Dibutyltin oxide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

02 Jul 2021 to 25 Feb 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of the study was to make a preliminary assessment of the systemic toxic potential of Dibutyltin Oxide (DBTO) in a 21 day oral gavage study in rats, to select a suitable high dose for a subsequent OECD 422 screening study.
Three groups, each comprising three male and three female Sprague Dawley rats received DBTO at doses of 0.75, 3 or 5 mg/kg/day for 21 days by oral gavage. The original intention of the study was to dose for 14 consecutive days but the treatment period was extended to better investigate toxicity within the study. During the study, clinical condition, body weight, food consumption, water consumption (by visual assessment), organ weight and macropathology investigations were undertaken.

GLP compliance:

no

Remarks:

No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles.

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD) rat

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Sprague Dawley (Crl:CD(SD)) strain was used because of the historical control data (HCD) available at this laboratory and it was also consistent with the strain used in previous studies with Dibutyltin Oxide.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Males: 69 to 75 days, Females: 83 to 89 days
- Weight at study initiation: Males: 323 to 349 g, Females: 234 to 255 g
- Housing:
Cages: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution: Males and females were blocked by sex and the cages constituting each group were dispersed in a battery so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable.
Three of the same sex per cage and wood based bedding which was changed at appropriate intervals each week. Aspen wood based products and plastic shelter were provided to each cage throughout the study and replaced when necessary.
- Diet (e.g. ad libitum): SDS VRF1 Certified, pelleted diet ad libitum. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes ad libitum. Bottles were changed at appropriate intervals.
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier. Certificates of analysis were also received from the suppliers of the wood based bedding and Aspen wood based products.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 40-70°C
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.

IN-LIFE DATES: From: 07 July 2021 (animal arrival) To: 02 August 2021 (Necropsy)

Route of administration:

oral: gavage

Details on route of administration:

In line with the OECD TG for the OECD 422, it is recommended that the test chemical be administered orally unless other routes of administration are considered more appropriate. When the oral route is selected, the test chemical is usually administered by gavage.

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
A series of formulations at the required concentrations were prepared by dilution of the relevant higher concentration with the vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance has a low water solubility. Therefore, a well-tolerated oil (peanut oil) was selected for preparation of a test item suspension.
- Concentration in vehicle: 0.15, 0.6, 1.0 mg/mL
- Amount of vehicle (if gavage): dose volume 5 mL/kg bw

Analytical verification of doses or concentrations:

no

Remarks:

Homogeneity/stability of formulations during storage were confirmed as part of another study: Formulations (0.1 - 5 mg/mL) were determined to be stable for: 24 hours at 15 to 25°C and 15 days at 2-8°C. No formulation analysis was performed on this study.

Duration of treatment / exposure:

21 days

Frequency of treatment:

Once daily, 7 days per week

Dose / conc.:

0.75 mg/kg bw/day (nominal)

Dose / conc.:

3 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3/sex/dose

Control animals:

yes, historical

Details on study design:

- Dose selection rationale:
Considering the prenatal developmental toxicity administering DBTO by gavage at a dose level of up to 6 mg/kg bw/day as the most relevant study (MPI Research Study Number: 399-252) to facilitate dose selection, the two deaths at the highest dose observed in this study can be clearly attributed to treatment due to a progressive decline in condition and body weight loss over 9 to 12 days of prior dosing. This dose is therefore unsuitable as a high dose, however, the absence of any effect of treatment at 3 mg/kg bw/day demonstrates a steep dose response. On this basis a dose level of 5 mg/kg bw/day is proposed as the highest dose for this preliminary study. It is expected that this dose level will induce toxicity in terms of an overall reduction of body weight gain and a reduction in the amount of food eaten, but no deaths.
An intermediate dose of 3 mg/kg bw/day is recommended which would be expected to produce a thymic effect to demonstrate a dose response. Although more classical evidence of toxicity would be ideal to demonstrate dose response at the intermediate level, dosing higher (4 mg/kg bw/day) is considered impractical due to dose and formulation accuracy limitations and the proximity to the highest dose of 5 mg/kg bw/day. A low dose of 0.75 mg/kg bw/day is recommended as the expected NOAEL. Dosing of 0.75 and 3 mg/kg bw/day will also allow direct comparison to the previous gavage studies administering DBTO.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of animal ill-health amongst the occupants.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.
Signs Associated with Dosing: Detailed observations were recorded daily at the following times in relation to dose administration:
- Pre-dose observation.
- As each animal is returned to its home cage.
- One to two hours after completion of dosing.
- As late as possible in the working day.
Clinical Signs: A detailed physical examination was performed on each animal on Days -3, 1, 4, 8, 11, 15, 18 and 22 to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded three days prior to the start of treatment (Day -3), before treatment commenced (Day 1), twice weekly throughout the study on Days 4, 8, 11, 15 and 18 and before necropsy (Day 22).

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
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for three days prior to the start of treatment (Day -3 to 1), and twice weekly throughout the study (Days 1 to 3, 4 to 7, 8 to 10, 11 to 14, 15 to 17 and 18 to 21).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals were subject to a necropsy. After a review of the history of each animal, a macroscopic examination of the tissues was performed. The thoracic and abdominal cavities were opened, the cranial cavity was opened only if observations indicated possible neurotoxic action. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY: No

Statistics:

Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data. Group mean values and standard deviations were frequently calculated using a greater number of decimal places than presented in the appendices.
Statistical analysis was not performed.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At routine physical examination there were no clinical signs observed that were considered to indicate any adverse effect of treatment on the clinical condition of animals at either 0.75, 3 or 5 mg/kg/day.
Instances of chin rubbing were observed following dose administration for one male receiving 3 mg/kg/day and all animals (both sexes) receiving 5 mg/kg/day. Chin rubbing is frequently observed when animals are dosed via the oral gavage route and is generally considered to reflect distaste of the dosing of formulations rather than any systemic effect of treatment.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled termination on Day 22.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall mean body weight gain (to Day 22) of males receiving 5 mg/kg/day was 41% lower than for males treated at 0.75 mg/kg/day, which was associated with slightly lower food intake. Although, all individual values at 5 mg/kg/day were lower than the range observed for males at 0.75 mg/kg/day, body weight gain for one male at 5 mg/kg/day was only just below this range. Overall mean body weight gain of males receiving 3 mg/kg/day was similar to that observed for males treated at 0.75 mg/kg/day but 1/3 individual values at 3 mg/kg/day was lower than the range observed for males at 0.75 mg/kg/day. In addition, compared to HCD the mean body weight gains for males receiving 5 mg/kg/day were below the 5% percentile for all gain intervals apart from body weight gains between Day 15 to 22.
Intergroup differences for overall body weight gains of females receiving 0.75, 3 or 5 mg/kg/day did not indicate any clear effect of treatment. However, occasional body weight losses were seen throughout the treatment period in some females from all dose groups and one female at 5 mg/kg/day had a poor overall body weight gain (to Day 22) of 4 g. Compared to HCD, 2/3 females receiving 5 mg/kg/day had body weight gains from Day 1 to 15 that were reduced by more than 50% compared to the HCD mean but this was below the 5% percentile for one female only.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption during treatment was lower than pre-treatment food intake at all dose levels and in both sexes.
Mean food consumption during the first three days of treatment for males receiving 3 or 5 mg/kg/day was lower than that observed for males receiving 0.75 mg/kg/day. For males at 5 mg/kg/day, lower food intake appeared to persist to some extent up to Day 18 of treatment. Assessment of food consumption against HCD is only possible for the first two weeks of treatment due to the lack of HCD for the third week on studies of this type. Compared to HCD, food consumption for males at 5 mg/kg/day was lower than the HCD mean but was either slightly below or equal to the 5% percentile up to Day 15 of treatment.
Intergroup differences in food consumption for females receiving 0.75, 3 or 5 mg/kg/day did not indicate any obvious effect of treatment. Compared to HCD, up to Day 15, food intake for females at all dose levels were within the 5% to 95% percentile range.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No visual effect was observed on fluid intake.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no intergroup differences on the kidney, liver, or spleen weights in both sexes after 21 days of treatment.
The thymus weights (absolute and % relative to terminal body weight) of both sexes receiving 3 or 5 mg/kg/day were lower than males or females treated at 0.75 mg/kg/day, with the extent of the mean thymus weight reductions being dose dependent. Mean reductions in thymus weights (% relative to terminal body weight) were 39% and 34% at 3 mg/kg/day and 40% and 46% at 5 mg/kg/day for males and females, respectively, compared to animals receiving 0.75 mg/kg/day. For males 3/3 and 2/3 individual values at 3 or 5 mg/kg/day, respectively, were lower than the range observed for males at 0.75 mg/kg/day. For females 2/3 and 3/3 individual values at 3 or 5 mg/kg/day, respectively, were lower than the range observed for females at 0.75 mg/kg/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic necropsy findings in males at all dose levels and females treated at 0.75 or 5 mg/kg/day.
One female receiving 3 mg/kg/day had a cyst on the left ovary, which is not considered related to treatment due to only one animal being affected in this dose group and with no similar finding in females treated at 5 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Conclusions:

It is concluded that, in the absence of any dose-limiting test item-related effects in this 21-day toxicity study, a dose level of 5 mg/kg/day would be suitable for use as the high dose level in the subsequent OECD 422 screening study. Although the extent of toxicity observed at 5 mg/kg/day suggests that a higher dose could be tolerated, as a dose of 6 mg/kg/day has previously been associated with treatment related deaths and total litter resorptions in a pre-natal development study, indicating a very steep dose response, the use of a high dose level greater than 5 mg/kg/day in an OECD 422 study is not recommended.

Executive summary:

The objective of the study was to make a preliminary assessment of the systemic toxic potential of Dibutyltin Oxide (an industrial chemical) in a 21 day oral gavage study in Sprague Dawley rats, to select a suitable high dose for a subsequent OECD 422 screening study.

Three groups, each comprising three male and three female Sprague Dawley rats received Dibutyltin Oxide at doses of 0.75, 3 and 5 mg/kg/day for 21 days by oral gavage. The original intention of the study was to dose for 14 consecutive days but the treatment period was extended to better investigate toxicity within the study.

During the study, clinical condition, body weight, food consumption, water consumption (by visual assessment), organ weight and macropathology investigations were undertaken.

Results

There were no treatment-related clinical signs and all animals survived to scheduled termination on Day 22.

Overall (Day 1 to 22) body weight gain was 41% lower in males receiving 5 mg/kg/day relative to males given 0.75 mg/kg/day. This was below the historical control data (HCD) range and was associated with a slight reduction of food intake. In addition, body weight gain was low for 1/3 males given 3 mg/kg/day, although, overall mean body weight gain of these males was similar to that observed at 0.75 mg/kg/day. Intergroup differences in female body weight gains did not indicate a clear effect of treatment, however, intermittent body weight losses were seen throughout the treatment period in some animals at all dose levels, but with no associated effects on food consumption.

After 21 days of treatment, absolute and body weight relative thymus weights were reduced in both sexes receiving 3 and 5 mg/kg/day, compared to animals receiving 0.75 mg/kg/day. Mean reductions in thymus weights (% relative to terminal body weight) were 39% and 34% at 3 mg/kg/day and 40% and 46% at 5 mg/kg/day for males and females, respectively, compared to animals receiving 0.75 mg/kg/day.

The macroscopic examination revealed no treatment-related findings.

