defined in Klimisch (1997). It was therefore considered justified to base conclusions of the toxicological nature of the substance on the findings of this study.

 

Oral (dietary) administration of control article (vehicle) or 5, 25 or 200 ppm

(equivalent to a nominal dose level of 0, 0.4, 1.8, or 11.8 mg/kg/day) Dioctyltin Oxide

to Han Wistar rats for up to two consecutive generations resulted in test article-related

effects at all dose levels.

Systemic effects in the F0 generation included changes in thymus following

administration of 200 ppm for both sexes and following administration of 25 ppm to

females. The kidney changes noted for F0 females were considered not adverse;

therefore, a no observed adverse effect level (NOAEL) was established as 25 ppm for

systemic toxicity for males and 5 ppm for females for the F0 generation.

Marginally lower mating and fertility indices were noted in the F1 generation

following administration of 200 ppm, however, this was considered nonadverse,

therefore the no observed effect level (NOEL) for reproductive toxicity is 200 ppm.

Immunotoxicity effects in the F1 generation consisted of a reduction of TDAR

coupled with slight reductions in circulating blood T lymphocyte numbers at all dose

levels, compared with controls, with the effect more prominent for males. In the

absence of any associated changes in hematology parameters, these findings were

considered nonadverse. As such, the NOAEL is 200 ppm for developmental

immunotoxicity.

Based on lower body weights, delayed sexual maturation which resulted as a

secondary effect to the lower mean body weights, in the F1 generation, and smaller

litter sizes and pup mortality in the F2 generation following administration of

200 ppm, the NOEL for offspring growth and development is established as 25 ppm.

No evidence of any endocrine disturbance was noted in this study.

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 November 2003 to 8 March 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted in accordance with the standardised testing guideline OECD 422 and in accordance with GLP with no deviations thought to affect the quality of the presented data. The study was reported to a high standard, sufficient to assess the reliability of the data presented.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10-11 weeks
- Weight at study initiation: The weight variation of the animals for each sex did not exceed 20 %.
- Diet: ad libitum
- Water: tap water ad libitum in polypropylene bottles

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 30-70 5
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

DOSE-RANGE FINDING TEST IN-LIFE DATES: From: 12 November 2003 To: 26 November 2003
MAIN TEST IN-LIFE DATES: From: 14 January 2004 To: 8 March 2004
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): The experimental diets were prepared once shortly before the study
- Mixing appropriate amounts with (Type of food): The test substance was weighed and placed in a small grinder. The tray was rinsed with food which was then also added to the grinder and mixed for 2 x 30 seconds. This mixture was moved to a Stephan cutter and the grinder was rinsed with food and moved to the cutter. Approximately 3 kg weighed food was mixed into the cutter for 2 x 2 minutes and moved. This was then moved to the Lödige cutter. The Stephan cutter was rinsed with approximately 3 kg of food and that was also moved to the Lödige cutter. Mixing was continued in the Lödige cutter for 2 minutes with the total amount of food.
- Storage temperature of food: <-18 °C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The feed was checked for homogenous distribution, stability and concentration of the test substance for all the doses in the dose-range finding study. The same dose preparation for the dose-range finding study was also used for the main test. The homogenous distribution and achieved concentration of the low dose in the main test was also analysed.
Directly after preparation of the diet for the dose-range finding study, samples for homogeneity and stability were taken. Five samples were taken (approximately 50 g each) to examine the homogeneity of the dose from the top centre, middle centre, bottom centre, left centre and right centre of the mixer. Secondly samples (around 50 g) were taken from the top centre part of the mixer to measure the stability. The samples taken for measurement of the homogeneity were also used for dose confirmation. In addition the content (achieved concentration) of the test substance in the batch of diet used in the main study. Diet samples were taken for analysis immediately after preparation and stored at – 18 °C.
Samples of the 0, 25, 75, 200 and 500 mg/kg diets from the dose-range finding study and doses of 0, 25 and 250 mg/kg from the main study as well as all related calibration samples were derivatised.
A calculated amount of internal standard solution (MHT, DHT and TTPT in methanol) was added to 2.0 g of diet in a 50 mL Corning tube. 10 mL of 100 % acetic acid was then added and the Corning tube was closed and shaken for 60 minutes (250 rpm). 10 mL of acetate buffer solution (pH 4.5) was added along with 10 mL methanol. 2.0 mL of 20 % (m/V) aqueous STEB solutions was added, followed by 10 mL hexane (containing naphthalene, approximately 0.1 mg/L). The tube was shaken for 15 minutes (250 rpm) then placed in an oven at 60 °C for 15 minutes. After phase separation, the hexane layer (approximately 3 mL) was removed and washed with 3 mL of 2 mol/L HCl (30 minutes shaking at 250 rpm). The hexane top layer was diluted with hexane: hexane extracts from sample with dose levels of 0, 25 and 75 mg/kg were diluted five times, the higher doses were diluted fifty times. The resulting solutions were transferred into an amber coloured glass vial and anaylysed using GC-MS.
The procedure for the samples from the main study at doses of 0 and 5 mg/kg followed the same derivatisation procedure, except the initial quantity of the diet was 5.0 g not 2.0 g
The concentration of organotin compounds in the extracts was determined using GC-MS. For calculation of the amount of DOTO in the samples, the peak area of DHT was used as an internal standard. Quantitiation was achieved using the calibration graphs constructed from the calibration solutions.
The following conditions were used:
- Column: Fused silica HP5 MS, 30 m, 0.25 mm ID, 0.25 µm film
- Precolumn: fused silica HP5 MS, 2.5 m, 0.25 mm ID, 0.25 µm film
- Column temperature: after 3 minutes 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: DOT m/z = 375*; 263; 151, DHT 347*; 249; 179
Mass fragments marked with an asterisk were used for quantitation
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until pregnancy occurred
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
Duration of treatment / exposure:
Treated food was available ad libitum
Frequency of treatment:
Daily
Duration of test:
28 days
No. of animals per sex per dose:
22 males and 22 females in the 14-day dose range finding study (5 groups of 4 male and 4 female rats)
52 males and 52 females in the main study (4 groups of 12 male and 12 female rats)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses were selected on the results of the range finding test
- Rationale for animal assignment: Randomised
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Every morning throughout the study, and a second observation in the afternoon of working days.
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the first exposure and then once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female rats were taken on day -2 (randomisation) and on days 0 (first day of dosing), 7 and 13 of the premating period.
Males were weighed weekly during the mating period until sacrifice. Females were weighed during mating (day 0, 7 and 13) and mated females were weighed on day 0, 7, 14 and 21 during presumed gestation and on day 1 and 4 of lactation. All animals were weighed at sacrifice.

