Dibutyltin oxide

Endpoint summary

Description of key information

The following key study on the registered substance DBTO has been submitted to address repeated-dose toxicity:

Morley, 2023: OECD TG 422, rat, 0.75, 3 or 5 mg DBTO/kg/day by oral gavage, NOAEL for systemic toxicity =3 mg/kg/day. Two females receiving 5 mg/kg/day were in bad health condition and thus euthanized for welfare reasons.

 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

3 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available study is of good quality, it was conducted according to OECD TG and in accordance with GLP

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is a repeated-dose toxicity study available for DBTO: 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 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 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.

 

Considerations on target organ toxicity

Based on studies with DBTO, it is unclear whether the substance can affect the immune system, particularly the thymus, after repeated exposure.

In a pre-natal developmental toxicity study with DBTO according to OECD 414 (Schroeder, 2017), lower maternal thymus weights were observed at all test material-treatment levels (0.75, 3.0, and 6.0 mg/kg/day) and an increased incidence of small thymus was observed macroscopically in the 6.0 mg/kg/day animals. The authors stated that that the toxicological significance of test material-related effects on thymus weights (decrease) in the absence of histopathological examination is unclear.

In a combined repeated dose toxicity and reproductive/ developmental toxicity screening study according to OECD 422 (Morley, 2022) male and female rats were dosed with 0.75, 3 or 5 mg DBTO/kg/day. It was reported that absolute and body weight relative mean thymus weights were lower in animals administered 3 or 5 mg/kg/day, achieving statistical significance in males. There was a higher incidence of thymic involution and atrophy seen in both sexes receiving 3 or 5 mg/kg/day, which correlated with the reduced organ weights recorded at necropsy. The authors discussed the findings in the thymus as non-adverse.

Justification for classification or non-classification

The test substance is classified as STOT Rep. Exp. 1 according to Regulation (EC) No. 1272/2008 with hazard statement: H372 (Causes damage to the immune system through prolonged or repeated exposure) based on studies on DBTO that have reported findings on the thymus. Although the toxicological relevance of these findings could not be further assessed in the context of these studies, DBTO is classified as STOT Rep. Exp. 1 H372 as a precautionary measure.