Conclusion

It is concluded that, in the absence of any dose-limiting test item-related effects in this 21-day toxicity study, a dose level of 5 mg/kg/day would be suitable for use as the high dose level in the subsequent OECD 422 screening study. Although, the extent of toxicity observed at 5 mg/kg/day suggests that a higher dose could be tolerated, as a dose of 6 mg/kg/day has previously been associated with treatment related deaths and total litter resorptions in a pre-natal development study, indicating a very steep dose response, the use of a high dose level greater than 5 mg/kg/day in an OECD 422 study is not recommended.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD) rat

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] rat was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Males: 70 to 76 days old, Females: 84 to 90 days old
- Weight at study initiation: Males: 326 to 395 g, Females: 230 to 294 g
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used throughout the study except during pairing.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.
The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week. A soft white untreated wood block (aspen wood based product) and plastic shelter were provided to each cage throughout the study (except during late gestation and lactation) and replaced when necessary and at the same time as the cages, respectively. Approximately two handfuls of paper shavings were provided to each cage as nesting material from Day 20 after mating and throughout lactation. Shavings were replaced at the same frequency as the bedding

Number of animals per cage:
Pre-pairing: up to four animals of one sex
Pairing: one male and one female
Males after mating: up to four animals
Gestation: one female
Lactation: one female + litter
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet, ad libitum. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum. Bottles were changed at appropriate intervals.
- Acclimation period: Males: Six days before commencement of treatment, Females: 20 days before commencement of treatment.


DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen wood based product.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.

IN-LIFE DATES: From: Animal arrival (Females: 18 August 2021, Males: 01 September 2021) To: F0 necropsy (Males: 11 to 12 October 2021, Females: 26 to 30 October 2021)

Route of administration:

oral: gavage

Details on route of administration:

In line with the OECD TG for the OECD 422, it is recommended that the test chemical be administered orally unless other routes of administration are considered more appropriate. When the oral route is selected, the test chemical is usually administered by gavage.

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared weekly or more frequently: The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste.
Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
A series of formulations at the required concentrations were prepared by dilution of the relevant higher concentration with the vehicle up to 06 October 2021. Formulations at the required concentrations were prepared by individual weighings of the test substance from 07 October 2021.
The method of preparation was changed to weighing DBTO individually for each dose group as it was considered that preparing the formulations by dilution may have been a contributing factor for the out of specification results observed on some occasions, particularity at 0.15 mg/mL.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance has a low water solubility. Therefore, a well-tolerated oil (peanut oil) was selected for preparation of a test item suspension.
- Concentration in vehicle: 0.15, 0.6, 1.0 mg/mL
- Amount of vehicle (if gavage): dose volume 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of formulations during storage were determined as part of another study. In that study the concentration of Dibutyltin oxide in the final solution in the range of 0.1 to 5 mg/mL was quantified by liquid chromatography using tandem mass spectrometric detection (LC-MS/MS) and were determined to be stable for: 24 hours at ambient temperature (15 to 25°C) and 15 days when stored refrigerated (2 to 8°C).
Samples of each formulation prepared for administration in Weeks 1 to 6 and the final week of treatment were analyzed for achieved concentration of the test item. Additional concentration measurements were included in the Study as results obtained were on occasions, particularly at 0.15 mg/mL, out of specified acceptance limits.
In addition, formulations from Week 1 and the final week of treatment were analysed for homogeneity of formulations by taking samples from the top, middle and bottom of formulations from Groups 2 to 4.

Duration of treatment / exposure:

Males: 15 days before pairing up to necropsy after a minimum of four weeks. Females: 15 days before pairing, then throughout pairing and gestation until Day 12 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

0.75 mg/kg bw/day (nominal)

Dose / conc.:

3 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: It was concluded that, in the absence of any dose-limiting test item-related effects in the 21-day toxicity dose-range finding study, a dose level of 5 mg/kg/day would be suitable for use as the high dose level in the subsequent OECD 422 screening study. Although, the extent of toxicity observed at 5 mg/kg/day suggests that a higher dose could be tolerated, as a dose of 6 mg/kg/day has previously been associated with treatment related deaths and total litter resorptions in a pre-natal development study, indicating a very steep dose response and therefore the use of a high dose level greater than 5 mg/kg/day in an OECD 422 study is not recommended. For more details please refer to 'Any other information on material and methods'.
- Rationale for selecting satellite groups: Spare animals were removed from the study room after treatment commenced. Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation
Unexpected death: one male
Irregular estrous cycle: four females
Acyclic estrous cycle: three females
- Dose range finding studies: see cross-reference

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.
Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males: Week 1 – daily; Week 2 to 4 - twice weekly (middle and end of week); Week 5 onwards - once each week
F0 females: Week 1 - daily; Week 2 - twice (middle and end of week); Gestation phase - Days 0, 7, 14 and 20; Lactation phase - Days 1, 6 and 12
Detailed observations were recorded at the following times in relation to dose administration: Pre-dose observation, one to two hours after completion of dosing, and as late as possible in the working day

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males: Weekly during acclimatization. Before dosing on the day that treatment commenced (Week 0) and weekly thereafter. On the day of necropsy.
F0 females: Weekly during acclimatization. Before dosing on the day that treatment commenced (Week 0) and weekly before pairing. Days 0, 7, 14 and 20 after mating and Days 1, 4, 7, 11 and 13 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Weekly, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4. For females after mating food consumption was performed to match the body weight recording: Days 0-7, 7-14, 14-20 after mating.
Days 1-4, 4-7 and 7-13 of lactation.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: The five lowest numbered surviving males per group and the first five surviving lactating females in each group
- Parameters examined were: see any other information on materials and methods incl. tables below

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were at termination
- Animals fasted: No
- How many animals: The five lowest numbered surviving males per group and the first five surviving lactating females in each group
- Parameters examined were: see any other information on materials and methods incl. tables below

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: at termination (all F0 males and females), on day 4 of age (F1 offspring: up to two females per litter), and on days 13 of age (F1 offspring, two males and two females per litter, where possible)
- Animals fasted: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation. During Week 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing)
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: see description under any other information on materials and methods incl. tables

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see 'Any other information on materials and methods incl. tables')

HISTOPATHOLOGY: Yes (see 'Any other information on materials and methods incl. tables')

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Time of Necropsy
F0 males: During Week 5 after at least four weeks of treatment.
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 13 of lactation.
F1 offspring: Day 13 of age.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non significant, are presented on the relevant tables. For some parameters, including estrous cycles before treatment, pre coital interval, mating performance and stage of estrous cycle at termination, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. Data relating to food consumption (except during gestation and lactation) were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
See further details under 'Any other information on materials and methods incl. tables.'

Clinical signs:

no effects observed

Description (incidence and severity):

For animals surviving to scheduled termination (Week 5 for males and Day 13 of lactation for females) there were no adverse treatment-related clinical signs seen.

Following dose administration, two males receiving 5 mg/kg/day were observed with decreased activity, piloerection, partially closed eyelids and hunched posture on Day 12 of treatment. Piloerection was also seen on some occasions in two females receiving 0.75 mg/kg/day and one control female during either gestation or lactation. In addition, instances of salivation or excessive salivation were seen in one male receiving 3 mg/kg/day and in one male at 5 mg/kg/day and in one, two or three females receiving 0.75, 3 or 5 mg/kg/day, respectively, during either gestation or lactation.
These findings were not considered toxicologically relevant (see 'Discussion' under 'Overall remarks')

Mortality:

mortality observed, treatment-related

Description (incidence):

Animal 4F 123 (5 mg/kg/day) was euthanized for welfare reasons on Day 22 of gestation. The signs seen in this animal were underactivity, piloerection, hunched posture and with red discharge from the vagina. Necropsy findings were restricted to the stomach with dark depressions on the glandular mucosa. Stress-related changes were also seen including marked involution/atrophy of the thymus, decreased cellularity of the white pulp of the spleen, increased apoptosis of the mesenteric lymph nodes, diffuse cortical hypertrophy of the adrenal cortex, and a slight, focal erosion of the glandular stomach which correlated with the depressions seen at necropsy. The cause of death of this animal was undetermined, however absence of any clear indication for a dosing trauma suggests relation to the test item. Minimal hyperplasia of the limiting ridge of the stomach was also evident.

Animal 4F 127 (5 mg/kg/day) was dispatched to necropsy for welfare reasons mid parturition on Day 22 of gestation. The signs seen in the female were decreased activity, piloerection, red aqueous discharge from the vaginal area and dull eyes. This female gave birth to six live pups and 1 dead pup. Macroscopic necropsy findings for this animal included thickened meninges, abnormal dark contents in the cecum, colon, ileum and jejunum, dark depressions on the glandular mucosa and red fluid in the vagina. Also, many organs were observed to be pale. Stress-related changes including slight involution/atrophy of the thymus, decreased cellularity of the white pulp of the spleen, were evident histopathologically, and a minimal, focal erosion of the glandular stomach correlating with the depressions seen at necropsy was also seen. No histopathological cause of death was identified, and the uterus, cervix and vagina were not available for histopathological evaluation. The cause of death was attributed to the animal’s poor clinical condition. Slight hyperplasia of the limiting ridge of the stomach was also evident.

Animal 3F 135 (3 mg/kg/day) was dispatched to necropsy due to signs seen prior to dosing on gestation Day 6. Signs seen in this animal were decreased activity, rapid breathing, piloerection, hunched posture and chromodacryorrhea. Macroscopic examination revealed a perforated esophagus and in the thoracic cavity there was abnormal red fluid as well as adhesions affecting multiple organs. Inflammation and adhesions of the thoracic cavity involving multiple organs including the lungs, heart and thymus were seen histopathologically, and other stress-related changes including severe involution/atrophy of the thymus, focal erosion of the glandular stomach, decreased cellularity of the white pulp of the spleen, and diffuse cortical hypertrophy of the adrenal cortex were also evident. The findings are consistent with dosing trauma, and an accidental cause of death was confirmed. Minimal hyperplasia of the limiting ridge of the stomach was also evident.

Animal 3F 147 (3 mg/kg/day) was euthanized in the animal room prior to dosing on gestation Day 6 due to the severity of the signs seen that consisted of abnormally cold to touch, laboured breathing, dull eyes, uncoordinated gait, brown staining around the muzzle and chromodacryorrhea. At necropsy this animal was found with thin clear/oily fluid in the thoracic cavity and adhesions involving multiple organs. Inflammation and adhesions of the thoracic cavity involving multiple organs including the heart and thymus were seen histopathologically, and other stress-related changes including marked involution/atrophy of the thymus, decreased cellularity of the white pulp of the spleen, increased apoptosis of the mesenteric lymph nodes and diffuse cortical hypertrophy of the adrenal cortex were also evident. The findings are consistent with dosing trauma, and an accidental cause of death was confirmed. Slight hyperplasia of the limiting ridge of the stomach was also evident.

Animal 4M 28 (5 mg/kg/day) was euthanized for welfare reasons due to excessive body weight loss on Day 25 of treatment prior to dosing. Prior to dispatch the animal was observed to be thin and ungroomed. Findings at necropsy included abnormal dark contents in the cecum, colon, ileum, jejunum, rectum and stomach, which also had depressions. A small thymus was noted for this animal and the kidneys were pale. Histopathological evaluation revealed moderate necrosis of the liver with associated bile duct hyperplasia and inflammation. Other changes
included degenerative/regenerative changes of the medullary epithelium of the kidneys with associated cortical tubular basophilia, vacuolation and dilatation, and unilateral pelvic dilatation. Stress-related changes including marked involution/atrophy of the thymus, and diffuse cortical hypertrophy of the adrenal cortex were evident histopathologically, and a slight, focal erosion of the glandular stomach was also seen. The kidney and thymus findings correlated with the observations noted at necropsy. Slight hyperplasia of the limiting ridge of the stomach and associated hyperplasia of the glandular and nonglandular regions of the stomach were also seen. The cause of death for this animal was considered to be the hepatic lesions. No other animal showed similar hepatic changes, and no effects on liver organ weights were seen in animals surviving to scheduled sacrifice so this death was not considered to be related to the test item.

Given the lack of test item-related effects on reproductive parameters in animals of the same dose level, and the lack of any organ weight, necropsy or histopathological changes in the female reproductive system of animals surviving to scheduled sacrifice, no clear cause of death could be identified for animals 4F 123 and 4F 127. However, the absence of any clear indication for an accidental dosing trauma and the need for euthanisation at the end of gestation in two animals of the same treatment group (highest dose level) strongly suggests a relationship to the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect of treatment on body weights for males and for females before mating, during gestation or during lactation.