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, also calculated as g/animal/day

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the premating period
- Anaesthetic used for blood collection: Yes, CO2/02 anaesthesia
- Anit-coagulant: K2-EDTA
- Animals fasted: Yes, overnight
- How many animals: 5 rats/group
- Parameters checked:
Haemoglobin
Packed cell volume
red blood cell count
reticulocytes
Total white blood cell count
Prothrombin time
Thrombocyte count
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the premating period
- Anaesthetic used for blood collection: Yes, CO2/02 anaesthesia
- Animals fasted: Yes, overnight
- How many animals: 5 rats /group
- Parameters checked:
Fasting glucose
Alkaline phosphatase activity (ALP)
Aspartate aminotransferace activity (ASAT)
Alanine aminotransferace activity (ALAT)
Gamma glutamyl transferase activity (GGT)
Total protein
Albumin
Ratio albumin to globulin
Urea
Creatinine
Bilirubin (total)
Cholesterol (total)
Triglycerides
Phospholipids
Calcium (Ca)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Inorganic phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Arena testing was performed prior to the first exposure and then once weekly until the end of dosing and in females until the end of lactation. Males and females selected for the functional observation battery test (FOB) and spontaneous activity measurements were excluded from the final arena testing.
At the end of the study, FOB test and spontaneous motor activity measurements were performed on day 27 for males and on post natal day for for females.
- Dose groups that were examined: All animals were subject to the arena testing. For the other two tests, 5 animals were randomly selected from each dose group.
- Battery of functions tested:
Autonomic: lacrimation, salivation, pupil response to light, palpebral closure, piloerection, defecation and urination
Neuromuscular: gait, mobility, forelimb and hindlimb gripstrength, landing foot splay, righting reflex
Sensorimotor: response to tail pinch, click, tough and approach of a visual object
Convulsive: clonic and tonic movements
Excitability: ease of removal, handling reactivity, arousal and vocalisations
Activity: rearing and motor activity
Physiological: body temperature

SACRIFICE
- Maternal animals: Sperm positive females that were not pregnant were killed 25 days after copulation, mothers with litters were killed on post natal day 4.

