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REACH

Dibutyltin oxide

EC number: 212-449-1 | CAS number: 818-08-6

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

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Administrative data

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Effects on fertility

Description of key information

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

Morley, 2023: OECD TG 422, rat, 0.75, 3 or 5 mg DBTO/kg/day by oral gavage, NOAEL for systemic toxicity and reproductive performance =3 mg/kg/day

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same 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:

GLP compliance:

yes

Limit test:

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)

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.

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 materials and methods'

Positive control:

none

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

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: 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.

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.

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

Critical effects observed:

not specified

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):

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

Dose descriptor:

NOAEL

Remarks:

development

Generation:

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

Critical effects observed:

not specified

Reproductive 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 (%)
Week	Nominal inclusion (mg/mL)	Top 1	Top 2	Middle 1	Middle 2	Bottom 1	Bottom 2	Mean (mg/mL)	RME (%)	CV (%)
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

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

0.15

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 (%)
Week	Nominal inclusion (mg/mL)	Middle 1	Middle 2	Mean (mg/mL)	RME (%)	CV (%)
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 (%)
Week	Nominal inclusion (mg/mL)	Middle 1	Middle 2	Mean (mg/mL)	RME (%)	CV (%)
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 (%)
Week	Nominal inclusion (mg/mL)	Top 1	Top 2	Middle 1	Middle 2	Bottom 1	Bottom 2	Mean (mg/mL)	RME (%)	CV (%)
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
Sex	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
Sex	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
Sex	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 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.

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 3 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: The data are of good quality; the available study is according to an OECD Test Guideline and GLP-complaint.

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

Effects on developmental toxicity

Description of key information

The following GLP compliant studies conducted according to recent OECD TG with the registered substance have been submitted to address the developmental toxicity/teratogenicity endpoint:

Key study: Schroeder (2017): OECD TG 414, GLP, rat, 0.75, 3.0, and 6.0 mg DBTO/kg/day, NOAEL for maternal and developmental toxicity = 3.0 mg/kg/day. At dose levels of 0.75, 3.0, and 6.0 mg/kg/day, the test material was not teratogenic in the rat.

Supporting study: Morley, 2023: OECD TG 422, rat, 0.75, 3 or 5 mg DBTO/kg/day by oral gavage, NOAEL for developmental toxicity =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

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 January 2017 to 31 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

January 2001

Deviations:

yes

Remarks:

The dose interval was extended allowing exposure through the pre-implantation period by dosing from gestation day 0 through to 19. To evaluate the known target organ toxicity, the thymus gland was collected and weighed for each dam.

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Hsd:Sprague Dawley® SD®

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 9 to 9.5 weeks of age at time of receipt.
- Weight at study initiation: 158 to 215 g, at randomisation.
- Housing: The animals were individually housed in solid bottom cages with nonaromatic bedding.
- Diet: Ad libitum
- Water: Ad libitum tap water was available via an automatic watering system.

ENVIRONMENTAL CONDITIONS
- Temperature: 68 to 79 °F
- Humidity (%): 30 to 70 %
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was provided for approximately 12 hours per day.

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations of the test material were prepared by mixing the appropriate amount of vehicle with the appropriate amount of test material at nominal concentrations of 0.15, 0.6, and 1.2 mg/mL. Formulations were prepared as needed and were stored at room temperature.
The control group received the vehicle in the same manner as the treated groups. The vehicle and test material formulations were continually stirred prior to and throughout dose administration. Individual doses were based on the most recent body weights.

VEHICLE
- Amount of vehicle: Dose volume of 5 mL/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing formulations prepared for the study were evaluated for homogeneity and concentration. Appropriate samples were collected using a positive displacement pipette, while stirring, and placed into amber glass scintillation vials. Upon receipt of the authorisation to finalise the main study report, the retained backup samples were discarded.
Dosing formulation samples were collected as follows:
Concentration/homogeneity analysis: 0.5 mL from 0.15 and 1.2 mg/mL dose concentrations were sampled at weekly intervals. Six samples were collected from each of the top, middle and bottom, with two analysed and four back up samples.
The averaged result from homogeneity analysis served as concentration verification.
Concentration analysis: 0.5 mL from 0, 0.15, 0.6 and 1.2 mg/mL dose concentrations were sampled at weekly and 3-week intervals. Six samples were collected from the middle stratum, with two analysed and four back up samples.
The samples, including backup samples, were stored at ambient temperature pending analyses or final disposition.
Stability analysis of the dosing formulations was not conducted because the stability of the dosing formulations at the concentrations used on study has been established for 13 days at ambient temperature.