Males receiving 0.75, 3 or 5 mg/kg/day gained more weight than control animals during the final week of treatment that attained statistical significance, but overall (Week 0-5) body weight gains were unaffected. The statistically significant increased body weight gain observed during Weeks 4 to 5 in treated males was due to low control group mean body weight gain during that week and, therefore, the statistical differences in treated males was considered incidental.

Females receiving 3 mg/kg/day gained more weight than control animals during gestation Days 14-20 (25% increase) with overall (gestation Days 0-20) body weight gains being increased by 20%, statistical significance was attained but there was no dose response in both instances. Females receiving 5 mg/kg/day gained less body weight than control immediately after mating (59% of controls; gestation Days 0-7), but statistical significance was not attained, and subsequent body weight gain was similar or slightly superior to control. In addition, early in lactation (Days 1-4) females receiving 5 mg/kg/day gained statistically significant more weight than controls animals, but no effect was apparent for overall weight gain during lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food intake was unaffected by treatment in males and females before mating, in females after mating or during lactation.

During Week 4, food intake for males receiving 5 mg/kg/day was statistically significantly higher than controls (16% increase). This finding was not considered toxicologically relevant (see 'Discussion' under 'Overall remarks')

Food efficiency:

not examined

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):

The hematological examination of peripheral blood performed in males in Week 5, when compared with controls, revealed low erythrocyte cell counts and high reticulocyte counts in males receiving 3 or 5 mg/kg/day, the extent of which exhibited dose-response relationships and attained statistical significance except reticulocyte counts at 3 mg/kg/day. In addition, males receiving 5 mg/kg/day had slightly low statistically significant haemoglobin concentrations. These changes were considered treatment-related.
These findings, in the absence of any supporting histopathological change, were considered non-adverse. For more details please refer to the 'Discussion' under 'Overall remarks'.

Other statistically significant differences, when compared with controls, were low platelet counts in males receiving 0.75 or 3 mg/kg/day, however this was considered not treatment-related as differences from controls were minor, lacked dose-relationship with no similar finding in males at 5 mg/kg/day. This finding was therefore considered incidental and attributed to normal biological variation.
There were no statistically significant differences, when compared with controls, in the hematological examination of peripheral blood performed in females on Day 13 of lactation. All differences from controls were minor and therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma performed in males in Week 5 revealed, when compared with controls, low bilirubin concentration at 5 mg/kg/day which attained statistical significance. In addition, plasma calcium concentrations were statistically significantly higher than controls in males receiving 3 or 5 mg/kg/day, but this lacked a dose-response relationship.
In females receiving 0.75, 3 or 5 mg/kg/day phosphorus concentrations were higher than that of controls, the extent of which was dose related though only the value at 5 mg/kg/day achieved statistical significance.
These clinical pathology changes, in the absence of any supporting histopathological change, were considered non-adverse (please refer to 'Discussion' under 'Overall remarks').

All other differences from controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.

Endocrine findings:

no effects observed

Description (incidence and severity):

No statistically significant differences in serum TSH concentrations were observed in male F0 Adults and F1 Day 13 Offspring administered up to 5 mg/kg/day compared to the control.

Assessment of TSH levels in female rats is still ongoing. Currently available results indicate no clear, or statistically significant, differences in serum TSH concentrations in female F0 Adults and Day 13 Offspring due to the test article. Preliminary data indicate a statistically significant increase in serum TSH concentrations (% change from control mean of 66.4%) in female F1 Day 4 Offspring administered 5 mg/kg/day compared to the control, however individual and mean TSH levels fell within the HCD range, therefore this modest increase was likely due to biological variation and not treatment-related.

No statistically significant differences in serum T4 concentrations were observed in F0 adult males and females, female offspring on Day 4 of age and male offspring on Day 13 of age. Serum T4 concentration from female offspring on Day 13 age from females treated at 5 mg/kg/day were statistically significantly lower than controls.

Currently, the changes in TSH and T4 are considered minor given the high variability in endogenous TSH and T4 concentrations and are thus considered non-adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no effects on sensory reactivity, grip strength or locomotor activity.

Sensory reactivity values for males during Week 5 of treatment and females at Day 7-9 of lactation in all treated groups were generally similar to control animals of the same sex. There were some minor inter-group differences in the grip strengths, but none achieved statistical significance and there was no dose-relationship.

Group mean high (rearing activity) and low (cage floor activity) beam activity scores for males in all treated groups during Week 5 of treatment showed no effects of treatment with scores similar to those in the Control group. Group mean high and low beam activity scores for females at Day 7-9 of lactation showed some inter and intra-group variation and some isolated statistical significances were achieved at the 12-minute interval scores (high beams) for females in all treated groups and at the 24 minute interval scores (low beams) for females receiving 3 or 5 mg/kg/day. However, no statistical significances were attained in the total mean activity scores for either high or low beams and there was no dose relationship. These differences in activity for females were therefore considered to be attributed to natural variation.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and body weight relative mean thymus weights were lower in animals administered 3 or 5 mg/kg/day, achieving statistical significance in males. Liver weights (% relative to body weights) were increased in females receiving 3 (12% increase) or 5 mg/kg/day (16% increase) and % relative kidney weights were increased (13% increase) in females receiving 5 mg/kg/day.
Changes in weights of liver and kidney were not considered adverse (please refer to 'Any other information on results incl. tables' and Overall remarks')

All other differences in organ weight parameters were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Thickening of the limiting ridge of the stomach was seen in males administered 3 or 5 mg/kg/day.

All other macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item related.

For more details please refer to 'Any other information on results incl. tables' and 'Overall remarks'

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A higher incidence and severity of vacuolation of the adrenal cortex was seen in males administered 5 mg/kg/day. This is a common response to the administration of xenobiotics (Gopinath & Mowat, 2014).


Hyperplasia of the limiting ridge of the stomach was seen in both sexes in all treated groups with a dose relationship in incidence and/or severity, and a higher incidence and severity in males compared to females. The changes in the males correlated with necropsy findings of thickening of the limiting ridge in animals administered 3 or 5 mg/kg/day. These findings were accompanied by diffuse hyperplasia of the nonglandular region of the stomach in occasional animals administered 5 mg/kg/day, and in males administered 5 mg/kg/day by diffuse hyperplasia of the glandular region of the stomach. All of these changes are considered relatively common in oral gavage studies where the test item has irritant potential (Nolte et al., 2016).


Higher incidences of thymic involution/atrophy were seen in animals administered 3 or 5 mg/kg/day, correlating with the reduced organ weights reported at necropsy at these doses. This change is a known effect of organotin compounds (Boyer, 1989). In addition, involution/atrophy is commonly seen in rodents in toxicity studies, and in the absence of an indication of an immune effect is usually attributed to stress (Willard-Mack et al., 2019).

All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item related.

The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

For more information please refer to 'Any other information on results incl. tables'

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Critical effects observed:

not specified

Formulation Analysis

The mean achieved concentrations for formulations prepared for Week 1 of dosing (Text Table 6.1) were satisfactory (within – 15%/+10% of nominal) except for Group 2 (0.15 mg/mL). As the result for Group 2 was below nominal, the residual dose from Day 4 of dosing was retained and analysed for the concentration of DBTO, which confirmed the initial result obtained. Therefore, the overall mean achieved concentration for Week 1 was 0.119 mg/mL at 20.7% below nominal. Samples taken from the top, middle and bottom strata of formulations for Week 1 confirmed that formulations were homogenous at all concentrations. 

 

Text Table  6.1: Week 1 Achieved Concentration and Homogeneity for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)						Mean (mg/mL)	RME (%)	CV (%)
		Top 1	Top 2	Middle 1	Middle 2	Bottom 1	Bottom 2
1	0	-	-	ND	ND	-	-	-	-	-
	0.15	0.123	0.116	0.120	0.103	0.118	0.107	0.119	-20.7	6.40
		0.114*	0.129*	0.111*	0.126*	0.122*	0.122*
		-	-	0.121**	0.127**	-	-
	0.6	0.530	0.555	0.548	0.570	0.543	0.510	0.543	-9.5	3.83
	1	1.04	1.07	0.916	0.984	0.827	0.972	0.968	-3.2	9.06

*  Results for analysis of contingency samples

**  Sample taken from dose pot, post dosing on Day 4

 

 

As the residual dose from Day 4 confirmed the low Week 1 achieved concentration from Group 2, an additional residual dose from Group 2 was retained post-dosing from Week 2 (Day 8) of the study and analyzed for achieved concentration (Text Table 6.2). The result obtained was very slightly low at 15.3% below nominal. In addition, samples taken from Group 2 formulations prepared for administration in Week 3 (Text Table 6.3) of the study (analyzed concurrently with the Week 2 residual dose) were low at 30% below nominal. Following this result the remaining doses prepared for Week 3 (Groups 2-4) were discarded

but were administered to the animals on Study Day 15. Instead, doses prepared for administration in Week 4 (Text Table 6.4) were used for dosing on study Days 16 to 19 during Week 3.

 

 

Text Table 6.2: Week 2 Achieved Concentration (Group 2 Dose Pot Analysis) for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)		Mean (mg/mL)	RME (%)	CV (%)
		Analysis 1	Analysis 2
2	0.15	0.125*	0.128*	0.127	-15.3	1.68

*  Sample taken from dose pot, post dosing on Day 8

 

Text Table 6.3: Week 3 Achieved Concentration (Group 2 Formulation Analysis) for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)		Mean (mg/mL)	RME (%)	CV (%)
		Middle 1	Middle 2
3	0.15	0.105	0.105	0.105	-30.0	0.0

Analysed concurrently with Week 2 Group 2 dose pot analysis

 

 

The mean achieved concentrations for formulations prepared on 22 September 2021 for Week 4 of dosing (dosed on Days 16-19, Week 3) were satisfactory except for Group 3 (0.6 mg/mL) at 19.5% below nominal.

 

 

Text Table 6.4: Week 4 Achieved Concentration (Formulations Prepared 22 September 2021) for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)		Mean (mg/mL)	RME (%)	CV (%)
		Middle 1	Middle 2
4	0.15	0.137	0.138	0.138	-9.3	+0.0
	0.6	0.486*	0.461*	0.483	-19.5	4.29
		0.510**	0.475**
	1	0.969	0.930	0.950	-5.0	2.90

* These original results were confirmed by redilution of initial dilution

** Results for analysis of the contingency vials

 

 

As doses intended for Week 4 were used partly in Week 3, Week 4 doses were subsequently re-formulated, sampled and analysed for achieved concentration and used for administration to the animals on study Days 20-28 (Text Table 6.5). The mean achieved concentrations for formulations prepared on 23 September 2021 for Week 4 of dosing were satisfactory except for Group 2 (0.15 mg/mL) at 18.0% above nominal.

 

Text Table 6.5: Week 4 Achieved Concentration (Formulations Prepared 23 September 2021) for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)		Mean (mg/mL)	RME (%)	CV (%)
		Middle 1	Middle 2
4	0.15	0.164	0.189	0.177	+18.0	10.0
	0.6	0.710*	0.640*	0.652	+8.6	5.97
		0.633**	0.626**
	1	1.07	1.03	1.05	+5.0	2.69

* These original results were confirmed by redilution of initial dilution

** Results for analysis of the contingency vials

 

 

The mean achieved concentrations for formulations prepared for Weeks 5 and 6 of dosing were satisfactory (Text Tables 6.6 and 6.7).