GROSS PATHOLOGY: Yes
The following were taken from all animals:
Ovaries
Uterus
Organs and tissues showing macroscopic abnormalities
The following were taken from 5 rats/group:
Adrenals
Axillary lymph node
Bone marrow (femur)
Brain
Caecum
Coagulation glands
Colon
Duodenum
Eyes
Heart
Jejunum
Lungs
Kidneys
Liver
Mammary gland (females only)
Mesentric lymph node
Parathyroids
Peyer's patches
Pituitary
Rectum
Sciatic nerve
Spinal cord
Spleen
Stomach
Thymus
Thyroids
Trachea
Urinary bladder

The following organs were weighed:
Adrenals
Brain
Heart
Kidneys
Liver
Spleen
Thymus

HISTOPATHOLOGY: Yes Microscopic examination was performed on the collected organs of all rats in the control and high-dose group.
The liver and ovaria of females and the thymus of the male and female rats in the low and mid-dose groups were also evaluated
The following tissues, though collected were not subject to histopathological examination:
Coagulation glands
Mammary gland (females only)
In addition, reproductive organs of females that were non-mated or non-pregnant of the mid and low dose groups were microscopically examined.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: Yes: all still born pups and pups that died during lactation
Statistics:
Clinical findings and histopathological changes of the maternal animals were evaluated using Fisher’s exact probability test.
Number of implantation sites, live and dead pups were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann Whitney U-test.
Bodyweight, bodyweight gain, organ weights, food consumption, red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values and organ weights were assessed by one-way ANOVA followed by Dunnett’s multiple comparison tests.
Reticulocytes and relative differential white blood cell counts were assessed using Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-tests.
The results of the functional observations were measured on different scales. Continuous measurements were analysed by one-way analysis of variance at each time point, if found to be statistically significant, a post-hoc group comparison was performed. Rank order data were analysed by Kruskal-Wallis analysis of variance at each test time point, followed by planned multiple comparisons between dose groups were a significant results occurred. Categorical data were assessed using Pearson chi-square analysis.
Motor activity data were assessed by one-way analysis of variance at each time point with a post-hoc group comparison performed on significant results.
All tests were two sided and the level of probability p<0.05 was considered as significant. Effects of treatment on habituation were analysed by repeated measures of analysis variance in five 6 minute time blocks. Statistical evaluations on pup variables were considered on a litter basis. Additional evaluations on a pup basis were performed to identify any specific dose-related effect that may have occurred.
Indices:
Sex ratio day n = (number of live male pups on day n/number of live pups on day n) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
adverse effects in the immune system (thymus)
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
thymus
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increase in liver weight, decrease of thymus weight
Gross pathological findings:
not specified
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not specified
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
only with high maternal toxicity
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
One female in the high dose group was found dead on gestation day 24; this animal was pregnant and 11 dead foetuses were found in the uterus.
The only finding during the gestation period (gestation day 21) was a sparsely haired animal in the 250 mg/kg group. During the lactation period, sparsely haired animals were noted in the control (n = 1), 5 mg/kg (n = 1) and in the 250 mg/kg group (n = 1). No other findings were noted.

BODY WEIGHT AND WEIGHT GAIN
Mean bodyweights of the dams of the high-dose group was statistically significantly decreased on gestation day 21 (8.5% lower than controls) and post-natal day 1 (9.3% lower than controls). Mean bodyweight changes of the dams of the high-dose group were more markedly affected during pregnancy, as the overall mean weight gain for the control animals accounted to 74.31 g, whereas that in the high-dose animals it was only 51.43 g (approx. 31% lower); during gestation day 14 to 21, the reduction in mean weight gain in the high-dose animals attained statistical significance, compared to controls, and was approximately 52% lower. Bodyweights and bodyweight change of the dams of all the treated groups were comparable to the controls group at all other times of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
During the gestation period and lactation period (gestation days 7-14 and 14-21 and post-natal days 1-4), food consumption (expressed as g/animal/day and g/kg bodyweight/day) of the dams in the high-dose group was significantly decreased. Considering the entire pregnancy period, high-dose animals ate approx. 13% less food than controls (in terms of g/kg/day), and approx. 24% less food than controls during the 4-day lactation period. No other treatment-related effects were observed.

- Females (listed as low, mid and high dose groups; 5, 25 and 250 mg/kg diet respectively)
premating days 0-7: 0.4, 1.7 and 17.4 mg/kg bodyweight/day
premating days 7-13: 0.3, 1.6 and 15.4 mg/kg bodyweight/day
gestation days 0-7: 0.4, 2.0 and 17.4 mg/kg bodyweight/day
gestation days 7-14: 0.4, 1.9 and 16.7 mg/kg bodyweight/day
gestation days 14-21: 0.3, 1.4 and 11.2 mg/kg bodyweight/day
Post-natal days 1-4: 0.5, 2.4 and 17.4 mg/kg bodyweight/day

HAEMATOLOGY
All treated groups were found to be comparable to controls

CLINICAL CHEMISTRY
Bilirubin (µmol/L) was found to be statistically significantly increased in the high-dose females. this finding were considered to be treatment related. Other effects such as the statistically significant increase in calcium in the 5 mg/kg group was not considered to be related to treatment. No other changes were observed.

NEUROBEHAVIOUR
No treatment-related effects were observed.

ORGAN WEIGHTS
The absolute and relative weight of the female animals of the high dose group was significantly decreased (-69 and -66%, respectively, compared to controls). In the mid-dose group the relative thymus weight was also statistically significantly decreased (-36% compared to controls).
In the female animals of the high-dose group, the relative kidney and liver weights were statistically significantly increased (+14 and +22%, respectively, compared to controls).
No other effects were observed.

GROSS PATHOLOGY
At necropsy, a decrease in thymic size was seen in all animals in the 250 mg/kg diet groups, 11 animals in the 25 mg/kg diet group, 7 animals in the 5 mg/kg diet group and 5 animals in the control group.
Examination of the female that was found dead revealed hydrothorax, haemorrhagic lungs, dilation of the vena cava and haemorrhagic discharge in the vagina, these were considered to be indicative of problems during parturition.

HISTOPATHOLOGY
Microscopic evaluation of the thymus revealed moderate to very severe lymphoid depletion in all animals of the 250 and 25 mg/kg diet groups. Lymphoid depletion was characterised by a decrease in the thymic lobules due to an extensive loss of cortical and medullary small lymphocytes. The distinction between the cortical and medullary areas was unclear. In the more extreme effects observed, the cortex was very small, or absent. The remaining lymphoid cells visible in the cortical areas were mainly lymphoblasts. Lymphoblastic cells and reticuloepithelial cells had increased, and/or higher numbers of these cells were visible due to the disappearance of small lymphocytes and the collapse of the thymic stroma. In 3 high-dose animals, lymphoid depletion was accompanied by lymphoid depletion in the PALS (periateriolar lymphocyte sheath areas) in the spleen. The macroscopically observed thymi in 5 control and 7 low dose females exhibited no microscopic abnormalities. In the thymi of the 2 control and 2 low dose females pregnancy/lactation involution was observed. The thymic lobules were decreased in size but exhibited normal structure with the histological appearance of age-involution. Increased glycomeric vacuolation, viz moderate versus very slight was seen in the liver of 4 high dose females and was considered to be a potential cause of the increased weight.
Examination of the reproductive organs revealed a statistically significant increased in the incidence of cysts in the ovaries of 8 high-dose females.
Key result
Dose descriptor:
NOAEL
Effect level:
0.3 - 0.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
only with high maternal toxicity
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
only with high maternal toxicity
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
VIABILITY
The number of females with liveborn pups was 11, 11, 11 and 8 for the control, low, mid and high dose respectively.
One female was found dead in the high dose group on gestation day 24; the animal was pregnant and 11 dead foetuses were discovered in the uterus. Stillborn pups were observed in the high dose in three litters. In the high-dose group 1 female with all stillborn pups was observed.
Pre-implantation loss was 8.4. 2.2. 12.3 and 6.2% for the control, low-, mid- and high-dose groups and was considered comparable.
Pos-implantation loss was 9.8, 11.6, 7.5 and 38.7% for the control, low-, mid- and high-dose groups respectively. The post-implantation loss recorded in the high-dose group was considered to be statistically significantly increased.
The number of pups delivered per litter was comparable in all groups, 9.8, 10.8, 9.6 and 8.0 in the control, low-, mid- and high-dose groups respectively. The number of liveborn pups was 108, 129, 104 and 64 in the control, low-, mid- and high-dose groups and was considered to be statistically significantly decreased in the high dose group.
The number of stillborn pups in the control, low- and mid-dose groups were 0, in the high-dose group a statistically significant increase was noted, with 8 stillborns recorded (6 of these from the same litter, 1 each in two additional litters).
Pup mortality on post-natal day 4 (PN 4) was comparable in all groups except the high dose groups in which there was a statistically significant increase. 1, 2, 2 and 22 mortalities (incidences 0.9, 1.6, 1.9 and 34.0%) were recorded in the control, low-, mid- and high-dose groups respectively.
Only in the high-dose group, 3 litters were lost entirely between post-natal day 0 and 4.
The number of live pups per litter on PN 1 (9.8, 10.8, 9.6 and 8.0) and PN 4 (9.7, 10.6, 9.4 and 7.0), was not found to be statistically significantly different even though the number of live pups was decreased in the high-dose group and PN1 and 4.
No difference was observed in the sex ratio

CLINICAL SIGNS (OFFSPRING)
On post-natal day 1, the number of runts was statistically significantly increased in the 250 mg/kg diet group. No other treatment related abnormalities were recorded.