 

Text Table 6.6: Week 5 Achieved Concentration for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)		Mean (mg/mL)	RME (%)	CV (%)
		Middle 1	Middle 2
5	0.15	0.141	0.149	0.145	-3.3	3.90
	0.6	0.508	0.553	0.531	-11.5	6.00
	1	1.02	0.968	0.994	-0.6	3.70

 

Text Table 6.7: Week 6 Achieved Concentration for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)		Mean (mg/mL)	RME (%)	CV (%)
		Middle 1	Middle 2
6	0.15	0.129	0.130	0.130	-13.3	0.55
	0.6	0.526	0.513	0.520	-13.3	1.77
	1	0.935	0.937	0.936	-6.4	0.15

Analysed concurrent with Week 5 samples and procedural recoveries

 

The mean achieved concentrations for formulations prepared for Week 7 of dosing were satisfactory for Group 3 (0.6 mg/mL) and were low for Group 2 (0.15 mg/mL) and slightly low for Group 4 (1 mg/mL) at 24.7% and 15.4% below nominal, respectively. Samples taken from the top, middle and bottom strata of formulations for Week 7 confirmed that formulations were homogenous at all concentrations.

 

Text Table 6.8: Week 7 Achieved Concentration and Homogeneity for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)						Mean (mg/mL)	RME (%)	CV (%)
		Top 1	Top 2	Middle 1	Middle 2	Bottom 1	Bottom 2
7	0	-	-	ND	ND	-	-	-	-	-
	0.15	0.125	0.110	0.117	0.108	0.106	0.114	0.113	-24.7	6.16
	0.6	0.563	0.550	0.561	0.578	0.542	0.562	0.559	-6.8	2.20
	1	0.904	0.875	0.758	0.882	0.782	0.876	0.846	-15.4	7.14

 

 

The mean recovery results obtained on each analytical occasion during the study were within ±10% of nominal (except for mean recovery for Week 1 at 110.4%) showing the continued accuracy of the method.

Formulations were initially prepared by dilution from higher concentrations and up to Day 19 of the study the quantitative dilution was performed using measuring cylinder. Measuring cylinders were used due to the total volumes (~400-500 mL) of formulations required for dosing each group for one week and the volumes required to quantitatively dilute from higher to lower concentrations. During the pre-study chemistry study (Study No. 8456479), that assessed stability and homogeneity of formulations, dilutions were performed using a syringe as the volumes required were lower than that required for administration to the animals for one week. Therefore, formulations administered to the animals from Study Day 20 to 35 were prepared by dilutions using a syringe while formulations were continuously stirred magnetically. It was assumed that diluting by syringe, while stirring, would mitigate the potential for DBTO suspensions becoming non-homogenous during the dilution transfer procedure. However, the dilution process change did not completely resolve concentrations being outside the applied acceptance limits as Group 2 formulations in Week 4 (Text Table 6.5) were at 18% above nominal. However, Week 5 formulations were within the acceptance limit at all concentrations, even though the preparation and sampling procedures were the same as used for preparing Week 4 doses. As the method of dilution did not completely resolve the achieved concentrations, a decision was made to change the formulation procedure by individually weighing DBTO for all dose groups. The change in the formulation method was employed for doses prepared for administration in Week 6 (Text Table 6.7) and all results obtained were satisfactory. However, for Week 7 the mean concentrations for Groups 2 and 4 were below the applied acceptance limit at 24.7% and 15.4% below the nominal, respectively. The results obtained for Group 4 were only slightly below, by 0.4%, the acceptance limit. Samples taken from the top, middle and bottom strata of formulations for Week 7 confirmed that formulations were homogenous at all concentrations.

All dose formulation preparation data were scrutinised, and no discrepancy could be identified; the correct data checks were carried out, materials and equipment inspections and procedures/methods were employed, and the correct test item weights, the correct volumes for dilution and vehicle (final) volumes were used. In addition, DBTO was not detected in the control formulations, demonstrating that there had been no inadvertent cross-contamination during the formulation procedures. Based on the evidence it was considered that the achieved concentrations, particularly for Group 2 (0.15 mg/mL), were on five out eight occasions either below or above the intended concentration, however, averaging the means from all analytical occasions yields an average formulation concentration of 0.13 mg/mL (12% below nominal). As this overall mean was only marginally lower than the intended concentration and that formulations were shown to be homogenous it was considered that animals at 0.75 mg/kg/day received the correct dose. Averaging the mean concentrations from all analytical occasions from Group 3 (0.6 mg/mL) and Group 4 (1 mg/mL) yields mean formulated concentrations of 0.55 mg/mL (9% below nominal) and 0.96 mg/mL (4% below nominal), respectively. For Groups 3 and 4, the analysed concentrations were below the acceptance criteria on only one out of six analytical occasions. Therefore, based on the evidence it was considered that the animals at 3 and 5 mg/kg/day received the correct dose. It is therefore considered not to affect the validity or integrity of the study, as the No Observed Adverse Effect Levels for toxicity established within this study were above the Group 2 dose level, where the measured dose concentrations were below nominal. The cause of the variability in nominal concentrations for DBTO formulations could not be identified but is likely, particularly for formulations at 0.75 mg/mL, due to the low concentration and small amount of DBTO required to formulate this concentration.

 

 

Organ Weights

Sex	Dibutyltin Oxide
	Males				Females
Dose Level (mg/kg/day)	0	0.75	3	5	0	0.75	3	5
Thymus
Absolute Weight (g)	0.337	0.317	0.200	0.200	0.148	0.137	0.121	0.125
Absolute Weight (% of control)	-	94	59**	59**	-	93	82	84
Body Weight Ratio (%)	0.0755	0.0675	0.0424	0.0427	0.0431	0.0389	0.0341	0.0378
Body Weight Ratio (% of control)	-	89	56**	57**	-	90	79	88
Liver
Absolute Weight (g)	15.966	15.532	16.558	17.386	14.136	15.563	16.469	15.971
Absolute Weight (% of control)	-	97	104	109	-	110	117	113
Body Weight Ratio (%)	3.52	3.32	3.51	3.71	4.13	4.41	4.61	4.81
Body Weight Ratio (% of control)	-	94	100	105	-	107	112**	116**
Kidney
Absolute Weight (g)	3.184	2.810	3.022	3.145	2.173	2.198	2.354	2.364
Absolute Weight (% of control)	-	88	95	99	-	101	108	109
Body Weight Ratio (%)	0.704	0.602	0.640	0.671	0.634	0.625	0.659	0.715
Body Weight Ratio (% of control)	-	86	91	95	-	99	104	113*

** = Statistically significant difference (absolute or relative) compared with respective control mean value

Liver weights were increased slightly in females receiving 3 or 5 mg/kg/day but there were no alterations in any of the plasma biomarkers of liver damage or microscopic changes, indicating that this difference in weight was not adverse.

Kidney weights were slightly increased in females at 5 mg/kg/day but there were no microscopic changes detected. Plasma biochemical findings in females (high phosphorus concentrations at all dose levels) and increased electrolyte (calcium) in males at 3 or 5 mg/kg/day are possibly related but in isolation, and in the absence of any microscopic correlate, these changes are not adverse.

 

Macropathology

Incidence of Test item-Related Macroscopic Findings – F0 Animals Killed at Scheduled Termination

Sex	Dibutyltin Oxide
	Males				Females
Dose Level (mg/kg/day)	0	0.75	3	5	0	0.75	3	5
Stomach
Number Examined	10	10	10	9	10	10	8	7
Thickened	0	0	2	5	0	0	0	0

 

Histopathology

Incidence and Severity of Test Item-Related Microscopic Findings – F0 Animals Killed at Scheduled Termination

Sex	Dibutyltin Oxide
	Males				Females
Dose Level (mg/kg/day)	0	0.75	3	5	0	0.75	3	5
Adrenals
Number Examined	5	5	5	5	5	0	0	5
Vacuolation, Cortical
Minimal	1	2	2	1	0	-	-	0
Slight	0	0	0	2	0	-	-	0
Moderate	0	0	0	2	0	-	-	0
Total	1	2	2	5	0	-	-	0
Stomach
Number Examined	5	5	5	6	5	5	5	5
Hyperplasia, Epithelial, Limiting Ridge
Minimal	0	2	3	1	0	1	3	4
Slight	0	0	1	5	0	0	0	0
Total	0	2	4	6	0	1	3	4
Hyperplasia, Epithelial, Nonglandular Region
Minimal	0	0	0	2	0	0	0	1
Hyperplasia, Epithelial, Glandular Region
Minimal	0	0	0	3	0	0	0	0
Slight	0	0	0	1	0	0	0	0
Total	0	0	0	4	0	0	0	0
Thymus
Number Examined	5	5	4	5	5	5	5	4
Involution/Atrophy
Minimal	0	0	0	3	0	0	3	0
Slight	0	0	2	1	0	0	1	0
Moderate	0	0	0	0	0	0	0	4
Total	0	0	2	4	0	0	4	4

 

Conclusions:

Based on the results obtained in this combined repeated dose toxicity and reproductive/ developmental toxicity screening study, it was concluded that the no observed adverse effect level (NOAEL) for systemic toxicity was 3 mg/kg/day due to the two deaths of females during parturition, even though these deaths were of uncertain relationship to treatment. However, the absence of any clear indication for an accidental dosing trauma and the need for euthanisation at the end of gestation in two animals of the same treatment group (highest dose level) strongly suggests a relationship to the test item. There was no evidence of endocrine adversity in any of the endocrine parameters evaluated.

Executive summary:

This combined repeated dose toxicity and reproductive/ developmental toxicity screening study was conducted according to OECD Test Guideline 422 (adopted 29 July 2016) and GLP. The purpose of this study was to assess the general systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of Dibutyltin Oxide (an industrial chemical) by oral gavage administration for at least four weeks. Here, only results of the F0 animals are reported (excluding parameters related to reproduction toxicity and developmental toxicity of the F1 offspring).

Three groups of ten male and ten female rats received Dibutyltin Oxide at doses of 0.75, 3 or 5 mg/kg/day by oral gavage administration at a dose volume of 5 mL/kg/day. Males were treated daily for 15 days before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for 15 days before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, peanut oil (arachis oil), over the same treatment periods and at the same volume dose as treated groups. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight as well as macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

 

Results of F0 animals
Oral administration of DBTO at doses of up to 5 mg/kg/day did in general not cause severe systemic toxicity. Although no deaths clearly related to treatment were observed, two females were killed at 5 mg/kg/day around the time of parturition and an association with treatment is considered likely, although a specific cause of death could not be identified in the context of this study. There were no treatment-related clinical signs or post-dosing observations, no effects on sensory reactivity, grip strength or locomotor activity and no adverse effects on body weight or food intake. In addition, there were no findings at macroscopic examination of the F0 females considered to be treatment-related. The evaluation of the results of the thyroid hormone analysis is not yet completed; preliminary results provide the following indications: No statistically significant differences in serum TSH and T4 concentrations were observed in male and female animals administered up to 5 mg/kg/day compared to the control.

There were no hematological changes detected in treated females. Changes in the biochemical composition of the plasma in males were low bilirubin concentrations at 5 mg/kg/day and high calcium concentrations at 3 or 5 mg/kg/day. Biochemical changes in females were restricted to high phosphorus concentrations at all dose levels. These clinical pathology changes, in the absence of any supporting histopathological change, were considered non-adverse.

Analysis of organ weights at scheduled termination revealed, when compared with controls, low absolute and body weight relative thymus weights in both sexes receiving 3 or 5 mg/kg/day, high liver weights in females receiving 3 or 5 mg/kg/day and high kidney weights in females at 5 mg/kg/day. Treatment-related macroscopic findings at scheduled termination were restricted to thickening of the limiting ridge of the stomach in males receiving 3 or 5 mg/kg/day. DBTO-related microscopic findings were observed in the adrenal cortex (vacuolation) of males at 5 mg/kg/day, stomach (hyperplasia of the limiting ridge) in both sexes at all dose levels, but with a higher incidence/severity in males, which was accompanied by diffuse hyperplasia of the nonglandular region of the stomach in some animals receiving 5 mg/kg/day, and in males administered 5 mg/kg/day by diffuse hyperplasia of the glandular region of the stomach. In addition, thymic involution/atrophy was seen in both sexes given 3 or 5 mg/kg/day. The findings in the thymus, stomach and adrenals were considered non-adverse. Although the findings in the stomach are considered non-adverse, a higher dose level than 5 mg/kg/day would have likely resulted in more animals requiring euthanisation for animal welfare reasons.

The measured concentration of formulations used to dose animals at 0.75 mg/kg/day were consistently lower than nominal concentrations throughout much of the study, indicating that animals may have received a lower dose level than intended. As the No Observed Adverse Effect Levels for toxicity established within this study were above the 0.75 mg/kg/day dose level, this occurrence had no impact on the integrity of the study or on the overall conclusions made.

Conclusion
Based on the results obtained in this combined repeated dose toxicity and reproductive/ developmental toxicity screening study, it was concluded that the no observed adverse effect level (NOAEL) for systemic toxicity was 3 mg/kg/day due to the two deaths of females during parturition, even though these deaths were of uncertain relationship to treatment. However, the absence of any clear indication for an accidental dosing trauma and the need for euthanisation at the end of gestation in two animals of the same treatment group (highest dose level) strongly suggests a relationship to the test item. There was no evidence of endocrine adversity in any of the endocrine parameters evaluated.

Data source

Reference

Reference Type:

other: draft study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyltin oxide

EC Number:

212-449-1

EC Name:

Dibutyltin oxide

Cas Number:

818-08-6

Molecular formula:

C8H18OSn

IUPAC Name:

dibutylstannanone

Test material form:

solid: particulate/powder

Details on test material:

Batch number: 2004RDX103
Molecular weight: 248.94
Appearance: White powder
Storage conditions: Ambient temperature (15 to 25°C), in the dark
Purity: 97.91%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD) rat

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] rat was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: (P) Males: 70 to 76 days old, Females: 84 to 90 days old
- Weight at study initiation: (P) Males: 326 to 395 g, Females: 230 to 294 g
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used throughout the study except during pairing.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.
The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week. A soft white untreated wood block (aspen wood based product) and plastic shelter were provided to each cage throughout the study (except during late gestation and lactation) and replaced when necessary and at the same time as the cages, respectively. Approximately two handfuls of paper shavings were provided to each cage as nesting material from Day 20 after mating and throughout lactation. Shavings were replaced at the same frequency as the bedding

Number of animals per cage:
Pre-pairing: up to four animals of one sex
Pairing: one male and one female
Males after mating: up to four animals
Gestation: one female
Lactation: one female + litter
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet, ad libitum
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum. Bottles were changed at appropriate intervals.
- Acclimation period: Males: Six days before commencement of treatment, Females: 20 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.
IN-LIFE DATES: From: Animal arrival (Females: 18 August 2021, Males: 01 September 2021) To: F0 necropsy (Males: 11 to 12 October 2021, Females: 26 to 30 October 2021)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared weekly or more frequently: The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste.
Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
A series of formulations at the required concentrations were prepared by dilution of the relevant higher concentration with the vehicle up to 06 October 2021. Formulations at the required concentrations were prepared by individual weighings of the test substance from 07 October 2021.
The method of preparation was changed to weighing DBTO individually for each dose group as it was considered that preparing the formulations by dilution may have been a contributing factor for the out of specification results observed on some occasions, particularity at 0.15 mg/mL.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance has a low water solubility. Therefore, a well-tolerated oil (peanut oil) was selected for preparation of a test item suspension.
- Concentration in vehicle: 0.15, 0.6, 1.0 mg/mL
- Amount of vehicle (if gavage): dose volume 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1 from within the same treatment groups.
- Length of cohabitation: Up to two weeks
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): separately, on the day when mating evidence was detected

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of formulations during storage were determined as part of another study. In that study the concentration of Dibutyltin oxide in the final solution in the range of 0.1 to 5 mg/mL was quantified by liquid chromatography using tandem mass spectrometric detection (LC-MS/MS) and were determined to be stable for: 24 hours at ambient temperature (15 to 25°C) and 15 days when stored refrigerated (2 to 8°C).
Samples of each formulation prepared for administration in Weeks 1 to 6 and the final week of treatment were analyzed for achieved concentration of the test item. Additional concentration measurements were included in the Study as results obtained were on occasions, particularly at 0.15 mg/mL, out of specified acceptance limits.
In addition, formulations from Week 1 and the final week of treatment were analysed for homogeneity of formulations by taking samples from the top, middle and bottom of formulations from Groups 2 to 4.

Duration of treatment / exposure:

Males: 15 days before pairing up to necropsy after a minimum of four weeks. Females: 15 days before pairing, then throughout pairing and gestation until Day 12 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

0.75 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

3 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

5 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: It was concluded that, in the absence of any dose-limiting test item-related effects in the 21-day toxicity dose-range finding study, a dose level of 5 mg/kg/day would be suitable for use as the high dose level in the subsequent OECD 422 screening study. Although, the extent of toxicity observed at 5 mg/kg/day suggests that a higher dose could be tolerated, as a dose of 6 mg/kg/day has previously been associated with treatment related deaths and total litter resorptions in a pre-natal development study, indicating a very steep dose response and therefore the use of a high dose level greater than 5 mg/kg/day in an OECD 422 study is not recommended. For more details please refer to 'Any other information on materials and methods'

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.
Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males: Week 1 – daily; Week 2 to 4 - twice weekly (middle and end of week); Week 5 onwards - once each week
F0 females: Week 1 - daily; Week 2 - twice (middle and end of week); Gestation phase - Days 0, 7, 14 and 20; Lactation phase - Days 1, 6 and 12
Detailed observations were recorded at the following times in relation to dose administration: Pre-dose observation, one to two hours after completion of dosing, and as late as possible in the working day

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males: Weekly during acclimatization. Before dosing on the day that treatment commenced (Week 0) and weekly thereafter. On the day of necropsy.
F0 females: Weekly during acclimatization. Before dosing on the day that treatment commenced (Week 0) and weekly before pairing. Days 0, 7, 14 and 20 after mating and Days 1, 4, 7, 11 and 13 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Weekly, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4. For females after mating food consumption was performed to match the body weight recording: Days 0-7, 7-14, 14-20 after mating.
Days 1-4, 4-7 and 7-13 of lactation.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit at least one typical 4-5 day cycles were not allocated to the study.
Dry and wet smears were taken as follows:
Dry smears: For 15 days before pairing using cotton swabs
Wet smears: Using pipette lavage during the following phases:
- For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
- After pairing until mating (for a maximum of 14 days).
- For four days before scheduled termination (nominally Days 10 to 13 of lactation).

Sperm parameters (parental animals):

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
- Clinical observations (Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate)
- Litter size (Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age)
- Sex ratio of each litter (Recorded on Days 1, 4, 7 and 13 of age)
- Individual offspring body weights (Days 1, 4, 7, 11 and 13 of age)
- Ano-genital distance (Day 1 - all F1 offspring)
- Nipple/areolae count (Day 13 of age - male offspring)
- F1 offspring on Day 4 of age:
Blood sampling
Externally normal offspring discarded without examination.
Externally abnormal offspring identified on dispatch to necropsy; examined externally, and retained pending possible future examination.

GROSS EXAMINATION OF DEAD PUPS: YES
- Premature deaths: Where possible, a fresh external macroscopic examination with an assessment of stomach for milk content was performed.

Postmortem examinations (parental animals):

SACRIFICE
F0 males: During Week 5 after at least four weeks of treatment.
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 13 of lactation.

GROSS NECROPSY
- A full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in corresponding Table under 'Any other information on methods' were prepared for microscopic examination and weighed, respectively.

OTHER
Females:
- Number of implantation sites
- Pre-Coital Interval
- Gestation length

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY / HISTOPATHOLOGY / ORGAN WEIGTHS
- Blood sampling (for Thyroid Hormone Analysis)
- All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormalities were retained in appropriate fixative.
- Thyroid glands were preserved from one male and one female in each litter.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non significant, are presented on the relevant tables. For some parameters, including estrous cycles before treatment, pre coital interval, mating performance and stage of estrous cycle at termination, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. Data relating to food consumption (except during gestation and lactation) were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
See further details under 'Any other information on materials and methods incl. tables.'

Reproductive indices:

Mating Performance and Fertility
Percentage mating (%) = Number of animals mating/Animals paired x 100
Conception rate (%) = Number of animals achieving pregnancy/Animals mated x 100
Fertility index (%) = Number of animals achieving pregnancy/Animals paired x 100

Gestation Length and Index
Gestation index (%) = Number of live litters born/Number pregnant x 100

Offspring viability indices:

Post-implantation survival index (%) = Total number of offspring born/Total number of uterine implantation sites x 100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = Number of live offspring on Day 1 after littering/Total number of offspring born x 100
Viability index (%) = Number of live offspring on Day 4 (before blood sampling)/Number live offspring on Day 1 after littering x 100
Lactation index (%) = Number of live offspring on Day 13 after littering /Number of live offspring on Day 4 (after blood sampling) x 100

Group mean values were calculated from individual litter values

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

For animals surviving to scheduled termination (Week 5 for males and Day 13 of lactation for females) there were no adverse treatment-related clinical signs seen.

Following dose administration, two males receiving 5 mg/kg/day were observed with decreased activity, piloerection, partially closed eyelids and hunched posture on Day 12 of treatment. Piloerection was also seen on some occasions in two females receiving 0.75 mg/kg/day and one control female during either gestation or lactation. In addition, instances of salivation or excessive salivation were seen in one male receiving 3 mg/kg/day and in one male at 5 mg/kg/day and in one, two or three females receiving 0.75, 3 or 5 mg/kg/day, respectively, during either gestation or lactation.
These findings were not considered toxicologically relevant (see 'Discussion' under 'Overall remarks')

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Animal 4F 123 (5 mg/kg/day) was euthanized for welfare reasons on Day 22 of gestation. The signs seen in this animal were underactivity, piloerection, hunched posture and with red discharge from the vagina. Necropsy findings were restricted to the stomach with dark depressions on the glandular mucosa. Stress-related changes were also seen including marked involution/atrophy of the thymus, decreased cellularity of the white pulp of the spleen, increased apoptosis of the mesenteric lymph nodes, diffuse cortical hypertrophy of the adrenal cortex, and a slight, focal erosion of the glandular stomach which correlated with the depressions seen at necropsy. The cause of death of this animal was undetermined, however absence of any clear indication for a dosing trauma suggests relation to the test item. Minimal hyperplasia of the limiting ridge of the stomach was also evident.



Animal 4F 127 (5 mg/kg/day) was dispatched to necropsy for welfare reasons mid parturition on Day 22 of gestation. The signs seen in the female were decreased activity, piloerection, red aqueous discharge from the vaginal area and dull eyes. This female gave birth to six live pups and 1 dead pup. Macroscopic necropsy findings for this animal included thickened meninges, abnormal dark contents in the cecum, colon, ileum and jejunum, dark depressions on the glandular mucosa and red fluid in the vagina. Also, many organs were observed to be pale. Stress-related changes including slight involution/atrophy of the thymus, decreased cellularity of the white pulp of the spleen, were evident histopathologically, and a minimal, focal erosion of the glandular stomach correlating with the depressions seen at necropsy was also seen. No histopathological cause of death was identified, and the uterus, cervix and vagina were not available for histopathological evaluation. The cause of death was attributed to the animal’s poor clinical condition. Slight hyperplasia of the limiting ridge of the stomach was also evident.



Animal 3F 135 was dispatched to necropsy due to signs seen prior to dosing on gestation Day 6. Signs seen in this animal were decreased activity, rapid breathing, piloerection, hunched posture and chromodacryorrhea. Macroscopic examination revealed a perforated esophagus and in the thoracic cavity there was abnormal red fluid as well as adhesions affecting multiple organs. Inflammation and adhesions of the thoracic cavity involving multiple organs including the lungs, heart and thymus were seen histopathologically, and other stress-related changes including severe involution/atrophy of the thymus, focal erosion of the glandular stomach, decreased cellularity of the white pulp of the spleen, and diffuse cortical hypertrophy of the adrenal cortex were also evident. The findings are consistent with dosing trauma, and an accidental cause of death was confirmed. Minimal hyperplasia of the limiting ridge of the stomach was also evident.