BODY WEIGHT (OFFSPRING)
On post-natal day 1 and 4, a statistically significant decrease in pup bodyweight in the high-dose group was recorded (day 1 mean value for males+females in the high dose group was 4.54 g, compared to 5.08 in the control group). The pup weight change (PN 1-4) was significantly decreased in the male pups of the high-dose group (mean body weight gain of 1.38 g, compared to 2.56 g in the controls). No effects were noted in the other treatment groups.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic evaluation of the stillborn pups revealed 3 partially cannibalized pups and 3 autolytic pups in the high-dose group; the latter pups had no abnormalities. In addition 2 stillborn pups with no abnormalities in the high-dose groups were examined.
Key result
Dose descriptor:
NOAEC
Effect level:
> 25 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Viability index day 1-4
Remarks on result:
other: effects only with high maternal toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Test material concentration in experimental diets

Nominal concentration (mg/kg)

Mean Nominal Measured Concentration (mg/kg)

Percent of Nominal

0 (#1)

<0.05

NA

0 (#2)

<0.05

NA

0 (#3)

<0.05

NA

5 (#1)

4.52

90

5 (#2)

4.93

99

5 (#3)

4.4

88

25 (#1)

24.9

100

25 (#2)

26.5

106

25 (#3)

26.3

105

250 (#1)

247

99

250 (#2)

240

96

250 (#3)

244

98

#3: repeated analysis of batch no. 2

 

Table 2: Summary of relevant treatment related findings

Parameter

Dose levels

5 mg/kg diet

25 mg/kg diet

250 mg/kg diet

Bodyweight: GD 21, PN 1 (females only)

 

 

Decreased

Bodyweight change: GD 14-21 (females only)

 

 

Decreased

Food consumption: PM 7-13 (males) GD 7-14, 14-21 and PN 1-4 (females only)

 

 

Decreased

Bilirubin (females only)

 

 

Increased

Alkaline phosphatase (males only)

 

 

Increased

Relative liver weight (females only)

 

 

Increased

Relative kidney weight (females only)

 

 

Increased

Absolute and/or relative thymus weight

 

Decreased

Decreased

Thymus: lymphoid depletion (males only)

 

 

Increased

Thymus: lymphoid depletion (females only)

 

Increased

Increased

Ovary: cysts (females only)

 

 

Increased

Liver: glycogenic vacuolation (females only)

 

 

Increased

Post-implantation loss

Increased

Number of stillborn pups

Increased

Pup mortality PN 4

Increased

Pup weight PN 1

Decreased

Number of runts PN 1

 

 

Increased

 

Table 3: Litter data (high dose group)

Parameter

High dose group

Control group

Number of females with liveborn pups

8

11

Post-implantation loss (%)

38.7*

9.8

Mean number of pups delivered

8.0

9.8

Mean number of live pups/litter PN1

8.0

9.8

Mean number of live pups/litter PN 4

7.0

9.7

Total number of stillborn pups

8*

0

Pup mortality PN 4 (%)

34*

0.9

Pup weight PN 1 (g)

4.5*

5.1

Pup weight PN 4 (g)

6.5

7.6

Percentage of runts PN 1 (%)

34*

2

* Statistically significantly different.

Conclusions:
Based on the effects noted in the thymus in the 25 mg/kg diet groups, the NOAEL for general toxicity was concluded to be the lowest group tested, 5 mg/kg diet which was equivalent to 0.3-0.5 mg/kg bw/day.
A NOAEL for developmental toxicity was not considered necessary because the reproductive effects observed were non-specific and considered to be related to metarnal toxicity.
Executive summary:

The developmental effects of the test substance was assessed in a repeated dose toxicity and reproductive and developmental screening study in rats. The study was performed in accordance with GLP and to the standardised guideline OECD 422. Only one death was noted during the study.

Based on the effects noted in the thymus in the 25 mg/kg diet groups, the NOAEL for general toxicity was concluded to be the lowest group tested, 5 mg/kg diet which was equivalent to 0.3-0.5 mg/kg bw/day.

A NOAEL for developmental toxicity was not considered necessary because the reproductive effects observed were non-specific and considered to be related to metarnal toxicity.

Endpoint:
developmental toxicity
Type of information:
other: according to CCH-D-2114340406-56-01/F the is to perform an OECD 414 study in a first species (rats or rabbits), oral route with the registered substance;
Adequacy of study:
supporting study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Pursuant to Articles 10(a)(vi) and/or (vii), 12(1)(d) and 13(4) of the REACH Regulation, a
technical dossier registered at 100 to 1000 tonnes per year shall contain as a minimum the
information specified in Annexes VII to IX of the REACH Regulation.
A “pre-natal developmental toxicity study” for a first species is a standard information
requirement as laid down in Annex IX, Section 8.7.2. of the REACH Regulation. Adequate
information on this endpoint needs to be present in the technical dossier for the registered
substance to meet this information requirement.
You have not provided any study record of a pre-natal developmental toxicity study in the
dossier that would meet the information requirement of Annex IX, Section 8.7.2.

Instead, you have sought to adapt this information requirement. The justification of the
adaptation given is "In accordance with Section 3 (Substance-Tailored Exposure-Driven
Testing) of Annex XI (General Rules for Adaptation of the Standard Testing Regime Set Out
In Annexes VII to X) of Regulation (EC) 1907/2006 (REACH), which states that testing in
accordance with Annex IX may be omitted, based on the exposure scenarios developed in
the Chemical Safety Report, it is considered justified to omit the developmental toxicity
study (required under point 8.7.2 of Annex IX). The results of the exposure assessment
conducted as a part of the Chemical Safety Report are considered to adequately
demonstrate the absence of or no significant exposure to the substance throughout the
lifecycle of the substance including its manufacture and all identified uses. As such further
testing for developmental effects is considered to be inappropriate".
However, ECHA notes that the information currently provided in the dossier does not meet
the specific rules for adaptation of Annex XI, section 3. According to Article 13(1) and
Section 3 of Annex XI of the REACH Regulation, testing in accordance with Annex IX may be
omitted based on a thorough and rigorous exposure assessment, provided that any one of
the three criteria of Section 3 of Annex XI is met and adequate justification and
documentation is provided. However, none of the criteria of that adaptation are currently
fulfilled.
The first criterion 3.2(a) requires “absence of or no significant exposure in all scenarios of
the manufacture and all identified uses”. Moreover, relevant PNECs or DNELs are to be
derived and exposure results are to be well below the derived PNECs or DNELs. According to
footnote (1) for 3.2.(a) (ii), "For the purpose of subparagraph 3.2(a)(ii), without prejudice
to column 2 of Section 8.7 of Annexes IX and X, a DNEL derived from a screening test for
reproductive/developmental toxicity shall not be considered appropriate to omit a prenatal
developmental toxicity study or a two-generation reproductive toxicity study". However, the
only DNEL in the dossier is based on a screening study, so criterion 3.2(a) cannot be
fulfilled.
ECHA considers that adequate and reliable documentation demonstrating the “absence of or
no significant exposure in all scenarios of the manufacture and all identified uses” has not
been provided. In several exposure scenarios for the combined routes, systemic long-term
the RCRs values were up to 0.503 and the PROCs indicate potential for exposure.
The second criterion 3.2(b) requires a demonstration that “throughout the life cycle strictly
controlled conditions as set out in Article 18(4)(a) to (f)” apply. As mentioned above, in
several exposure scenarios for the combined routes, systemic long-term the RCRs were not
demonstrating strictly controlled conditions as per Annex XI, section 3.2 (b). Strictly
controlled conditions are not demonstrated and therefore criterion 3.2(b) for exposurebased
adaptation is not satisfied. In particular, condition (a) as set out in Article 18(4) does
not appear to be fulfilled because it has not been demonstrated that the substance is
rigorously contained by technical means during its whole lifecycle.
The third criterion 3.2(c) sets out conditions which have to be fulfilled for a substance
incorporated in an article, particularly that the substance is not released during its life cycle,
that the likelihood of exposure of workers and general public under normal and foreseeable
circumstances is negligible and that the substance is handled under the conditions set out in
Article 18(4)(a) to (f) during all manufacturing and production stages including waste
management. Since the substance is not incorporated in an article within the meaning of
Article 3(3), this criterion does not apply to this case. Furthermore, strictly controlled
conditions as set out in Article 18(4)(a) to (f) are not demonstrated.
Upon receipt of the draft decision you submitted comments explaining that “Currently an
extended OECD TG 414 study (teratogenicity) in the rat is executed in order to meet USFDA
notification requirements.”. You also provided an overview of the study and indicated
that the main study will include: Teratogenicity according to OECD TG 414, Immunotoxicity,
Toxicokinetics (Mono-, Dioctyltin kation in food, plasma, feces, urine) and Zinc level
(plasma, food, water). You also indicated that a side study investigating an osteoporosis
marker (e.g. B-CTX, Osteocalcin, P1NP) and an endocrine marker, will also be performed.
ECHA acknowledged the comments and information provided. However, as the data for this
endpoint are not yet available, the draft decision was not amended. The compliance of the
ongoing study will be evaluated during the follow up process.
Therefore, the adaptations of the information requirement cannot be accepted.
You proposed to extend the pre-natal developmental toxicity study by including additional
examinations/parameters on immunotoxicity. ECHA notes, that it is at your discretion to
perform the intended additional examinations during the testing program and use the
results to ensure the safe use of the substance.
As explained above, the information available on this endpoint for the registered substance
in the technical dossier does not meet the information requirement. Consequently there is
an information gap and it is necessary to provide information for this endpoint.
According to the test method EU B.31/OECD 414, the rat is the preferred rodent species,
the rabbit the preferred non-rodent species and the test substance is usually administered
orally. ECHA considers these default parameters appropriate and testing should be
performed by the oral route with the rat or the rabbit as a first species to be used.
Therefore, pursuant to Article 41(1) and (3) of the REACH Regulation, you are requested to
submit the following information derived with the registered substance subject to the
present decision: Pre-natal developmental toxicity study (test method: EU B.31./OECD 414)
in rats or rabbits by the oral route.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not applicable
Remarks:
additional immunotixicity, tin in feces and plasma, zin in plasma
Principles of method if other than guideline:
additional immunotixicity, tin in feces and plasma, zin in plasma
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Route of administration:
oral: feed
Vehicle:
other: test substance was applicated in diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
yes, see attached report
Details on mating procedure:
not relevant for OECD 414
Dose / conc.:
0 mg/kg diet
Remarks:
0 mg/kg bw / say
Dose / conc.:
5 mg/kg diet
Remarks:
0.4 mg/kg bw / say
Dose / conc.:
25 mg/kg diet
Remarks:
1.8 mg/kg bw / say
Dose / conc.:
200 mg/kg diet
Remarks:
11.8 mg/kg bw / say
Control animals:
yes, plain diet
Details on study design:
Background Information:
In a previous dose-range finding study (Covance Study 8255410; Covance
Laboratories Ltd, 2015), tin (dioctylbis [2,4-pentanedionato-KO2, KO4]) in sesame oil
was administered to the rat by oral gavage (dose volume of 2 mL/kg) at dose levels of
4, 20, and 50 mg/kg/day.