Animal 3F 147 was euthanized in the animal room prior to dosing on gestation Day 6 due to the severity of the signs seen that consisted of abnormally cold to touch, laboured breathing, dull eyes, uncoordinated gait, brown staining around the muzzle and chromodacryorrhea. At necropsy this animal was found with thin clear/oily fluid in the thoracic cavity and adhesions involving multiple organs. Inflammation and adhesions of the thoracic cavity involving multiple organs including the heart and thymus were seen histopathologically, and other stress-related changes including marked involution/atrophy of the thymus, decreased cellularity of the white pulp of the spleen, increased apoptosis of the mesenteric lymph nodes and diffuse cortical hypertrophy of the adrenal cortex were also evident. The findings are consistent with dosing trauma, and an accidental cause of death was confirmed. Slight hyperplasia of the limiting ridge of the stomach was also evident.



Animal 4M 28 was euthanized for welfare reasons due to excessive body weight loss on Day 25 of treatment prior to dosing. Prior to dispatch the animal was observed to be thin and ungroomed. Findings at necropsy included abnormal dark contents in the cecum, colon, ileum, jejunum, rectum and stomach, which also had depressions. A small thymus was noted for this animal and the kidneys were pale. Histopathological evaluation revealed moderate necrosis of the liver with associated bile duct hyperplasia and inflammation. Other changes
included degenerative/regenerative changes of the medullary epithelium of the kidneys with associated cortical tubular basophilia, vacuolation and dilatation, and unilateral pelvic dilatation. Stress-related changes including marked involution/atrophy of the thymus, and diffuse cortical hypertrophy of the adrenal cortex were evident histopathologically, and a slight, focal erosion of the glandular stomach was also seen. The kidney and thymus findings correlated with the observations noted at necropsy. Slight hyperplasia of the limiting ridge of the stomach and associated hyperplasia of the glandular and nonglandular regions of the stomach were also seen. The cause of death for this animal was considered to be the hepatic lesions. No other animal showed similar hepatic changes, and no effects on liver organ weights were seen in animals surviving to scheduled sacrifice so this death was not considered to be related to the test item.



Given the lack of test item-related effects on reproductive parameters in animals of the same dose level, and the lack of any organ weight, necropsy or histopathological changes in the female reproductive system of animals surviving to scheduled sacrifice, no clear cause of death could be identified for animals 4F 123 and 4F 127. However, the absence of any clear indication for an accidental dosing trauma and the need for euthanisation at the end of gestation in two animals of the same treatment group (highest dose level) strongly suggests a relationship to the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect of treatment on body weights for males and for females before mating, during gestation or during lactation.

Males receiving 0.75, 3 or 5 mg/kg/day gained more weight than control animals during the final week of treatment that attained statistical significance, but overall (Week 0-5) body weight gains were unaffected. The statistically significant increased body weight gain observed during Weeks 4 to 5 in treated males was due to low control group mean body weight gain during that week and, therefore, the statistical differences in treated males was considered incidental.

Females receiving 3 mg/kg/day gained more weight than control animals during gestation Days 14-20 (25% increase) with overall (gestation Days 0-20) body weight gains being increased by 20%, statistical significance was attained but there was no dose response in both instances. Females receiving 5 mg/kg/day gained less body weight than control immediately after mating (59% of controls; gestation Days 0-7), but statistical significance was not attained, and subsequent body weight gain was similar or slightly superior to control. In addition, early in lactation (Days 1-4) females receiving 5 mg/kg/day gained statistically significant more weight than controls animals, but no effect was apparent for overall weight gain during lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food intake was unaffected by treatment in males and females before mating, in females after mating or during lactation.

During Week 4, food intake for males receiving 5 mg/kg/day was statistically significantly higher than controls (16% increase). This finding was not considered toxicologically relevant (see 'Discussion' under 'Overall remarks')

Food efficiency:

not examined

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):

The hematological examination of peripheral blood performed in males in Week 5, when compared with controls, revealed low erythrocyte cell counts and high reticulocyte counts in males receiving 3 or 5 mg/kg/day, the extent of which exhibited dose-response relationships and attained statistical significance except reticulocyte counts at 3 mg/kg/day. In addition, males receiving 5 mg/kg/day had slightly low statistically significant haemoglobin concentrations. These changes were considered treatment-related.
These findings, in the absence of any supporting histopathological change, were considered non-adverse. For more details please refer to the 'Discussion' under 'Overall remarks'.

Other statistically significant differences, when compared with controls, were low platelet counts in males receiving 0.75 or 3 mg/kg/day, however this was considered not treatment related as differences from controls were minor, lacked dose-relationship with no similar finding in males at 5 mg/kg/day. This finding was therefore considered incidental and attributed to normal biological variation.

There were no statistically significant differences, when compared with controls, in the hematological examination of peripheral blood performed in females on Day 13 of lactation. All differences from controls were minor and therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma performed in males in Week 5 revealed, when compared with controls, low bilirubin concentration at 5 mg/kg/day which attained statistical significance. In addition, plasma calcium concentrations were statistically significantly higher than controls in males receiving 3 or 5 mg/kg/day, but this lacked a dose-response relationship.
In females receiving 0.75, 3 or 5 mg/kg/day phosphorus concentrations were higher than that of controls, the extent of which was dose related though only the value at 5 mg/kg/day achieved statistical significance.
These clinical pathology changes, in the absence of any supporting histopathological change, were considered non-adverse (please refer to 'Discussion' under 'Overall remarks').

All other differences from controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant differences in serum TSH concentrations were observed in male F0 Adults and F1 Day 13 Offspring administered up to 5 mg/kg/day compared to the control.

Assessment of TSH levels in female rats is still ongoing. Currently available results indicate no clear, or statistically significant, differences in serum TSH concentrations in female F0 Adults and Day 13 Offspring due to the test article. Preliminary data indicate a statistically significant increase in serum TSH concentrations (% change from control mean of 66.4%) in female F1 Day 4 Offspring administered 5 mg/kg/day compared to the control, however individual and mean TSH levels fell within the HCD range, therefore this modest increase was likely due to biological variation and not treatment-related.

No statistically significant differences in serum T4 concentrations were observed in F0 adult males and females, female offspring on Day 4 of age and male offspring on Day 13 of age. Serum T4 concentration from female offspring on Day 13 age from females treated at 5 mg/kg/day were statistically significantly lower than controls.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no effects on sensory reactivity, grip strength or locomotor activity.

Sensory reactivity values for males during Week 5 of treatment and females at Day 7-9 of lactation in all treated groups were generally similar to control animals of the same sex. There were some minor inter-group differences in the grip strengths, but none achieved statistical significance and there was no dose-relationship.

Group mean high (rearing activity) and low (cage floor activity) beam activity scores for males in all treated groups during Week 5 of treatment showed no effects of treatment with scores similar to those in the Control group. Group mean high and low beam activity scores for females at Day 7-9 of lactation showed some inter and intra-group variation and some isolated statistical significances were achieved at the 12-minute interval scores (high beams) for females in all treated groups and at the 24 minute interval scores (low beams) for females receiving 3 or 5 mg/kg/day. However, no statistical significances were attained in the total mean activity scores for either high or low beams and there was no dose relationship. These differences in activity for females were therefore considered to be attributed to natural variation.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A higher incidence and severity of vacuolation of the adrenal cortex was seen in males administered 5 mg/kg/day. This is a common response to the administration of xenobiotics (Gopinath & Mowat, 2014).


Hyperplasia of the limiting ridge of the stomach was seen in both sexes in all treated groups with a dose relationship in incidence and/or severity, and a higher incidence and severity in males compared to females. The changes in the males correlated with necropsy findings of thickening of the limiting ridge in animals administered 3 or 5 mg/kg/day. These findings were accompanied by diffuse hyperplasia of the nonglandular region of the stomach in occasional animals administered 5 mg/kg/day, and in males administered 5 mg/kg/day by diffuse hyperplasia of the glandular region of the stomach. All of these changes are considered relatively common in oral gavage studies where the test item has irritant potential (Nolte et al., 2016).


Higher incidences of thymic involution/atrophy were seen in animals administered 3 or 5 mg/kg/day, correlating with the reduced organ weights reported at necropsy at these doses. This change is a known effect of organotin compounds (Boyer, 1989). In addition, involution/atrophy is commonly seen in rodents in toxicity studies, and in the absence of an indication of an immune effect is usually attributed to stress (Willard-Mack et al., 2019).

All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item related.

The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

For more information please refer to 'Any other information on results incl. tables'

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of treatment on estrous cycles. Before treatment commenced, all females allocated to the study showed normal four- or five day estrous cycles. During the two-week pre-mating period, there were several females in each DBTO treatment group that showed either an irregular cycle or that were acyclic, but the same was also seen in the Controls. Three control females were acyclic, three females receiving 0.75 mg/kg/day were acyclic, one female was irregular and one female acyclic at 3 mg/kg/day, and two females were irregular and one female was acyclic at 5 mg/kg/day. The number of females showing a regular 4- or 5-day cycle in all treated groups was similar to Controls. All females surviving to scheduled termination on Day 13 of lactation were in di-estrus; therefore, as expected, estrus cycles ceased during lactation.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no effect of treatment on mating performance, conception rate or fertility index. There was one female receiving 5 mg/kg/day that did not achieve pregnancy resulting in a slightly low conception rate and fertility index of Group 4 compared with Controls, however this difference was marginal.

There was no effect of treatment on pre-coital interval and gestation length. Only one female receiving 0.75 mg/kg/day and one female receiving 3 mg/kg/day did not mate in the first four days of pairing. The distribution of gestation lengths in all DBTO-treated groups was similar to Controls. The gestation index for females receiving 5 mg/kg/day was slightly low (0.78 fold of control mean), compared with controls, which achieved statistical significance. The low gestation index reflects the two females that were killed around the time of parturition and not any effect on litter resorptions or females failing to litter. Regardless, the gestation index for females receiving 5 mg/kg/day was within the historical control data (HCD) range and therefore considered non-adverse.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

3 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects on reproduction parameter observed.

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

3 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs seen in F1 offspring related to maternal treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The number of implantations, post-implantation survival index, post-natal litter size, the sex ratio (% males) of offspring, live birth index, viability index (Day 4) and lactation index (Day 13) were considered to be unaffected by maternal treatment. The mean numbers of implantations for females from DBTO-treated groups were slightly higher than the control, although no statistical significance or dose relationship was apparent. The higher number of implantations for treated females resulted in mean litter sizes for treated groups, being higher than control from birth to termination on Day 13. These differences in mean litter size were considered incidental and unrelated to maternal treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Both male and female offspring body weights from maternal females treated at 5 mg/kg/day were statistically significantly lower (10 and 9% reduction for male and female offspring, respectively) than offspring from control females on Day 1 of age. Whilst this may have reflected the slightly larger litter size apparent at 5 mg/kg/day, compared to control, mean offspring weights at 5 mg/kg/day were also lower than mean offspring weights observed for litters from the lower dose levels, where litter size was similar. It is therefore considered that this indicates an underlying effect of maternal treatment at this dose level. Subsequent offspring body weight gains for both sexes at this dose were slightly lower than that of the control throughout, although only differences for male body weight gain between Days 7 to 11 of age attained statistical significance. This resulted in overall body weight gain being reduced by 13-14% for male and female offspring and lower mean absolute body weights attaining statistical significance, when compared with control, for male offspring at 5 mg/kg/day on Days 11 and 13 of age and female offspring on Days 4 and 11 of age.