Six unscheduled deaths occurred during that study. Two females administered
20 mg/kg/day were found dead, and two females administered 20 or 50 mg/kg/day
were sacrificed due to the severity of clinical observations, which included thin
appearance, and hunched posture. Following administration of 20 or 50 mg/kg/day,
significant reductions in body weight gain were noted, and changes in clinical
pathology parameters and increased liver weights were noted at the end of the
dosing period. In addition, test article-related macroscopic changes in the thymus and
microscopic findings in the thymus and thyroid were noted in males and females at all
dose levels due to effects of the test article.

For the F1 offspring, the number of implantation sites was generally similar to the
number of pups born; this indicated no embryo-lethality occurred. No effect on pup
body weight was noticed at birth, although an increased number of stillborn pups was
evident in test article-treated groups. Based on findings from that study and on a
literature search carried out by the Sponsor, it is known that dioctyltin (DOT)
compounds have an effect on the immune system and are associated with thymic
atrophy (Tonk et al., 2011); this was the main finding at necropsy of the Covance
study.

For comparability with studies described in the literature, this study was carried out
using the oral (dietary) route. Dioctyltin oxide (DOTO) was offered to rats in the diet
at 5, 25, and 250 ppm, and data have been supplied to detail the dietary dosing
analysis, stability, and homogeneity (Waalkens-Berendsen, 2004). In addition, data,
which describes food consumption during the study in g/animal/day indicated that
there is no effect on food consumption when the diet is produced with these levels of
DOTO. The dose levels proposed for this study did not exceed those used and were
selected by the Sponsor based on that study and on a separate study published in 2011
using di-n-octyltin dichloride (Tonk et al., 2011).

Dioctyltin interacts with NR3C1, causing an acute, adverse effect in the immune
system. Twenty-four hours after a single dose, a decrease in CD4-/CD8- occurs. After
72 hours, decreased thymus weight and thymus atrophy occur. The suppression of the
immune system causes an increase in tissue and metallothionein zinc, followed by a
decrease in plasma zinc levels. Zinc is required for embryo development and growth.

The oral (dietary) route of administration was chosen because it is an acceptable and
commonly used route exposure for regulatory studies of this type. In addition, this
route was considered as other tests on dioctyl tin compounds have been carried out
using this route, and it is considered to reduce stress to the animal as the test article
may result in stomach irritation when given by oral gavage.
Maternal examinations:
Dioctyltin Oxide-related changes in the thymus weights were recorded in toxicity females, compared with concurrent controls.
Group mean thymus:body weight ratios and unadjusted thymus weights were lower for toxicity females administered 200 mg/kg diet, compared with concurrent controls
Ovaries and uterine content:
findings in other tissues [beyond thymus] were generally infrequent, of a minor nature and consistent with the usual pattern of findings in rats of this strain and age
Fetal examinations:
Minor increases in some variations were recorded in fetuses/litters from high dose group; these included sternebrae with misaligned ossification centres, incomplete ossification of cervical centrum (p<0.05) and split in xyphoid cartilage, with the latter also showing an increase in intermediate dose fetuses/litters. A dose-relationship was not apparent and these minor changes were considered to be of no toxicological significance.