Male and female offspring body weights from maternal females treated at 0.75 or 3 mg/kg/day were also slightly lower than offspring from control females on Day 1 of age, but differences did not attain statistical significance and probably reflected the slightly larger litter size apparent at these dose levels, compared to control. Subsequent offspring body weight gains for both sexes were generally slightly lower than that of the control throughout, although only differences for male body weight gain at 3 mg/kg/day between Days 7 to 11 of age attained statistical significance. This resulted in overall body weight gain being reduced by 8 or 5% for male and female offspring respectively at 0.75 mg/kg/day and 11 or 9% for male and female offspring respectively at 3 mg/kg/day. However, the differences from control observed for absolute bodyweights at these dose levels failed to attain statistical significance for either sex to termination at Day 13 of age. Effects on offspring body weight at 0.75 or 3 mg/kg/day were therefore considered not adverse.

For more details please refer to 'Discussion' under 'Overall remarks'

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

no effects observed

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

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Offspring ano-genital distances were unaffected by parental treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were observed.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic findings observed in F1 offspring killed or dying before scheduled termination were restricted to the absence of milk in the stomach and/or autolyzed abdominal contents in controls and at 0.75, 3 or 5 mg/kg/day. These findings are typically observed in animals of this age and the intergroup incidences did not indicate any effect of treatment.

Macroscopic findings observed in F1 offspring killed at scheduled termination were hair loss (patchy) in the skin and subcutis of offspring from litters No. 137 at 3 mg/kg/day and No. 124 at 5 mg/kg/day.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant differences in serum TSH concentrations were observed in male F0 Adults and F1 Day 13 Offspring administered up to 5 mg/kg/day compared to the control.

Assessment of TSH levels in female rats is still ongoing. Currently available results indicate no clear, or statistically significant, differences in serum TSH concentrations in female F0 Adults and Day 13 Offspring due to the test article. Preliminary data indicate a statistically significant increase in serum TSH concentrations (% change from control mean of 66.4%) in female F1 Day 4 Offspring administered 5 mg/kg/day compared to the control, however individual and mean TSH levels fell within the HCD range, therefore this modest increase was likely due to biological variation and not treatment-related.

No statistically significant differences in serum T4 concentrations were observed in F0 adult males and females, female offspring on Day 4 of age and male offspring on Day 13 of age. Serum T4 concentration from female offspring on Day 13 age from females treated at 5 mg/kg/day were statistically significantly lower than controls.

Currently, the changes in TSH and T4 are considered minor given the high variability in endogenous TSH and T4 concentrations and are thus considered non-adverse.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Two females receiving 3 mg/kg/day were euthanized for welfare reasons on Day 6 of gestation due to design traumas, two females receiving 5 mg/kg/day were euthanized for welfare reasons on Day 22 of gestation and one female receiving 5 mg/kg/day did not achieve pregnancy. Therefore, the F1 responses were assessed using ten control litters and ten, eight or seven litters at 0.75, 3 or 5 mg/kg/day, respectively.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

3 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Due to the smaller size at birth and reduced growth observed in offspring from maternal females treated at 5 mg/kg/day

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Formulation Analysis

The mean achieved concentrations for formulations prepared for Week 1 of dosing (Text Table 6.1) were satisfactory (within – 15%/+10% of nominal) except for Group 2 (0.15 mg/mL). As the result for Group 2 was below nominal, the residual dose from Day 4 of dosing was retained and analysed for the concentration of DBTO, which confirmed the initial result obtained. Therefore, the overall mean achieved concentration for Week 1 was 0.119 mg/mL at 20.7% below nominal. Samples taken from the top, middle and bottom strata of formulations for Week 1 confirmed that formulations were homogenous at all concentrations. 

 

Text Table  6.1: Week 1 Achieved Concentration and Homogeneity for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)						Mean (mg/mL)	RME (%)	CV (%)
		Top 1	Top 2	Middle 1	Middle 2	Bottom 1	Bottom 2
1	0	-	-	ND	ND	-	-	-	-	-
	0.15	0.123	0.116	0.120	0.103	0.118	0.107	0.119	-20.7	6.40
		0.114*	0.129*	0.111*	0.126*	0.122*	0.122*
		-	-	0.121**	0.127**	-	-
	0.6	0.530	0.555	0.548	0.570	0.543	0.510	0.543	-9.5	3.83
	1	1.04	1.07	0.916	0.984	0.827	0.972	0.968	-3.2	9.06

*  Results for analysis of contingency samples

**  Sample taken from dose pot, post dosing on Day 4

 

 

As the residual dose from Day 4 confirmed the low Week 1 achieved concentration from Group 2, an additional residual dose from Group 2 was retained post-dosing from Week 2 (Day 8) of the study and analyzed for achieved concentration (Text Table 6.2). The result obtained was very slightly low at 15.3% below nominal. In addition, samples taken from Group 2 formulations prepared for administration in Week 3 (Text Table 6.3) of the study (analyzed concurrently with the Week 2 residual dose) were low at 30% below nominal. Following this result the remaining doses prepared for Week 3 (Groups 2-4) were discarded

but were administered to the animals on Study Day 15. Instead, doses prepared for administration in Week 4 (Text Table 6.4) were used for dosing on study Days 16 to 19 during Week 3.

 

 

Text Table 6.2: Week 2 Achieved Concentration (Group 2 Dose Pot Analysis) for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)		Mean (mg/mL)	RME (%)	CV (%)
		Analysis 1	Analysis 2
2	0.15	0.125*	0.128*	0.127	-15.3	1.68

*  Sample taken from dose pot, post dosing on Day 8

 

Text Table 6.3: Week 3 Achieved Concentration (Group 2 Formulation Analysis) for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)		Mean (mg/mL)	RME (%)	CV (%)
		Middle 1	Middle 2
3	0.15	0.105	0.105	0.105	-30.0	0.0

Analysed concurrently with Week 2 Group 2 dose pot analysis

 

 

The mean achieved concentrations for formulations prepared on 22 September 2021 for Week 4 of dosing (dosed on Days 16-19, Week 3) were satisfactory except for Group 3 (0.6 mg/mL) at 19.5% below nominal.

 

 

Text Table 6.4: Week 4 Achieved Concentration (Formulations Prepared 22 September 2021) for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)		Mean (mg/mL)	RME (%)	CV (%)
		Middle 1	Middle 2
4	0.15	0.137	0.138	0.138	-9.3	+0.0
	0.6	0.486*	0.461*	0.483	-19.5	4.29
		0.510**	0.475**
	1	0.969	0.930	0.950	-5.0	2.90

* These original results were confirmed by redilution of initial dilution

** Results for analysis of the contingency vials

 

 

As doses intended for Week 4 were used partly in Week 3, Week 4 doses were subsequently re-formulated, sampled and analysed for achieved concentration and used for administration to the animals on study Days 20-28 (Text Table 6.5). The mean achieved concentrations for formulations prepared on 23 September 2021 for Week 4 of dosing were satisfactory except for Group 2 (0.15 mg/mL) at 18.0% above nominal.

 

Text Table 6.5: Week 4 Achieved Concentration (Formulations Prepared 23 September 2021) for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)		Mean (mg/mL)	RME (%)	CV (%)
		Middle 1	Middle 2
4	0.15	0.164	0.189	0.177	+18.0	10.0
	0.6	0.710*	0.640*	0.652	+8.6	5.97
		0.633**	0.626**
	1	1.07	1.03	1.05	+5.0	2.69

* These original results were confirmed by redilution of initial dilution

** Results for analysis of the contingency vials

 

 

The mean achieved concentrations for formulations prepared for Weeks 5 and 6 of dosing were satisfactory (Text Tables 6.6 and 6.7).

 

Text Table 6.6: Week 5 Achieved Concentration for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)		Mean (mg/mL)	RME (%)	CV (%)
		Middle 1	Middle 2
5	0.15	0.141	0.149	0.145	-3.3	3.90
	0.6	0.508	0.553	0.531	-11.5	6.00
	1	1.02	0.968	0.994	-0.6	3.70

 

Text Table 6.7: Week 6 Achieved Concentration for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)		Mean (mg/mL)	RME (%)	CV (%)
		Middle 1	Middle 2
6	0.15	0.129	0.130	0.130	-13.3	0.55
	0.6	0.526	0.513	0.520	-13.3	1.77
	1	0.935	0.937	0.936	-6.4	0.15

Analysed concurrent with Week 5 samples and procedural recoveries

 

The mean achieved concentrations for formulations prepared for Week 7 of dosing were satisfactory for Group 3 (0.6 mg/mL) and were low for Group 2 (0.15 mg/mL) and slightly low for Group 4 (1 mg/mL) at 24.7% and 15.4% below nominal, respectively. Samples taken from the top, middle and bottom strata of formulations for Week 7 confirmed that formulations were homogenous at all concentrations.

 

Text Table 6.8: Week 7 Achieved Concentration and Homogeneity for Dibutyltin oxide in Peanut oil

Week	Nominal inclusion (mg/mL)	Analysed concentration (mg/mL)						Mean (mg/mL)	RME (%)	CV (%)
		Top 1	Top 2	Middle 1	Middle 2	Bottom 1	Bottom 2
7	0	-	-	ND	ND	-	-	-	-	-
	0.15	0.125	0.110	0.117	0.108	0.106	0.114	0.113	-24.7	6.16
	0.6	0.563	0.550	0.561	0.578	0.542	0.562	0.559	-6.8	2.20
	1	0.904	0.875	0.758	0.882	0.782	0.876	0.846	-15.4	7.14

 

 

The mean recovery results obtained on each analytical occasion during the study were within ±10% of nominal (except for mean recovery for Week 1 at 110.4%) showing the continued accuracy of the method.

Formulations were initially prepared by dilution from higher concentrations and up to Day 19 of the study the quantitative dilution was performed using measuring cylinder. Measuring cylinders were used due to the total volumes (~400-500 mL) of formulations required for dosing each group for one week and the volumes required to quantitatively dilute from higher to lower concentrations. During the pre-study chemistry study (Study No. 8456479), that assessed stability and homogeneity of formulations, dilutions were performed using a syringe as the volumes required were lower than that required for administration to the animals for one week. Therefore, formulations administered to the animals from Study Day 20 to 35 were prepared by dilutions using a syringe while formulations were continuously stirred magnetically. It was assumed that diluting by syringe, while stirring, would mitigate the potential for DBTO suspensions becoming non-homogenous during the dilution transfer procedure. However, the dilution process change did not completely resolve concentrations being outside the applied acceptance limits as Group 2 formulations in Week 4 (Text Table 6.5) were at 18% above nominal. However, Week 5 formulations were within the acceptance limit at all concentrations, even though the preparation and sampling procedures were the same as used for preparing Week 4 doses. As the method of dilution did not completely resolve the achieved concentrations, a decision was made to change the formulation procedure by individually weighing DBTO for all dose groups. The change in the formulation method was employed for doses prepared for administration in Week 6 (Text Table 6.7) and all results obtained were satisfactory. However, for Week 7 the mean concentrations for Groups 2 and 4 were below the applied acceptance limit at 24.7% and 15.4% below the nominal, respectively. The results obtained for Group 4 were only slightly below, by 0.4%, the acceptance limit. Samples taken from the top, middle and bottom strata of formulations for Week 7 confirmed that formulations were homogenous at all concentrations.

All dose formulation preparation data were scrutinised, and no discrepancy could be identified; the correct data checks were carried out, materials and equipment inspections and procedures/methods were employed, and the correct test item weights, the correct volumes for dilution and vehicle (final) volumes were used. In addition, DBTO was not detected in the control formulations, demonstrating that there had been no inadvertent cross-contamination during the formulation procedures. Based on the evidence it was considered that the achieved concentrations, particularly for Group 2 (0.15 mg/mL), were on five out eight occasions either below or above the intended concentration, however, averaging the means from all analytical occasions yields an average formulation concentration of 0.13 mg/mL (12% below nominal). As this overall mean was only marginally lower than the intended concentration and that formulations were shown to be homogenous it was considered that animals at 0.75 mg/kg/day received the correct dose. Averaging the mean concentrations from all analytical occasions from Group 3 (0.6 mg/mL) and Group 4 (1 mg/mL) yields mean formulated concentrations of 0.55 mg/mL (9% below nominal) and 0.96 mg/mL (4% below nominal), respectively. For Groups 3 and 4, the analysed concentrations were below the acceptance criteria on only one out six analytical occasions. Therefore, based on the evidence it was considered that the animals at 3 and 5 mg/kg/day received the correct dose. It is therefore considered not to affect the validity or integrity of the study, as the No Observed Adverse Effect Levels for toxicity established within this study were above the Group 2 dose level, where the measured dose concentrations were below nominal. The cause of the variability in nominal concentrations for DBTO formulations could not be identified but is likely, particularly for formulations at 0.75 mg/mL, due to the low concentration and small amount of DBTO required to formulate this concentration.

 

 

Organ Weights

Sex	Dibutyltin Oxide
	Males				Females
Dose Level (mg/kg/day)	0	0.75	3	5	0	0.75	3	5
Thymus
Absolute Weight (g)	0.337	0.317	0.200	0.200	0.148	0.137	0.121	0.125
Absolute Weight (% of control)	-	94	59**	59**	-	93	82	84
Body Weight Ratio (%)	0.0755	0.0675	0.0424	0.0427	0.0431	0.0389	0.0341	0.0378
Body Weight Ratio (% of control)	-	89	56**	57**	-	90	79	88
Liver
Absolute Weight (g)	15.966	15.532	16.558	17.386	14.136	15.563	16.469	15.971
Absolute Weight (% of control)	-	97	104	109	-	110	117	113
Body Weight Ratio (%)	3.52	3.32	3.51	3.71	4.13	4.41	4.61	4.81
Body Weight Ratio (% of control)	-	94	100	105	-	107	112**	116**
Kidney
Absolute Weight (g)	3.184	2.810	3.022	3.145	2.173	2.198	2.354	2.364
Absolute Weight (% of control)	-	88	95	99	-	101	108	109
Body Weight Ratio (%)	0.704	0.602	0.640	0.671	0.634	0.625	0.659	0.715
Body Weight Ratio (% of control)	-	86	91	95	-	99	104	113*

** = Statistically significant difference (absolute or relative) compared with respective control mean value

Liver weights were increased slightly in females receiving 3 or 5 mg/kg/day but there were no alterations in any of the plasma biomarkers of liver damage or microscopic changes, indicating that this difference in weight was not adverse.

Kidney weights were slightly increased in females at 5 mg/kg/day but there were no microscopic changes detected. Plasma biochemical findings in females (high phosphorus concentrations at all dose levels) and increased electrolyte (calcium) in males at 3 or 5 mg/kg/day are possibly related but in isolation, and in the absence of any microscopic correlate, these changes are not adverse.

 

Macropathology

Incidence of Test item-Related Macroscopic Findings – F0 Animals Killed at Scheduled Termination

Sex	Dibutyltin Oxide
	Males				Females
Dose Level (mg/kg/day)	0	0.75	3	5	0	0.75	3	5
Stomach
Number Examined	10	10	10	9	10	10	8	7
Thickened	0	0	2	5	0	0	0	0

 

Histopathology

Incidence and Severity of Test Item-Related Microscopic Findings – F0 Animals Killed at Scheduled Termination

Sex	Dibutyltin Oxide
	Males				Females
Dose Level (mg/kg/day)	0	0.75	3	5	0	0.75	3	5
Adrenals
Number Examined	5	5	5	5	5	0	0	5
Vacuolation, Cortical
Minimal	1	2	2	1	0	-	-	0
Slight	0	0	0	2	0	-	-	0
Moderate	0	0	0	2	0	-	-	0
Total	1	2	2	5	0	-	-	0
Stomach
Number Examined	5	5	5	6	5	5	5	5
Hyperplasia, Epithelial, Limiting Ridge
Minimal	0	2	3	1	0	1	3	4
Slight	0	0	1	5	0	0	0	0
Total	0	2	4	6	0	1	3	4
Hyperplasia, Epithelial, Nonglandular Region
Minimal	0	0	0	2	0	0	0	1
Hyperplasia, Epithelial, Glandular Region
Minimal	0	0	0	3	0	0	0	0
Slight	0	0	0	1	0	0	0	0
Total	0	0	0	4	0	0	0	0
Thymus
Number Examined	5	5	4	5	5	5	5	4
Involution/Atrophy
Minimal	0	0	0	3	0	0	3	0
Slight	0	0	2	1	0	0	1	0
Moderate	0	0	0	0	0	0	0	4
Total	0	0	2	4	0	0	4	4

 

Applicant's summary and conclusion

Conclusions:

Based on the results obtained in this combined repeated dose toxicity and reproductive/ developmental toxicity screening study, it was concluded that the no observed adverse effect level (NOAEL) for systemic toxicity and reproductive performance was 3 mg/kg/day due to the two deaths of females during parturition, even though these deaths were of uncertain relationship to treatment. However, the absence of any clear indication for an accidental dosing trauma and the need for euthanisation at the end of gestation in two animals of the same treatment group (highest dose level) strongly suggests a relationship to the test item. The NOAEL for developmental toxicity was concluded to be 3 mg/kg/day, due to the smaller size at birth and reduced growth observed in offspring from maternal females treated at 5 mg/kg/day. There was no evidence of endocrine adversity in any of the endocrine parameters evaluated.

Executive summary:

This combined repeated dose toxicity and reproductive/ developmental toxicity screening study was conducted according to OECD Test Guideline 422 (adopted 29 July 2016) and GLP. The purpose of this study was to assess the general systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of Dibutyltin Oxide (an industrial chemical) by oral gavage administration for at least four weeks.

Three groups of ten male and ten female rats received Dibutyltin Oxide at doses of 0.75, 3 or 5 mg/kg/day by oral gavage administration at a dose volume of 5 mL/kg/day. Males were treated daily for 15 days before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for 15 days before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, peanut oil (arachis oil), over the same treatment periods and at the same volume dose as treated groups. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Results
Oral administration of DBTO at doses of up to 5 mg/kg/day did in general not cause severe systemic toxicity nor any adverse effect on reproduction. Although no deaths clearly related to treatment were observed, two females were killed at 5 mg/kg/day around the time of parturition and an association with treatment is considered likely, although a specific cause of death could not be identified in the context of this study. There were no treatment-related clinical signs or post-dosing observations, no effects on sensory reactivity, grip strength or locomotor activity and no adverse effects on body weight or food intake. In addition, there were no findings at macroscopic examination of the F0 females or their offspring considered to be treatment-related and offspring survival or offspring ano-genital distances were unaffected by maternal treatment. The evaluation of the results of the thyroid hormone analysis is not yet completed; preliminary results provide the following indications:

No statistically significant differences in serum TSH concentrations were observed in male animals analysed (F0 Adults and F1 Day 13 Offspring) administered up to 5 mg/kg/day compared to the control. No treatment-related, statistically significant differences in serum TSH concentrations were observed in female F0 Adults and Day 13 Offspring. A statistically significant increase in serum TSH concentrations (1.66-fold increased mean compared to control mean) was observed in female F1 Day 4 Offspring administered 5 mg/kg/day compared to the control, however individual and mean TSH levels fell within the HCD range, therefore this modest increase was likely due to biological variation and not treatment-related.

No statistically significant differences in serum T4 concentrations were observed in F0 adult males and females, female offspring on Day 4 of age and male offspring on Day 13 of age. Serum T4 concentration from female offspring on Day 13 age from females treated at 5 mg/kg/day were statistically significantly lower than controls.

 

Estrous cycles, pre-coital interval, gestation length, mating performance, conception rate, or fertility index were unaffected by treatment. The gestation index for females receiving 5 mg/kg/day was slightly low (0.78-fold of control mean), compared with controls, which achieved statistical significance. The low gestation index reflects the two females that were killed around the time of parturition and not any effect on litter resorptions or females failing to litter. Regardless, the gestation index for females receiving 5 mg/kg/day was within the historical control data (HCD) range and therefore considered non-adverse. Hematological investigations for the males revealed low erythrocyte and high reticulocyte cell counts at 3 or 5 mg/kg/day and low haemoglobin concentrations at 5 mg/kg/day. There were no hematological changes detected in treated females. Changes in the biochemical composition of the plasma in males were low bilirubin concentrations at 5 mg/kg/day and high calcium concentrations at 3 or 5 mg/kg/day. Biochemical changes in females were restricted to high phosphorus concentrations at all dose levels. These clinical pathology changes, in the absence of any supporting histopathological change, were considered non-adverse.

Analysis of organ weights at scheduled termination revealed, when compared with controls, low absolute and body weight relative thymus weights in both sexes receiving 3 or 5 mg/kg/day, high liver weights in females receiving 3 or 5 mg/kg/day and high kidney weights in females at 5 mg/kg/day. Treatment-related macroscopic findings at scheduled termination were restricted to thickening of the limiting ridge of the stomach in males receiving 3 or 5 mg/kg/day. DBTO-related microscopic findings were observed in the adrenal cortex (vacuolation) of males at 5 mg/kg/day, stomach (hyperplasia of the limiting ridge) in both sexes at all dose levels, but with a higher incidence/severity in males, which was accompanied by diffuse hyperplasia of the nonglandular region of the stomach in some animals receiving 5 mg/kg/day, and in males administered 5 mg/kg/day by diffuse hyperplasia of the glandular region of the stomach. In addition, thymic involution/atrophy was seen in both sexes given 3 or 5 mg/kg/day. The findings in the thymus, stomach and adrenals were considered non-adverse. Although the findings in the stomach are considered non-adverse, a higher dose level than 5 mg/kg/day would have likely resulted in more animals requiring euthanisation for animal welfare reasons.

For the F1 responses, the number of implantations, post-implantation loss, post-natal litter size, the sex ratio of offspring, live birth index, viability index on Day 4 of age, lactation index on Day 13 of age and the clinical condition of offspring were unaffected by maternal treatment. Offspring from maternal females at 5 mg/kg/day were smaller on the day of partum with subsequent offspring body weight gains for both sexes being lower than that of control offspring throughout to Day 13 of age. Overall body weight gains (Days 1 to 13 of age) were reduced by 13-14% for male and female offspring, when compared with controls. The effects on offspring body weight performance at 5 mg/kg/day was considered adverse. Male and female offspring body weights from maternal females treated at 0.75 or 3 mg/kg/day were also slightly lower than offspring from control females on Day 1 of age, probably reflecting the slightly larger litter size at these dose levels. Subsequent offspring body weight gains for both sexes were generally slightly lower than that of the control throughout, although only differences for male body weight gain at 3 mg/kg/day between Days 7 to 11 of age attained statistical significance. Overall, the magnitude of these differences at 0.75 or 3 mg/kg/day were considered to be insufficient for this to be regarded as adverse.
Ano-genital distances of offspring were unaffected by parental treatment and no nipples were detected in male offspring on Day 13 of age. There were no macroscopic findings in the offspring killed or died before scheduled termination or at scheduled termination that were considered to be maternally treatment-related.
The measured concentration of formulations used to dose animals at 0.75 mg/kg/day were consistently lower than nominal concentrations throughout much of the study, indicating that animals may have received a lower dose level than intended. As the No Observed Adverse Effect Levels for toxicity established within this study were above the 0.75 mg/kg/day dose level, this occurrence had no impact on the integrity of the study or on the overall conclusions made.

Conclusion
Based on the results obtained in this combined repeated dose toxicity and reproductive/ developmental toxicity screening study, it was concluded that the no observed adverse effect level (NOAEL) for systemic toxicity and reproductive performance was 3 mg/kg/day due to the two deaths of females during parturition, even though these deaths were of uncertain relationship to treatment. However, the absence of any clear indication for an accidental dosing trauma and the need for euthanisation at the end of gestation in two animals of the same treatment group (highest dose level) strongly suggests a relationship to the test item. The NOAEL for developmental toxicity was concluded to be 3 mg/kg/day, due to the smaller size at birth and reduced growth observed in offspring from maternal females treated at 5 mg/kg/day. There was no evidence of endocrine adversity in any of the endocrine parameters evaluated.