Any other intergroup differences, including those achieving statistical significance, were considered to be due to normal biological variation
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):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Description (incidence and severity):
no data reported in raw draft data
Clinical biochemistry findings:
not examined
Description (incidence and severity):
no data reported in raw draft data
Urinalysis findings:
not examined
Description (incidence and severity):
no data reported in raw draft data
Behaviour (functional findings):
no effects observed
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
The results indicated that test article-related changes were limited to a slight to marked, transient, increase in circulating CD25 positive B and T lymphocytes noted at the 11.8 mg/kg/day dose level only. Results at necropsy indicate that this finding was reversible.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Dioctyltin Oxide-related changes in the thymus weights were recorded in toxicity females, compared with concurrent controls.
Group mean thymus:body weight ratios and unadjusted thymus weights were lower for toxicity females administered 200 mg/kg diet, compared with concurrent controls
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Upon macroscopic examination, Dioctyltin Oxide-related findings were noted in the thymus.
Small thymus was recorded in some toxicity females administered 200 mg/kg diet, which generally correlated with microscopic findings.
No other macroscopic findings considered related to Dioctyltin Oxide were recorded.
Other tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Upon microscopic examination, Dioctyltin Oxide-related findings were recorded in the thymus of toxicity animals.
An increased incidence and severity of thymus atrophy was recorded in most females administered 200 mg/kg diet
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Description (incidence and severity):
no data reported in raw draft data
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): not relevant in an OECD 414 study
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
see attached summary and raw fata fetel
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): see attached summary and raw fata fetel
Reduction in number of live offspring:
not examined
Description (incidence and severity):
see attached summary and raw fata fetel
Changes in sex ratio:
no effects observed
Description (incidence and severity):
see attached summary and raw fata fetel
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
see attached summary and raw fata fetel
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
see attached summary and raw fata fetel
External malformations:
no effects observed
Description (incidence and severity):
see attached summary and raw fata fetel
Skeletal malformations:
no effects observed
Description (incidence and severity):
see attached summary and raw fata fetel
Visceral malformations:
no effects observed
Description (incidence and severity):
see attached summary and raw fata fetel
Other effects:
not examined
Description (incidence and severity):
no data reported in raw draft data
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 11.8 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
The LOAEL for acute immune toxicity for Dioctyltin oxide was determined to be 11.8 mg / kg bw7day thus a NOAEL of 1.8 mg/kg bw7day results foaternnal toxicity, with decrease of thymus. No adverse effects relating to reproduction/development
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
11.8 mg/kg bw/day
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The key study Barraclough, Dioctyltin Oxide: Oral (Dietary) Extended One-Generation Reproductive Toxicity Study in the Rat (OECD 443), was performed in accordance with GLP and to the standardised guideline OECD 443. The study was performed to a high standard and the reporting was to a sufficient level to assess the quality of the data presented. The study was therefore awarded a reliability score of 1 in accordance with the criteria for assessing data quality as defined in Klimisch (1997). It was therefore considered justified to base conclusions of the toxicological nature of the substance on the findings of this study.

 

Oral (dietary) administration of control article (vehicle) or 5, 25 or 200 ppm

(equivalent to a nominal dose level of 0, 0.4, 1.8, or 11.8 mg/kg/day) Dioctyltin Oxide

to Han Wistar rats for up to two consecutive generations resulted in test article-related

effects at all dose levels.

Systemic effects in the F0 generation included changes in thymus following

administration of 200 ppm for both sexes and following administration of 25 ppm to

females. The kidney changes noted for F0 females were considered not adverse;

therefore, a no observed adverse effect level (NOAEL) was established as 25 ppm for

systemic toxicity for males and 5 ppm for females for the F0 generation.

Marginally lower mating and fertility indices were noted in the F1 generation

following administration of 200 ppm, however, this was considered nonadverse,

therefore the no observed effect level (NOEL) for reproductive toxicity is 200 ppm.

Immunotoxicity effects in the F1 generation consisted of a reduction of TDAR

coupled with slight reductions in circulating blood T lymphocyte numbers at all dose

levels, compared with controls, with the effect more prominent for males. In the

absence of any associated changes in hematology parameters, these findings were

considered nonadverse. As such, the NOAEL is 200 ppm for developmental

immunotoxicity.

Based on lower body weights, delayed sexual maturation which resulted as a

secondary effect to the lower mean body weights, in the F1 generation, and smaller

litter sizes and pup mortality in the F2 generation following administration of

200 ppm, the NOEL for offspring growth and development is established as 25 ppm.

No evidence of any endocrine disturbance was noted in this study.

Justification for classification or non-classification

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