All analytical work was conducted using an analytical method developed and validated under MPI Research. Dose formulations were verified to be the concentrations stated within the acceptable range of variability, for homogeneity and concentration analytical results.
Reference Standard: Test material (Correction Factor: 1.028 for purity)
LC-MS Conditions: Liquid chromatography system equipped with a Thermo BETASIL C18 column, 50 x 2.1 mm, with a gradient flow of 88:12 water:acetic acid with 0.05 % trimethylamine (TEA) (mobile phase A), and 88:12 isopropyl alcohol (IPA):acetic acid with 0.05 % TEA (mobile phase B) at a flow rate of 0.6 mL/minute. The analyte was detected using an LC-MS/MS system operated in the positive ionisation mode at a mass to charge (m/z) ratio of 293 for the precursor ion and 251 for the product ion.
Analysis Description: Prior to analysis, samples were diluted with 88:12 IPA:acetic acid (diluent) to within the range of the calibration curve. Vehicle samples were diluted with diluent using a dilution factor of 10. An aliquot of each sample was injected into the LC-MS/MS system for analysis.
Regression Type: Linear, unweighted
Study Sample Storage Conditions: Ambient, protected from light.
Storage Stability: All samples were analysed within the established storage stability.

Run Acceptance Criteria
System Suitability Test Standards: Injection repeatability (peak area and retention time) ≤10% RSD (relative standard deviation)
Calibration Standards:
1. Accuracy within ±10 % of the nominal concentration
2. Coefficient of determination (R^2) ≥0.99
Performance Check Standards (Same preparation as the System Suitability Standard):
1. Periodically injected so that no more than 10 samples are bracketed by performance check standards. Samples are considered valid if bracketed by passing performance check standard injections.
2. Accuracy within ±10 % of the nominal concentration
Blank Injections: ≤20 % of the limit of quantitation (LOQ)

Assessments
- Homogeneity:
1. Average concentration within ±15 % of the nominal concentration
2. Precision ≤15 % RSD
- Concentration:
1. Average concentration within ±15 % of the nominal concentration
2. Precision ≤15 % RSD
3. Vehicle (control) samples < effective limit of quantitation (ELOQ)

Results:
Analytical run 1: System sustainability test (SST) injection 1: Retention time (tR): 1.816 min, peak area 7596
Analytical run 1: SST injection 2: tR: 1.823 min, peak area 7537
Analytical run 1: SST injection 3: tR: 1.816 min, peak area 7456
Mean for analytical run 1: tR: 1.816 min, peak area 7530
% Relative standard deviation (RSD): tR 0.218 min, peak area 0.933

Analytical run 2: SST injection 1: tR: 1.806 min, peak area 11684
Analytical run 2: SST injection 2: tR: 1.812 min, peak area 11658
Analytical run 2: SST injection 3: tR: 1.806 min, peak area 11601
Mean for analytical run 2: tR: 1.808 min, peak area 11648
% RSD: tR 0.219 min, peak area 0.367

Analytical run 1: Performance check 1: 9.987354 μg/mL nominal concentration, 9.850389 μg/mL calculated concentration; 98.6 % recovery
Analytical run 1: Performance check 2: 9.987354 μg/mL nominal concentration, 9.646217 μg/mL calculated concentration; 96.6 % recovery
Analytical run 1: Performance check 3: 9.987354 μg/mL nominal concentration, 9.878215 μg/mL calculated concentration; 98.9 % recovery

Analytical run 3: Performance check 1: 9.994358 μg/mL nominal concentration, 9.866436 μg/mL calculated concentration; 98.7 % recovery
Analytical run 3: Performance check 2: 9.994358 μg/mL nominal concentration, 10.063415 μg/mL calculated concentration; 100.7 % recovery

Conclusion:
A total of 32 samples were analysed (24 reported results) for the test material in dosing formulations using a validated method. The analytical data demonstrated acceptable performance of the method for all reported results.
Deviation: Analytical Method Deviation: In analytical run 2, the performance check 2 failed to meet acceptance criterion for recovery (±10 %), therefore the results of the run were not reportable as samples were not bracketed by passing performance check injections. The backup samples were analysed in run 3 (on a different instrument) and the run met all acceptance criteria.

Details on mating procedure:

- Impregnation procedure: Purchased timed pregnant.

Duration of treatment / exposure:

GD 0 to 19

Frequency of treatment:

Once daily at approximately the same time each day (± 2 h from the GD 0 dose).

Duration of test:

GD 0 to 19

Dose / conc.:

0.75 mg/kg bw/day

Dose / conc.:

3 mg/kg bw/day

Dose / conc.:

6 mg/kg bw/day

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral route is one of the potential routes of human exposure to this test material.
The dose levels were selected by the Sponsor on the basis of available data from a previously conducted pilot developmental toxicity study. The pilot study showed that at the dose level of 9.5 mg/kg/day, 40 % of the dams were unable to tolerate the dosing schedule and had to be euthanised. In addition, dams exposed to 9.5 mg/kg/day weighed significantly less than controls in the early stages of gestation. The severity of these endpoints on the dam was the basis for selecting the high dose of 6 mg/kg/day, with the middle and low doses of 3.0 and 0.75 mg/kg/day being selected as following a geometric progression factor of 4 and 2, respectively.
- Rationale for animal assignment: Using a standard, by weight, randomidation procedure, 100 female animals (weighing 158 to 215 g, at randomisation) were assigned to the control and treatment groups. All animals were given a detailed clinical examination and body weights were recorded prior to selection and before dosing on GD 0. The Study Director reviewed GD 0 body weight and detailed observation data for all animals and gave final approval for assignment to study.
Animals assigned to study had body weights within ± 20 % of the mean body weight. Extra animals obtained, but not placed on study, were euthanised by carbon dioxide inhalation followed by an MPI Research SOP approved method to ensure death. The carcasses were discarded.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations: All animals were observed for morbidity, mortality, injury, and the availability of food and water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from GD 0 through 20 (75 minutes, ±15 minutes, post-dose on dosing days), each animal was removed from the cage and given a detailed clinical examination. On occasion, clinical observations were recorded at unscheduled intervals. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration.
Veterinary Observations: On occasion, veterinary observations were conducted during the course of the study. All treatments and observations were recorded. The medical treatments and observations are not reported but are maintained in the study file.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights for all animals were measured and recorded on GD 0, 3, 6, 9, 12, 15, 18, and 20. Individual body weight change was calculated for the following GD intervals: GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-20 and 0-20. Adjusted body weight (GD 20 body weight minus gravid uterine weight) and adjusted body weight change (GD 0 to 20) were also calculated.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was measured and recorded on the corresponding body weight days and calculated for the same intervals.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: On GD 20, each surviving female was euthanised by carbon dioxide inhalation followed by an SOP approved method to ensure death and immediately subjected to a cesarean section.
- Organs examined: Maternal necropsies were conducted without knowledge of the treatment groups in order to minimise bias. The skin was reflected from a ventral midline incision to examine mammary tissue and locate any subcutaneous masses. The abdominal cavity was then opened, and the uterus was exposed. The uterus was excised, and the gravid uterine weight was recorded. Beginning at the distal end of the left uterine horn, the location of viable and nonviable foetuses, early and late resorptions for each uterine horn, and the total number of implantations were recorded. The number of corpora lutea on each ovary was also recorded.
The foetuses were removed by making a dorsal incision longitudinally along both uterine horns. The embryonic membrane of each foetus was gently removed, and each foetus was pulled away from the placenta, fully extending the umbilical cord. The placentae were examined grossly.
Uteri from females that appeared nongravid were opened and placed in 10 % ammonium sulfide solution for detection of implantation sites. If no foci were detected, the female was considered to be nonpregnant.
A complete necropsy was performed on all euthanised in extremis and surviving dams under procedures approved by a veterinary pathologist. Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced the pregnancy. The presence of lesions or other abnormal conditions in the dam were noted and described in the study records. Additionally, the thymus gland from each animal was collected, weighed, and preserved in 10 % neutral buffered formalin for possible further evaluation.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Each implant was categorised according to the following criteria. Viable foetuses responded to touch. Nonviable foetuses did not respond to touch and had no signs of autolysis. Late resorptions were characterised by recognisable foetal form, but undergoing autolysis. Early resorptions were characterised as implantation sites that had no recognisable foetal characteristics.

Fetal examinations:

Foetal examinations were conducted without knowledge of the treatment groups in order to minimise bias. Each foetus was individually weighed, sexed, tagged, and examined for external malformations and variations. Foetuses were then euthanised by intraperitoneal injection of euthanasia solution.
Approximately one-half of the foetuses in each litter were placed in Bouin’s solution and the remaining foetuses were fixed in alcohol.

- External examinations: Yes: All per litter.
- Soft tissue examinations: Yes: Approximately half per litter. All foetuses fixed in Bouin’s solution were examined for soft tissue defects using the Wilson razor-blade sectioning technique.
- Skeletal examinations: Yes: Approximately half per litter. The foetuses fixed in alcohol were macerated in potassium hydroxide, stained with Alizarin Red S, and cleared with glycerin by a method similar to that described by Dawson for subsequent skeletal examination.
- Head examinations: No

Foetal findings were classified as malformations or developmental variations under procedures approved by a developmental toxicologist. On occasion, additional information to clarify or identify a visceral or skeletal observation was documented. These comments are not reported, but are maintained in the study data. Mechanical artifacts (i.e., tail removed and discarded) occurred during examination and processing of the foetuses. These artifacts are not reported, but are maintained in the study data.

Statistics:

The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint by sex and group.

Parental In-life Data
Group pair-wise comparisons: Gestation body weights, gestation body weight changes, gestation food consumption, adjusted body weights, adjusted body weight changes (GD 0 - 20) and thymus weight (absolute and relative to adjusted GD 20 bodyweight).

Uterine and Ovarian Examinations
Fischer’s exact test: Pregnancy index, malformations by finding and exam type (external, visceral and skeletal)-litter incidence and variations by finding and exam type (external, visceral and skeletal)-litter incidence. Foetal and litter incidences are reported, but only the litter incidences were statistically analysed.
Group pair-wise comparison: Gravid uterine weights, corpora lutea/dam, total implantations/dam, litter size/dam, viable foetuses/dam, total number resorptions/dam, number early resorptions/dam and number late resorptions/dam.
Arcsin-square-root transformation: Foetal sex ratio (% males/litter), % pre-implantation loss and % post-implantation loss.
Descriptive statistics: Non-viable foetuses/dam.
Covariate analysis: Mean foetal body weights.

Indices:

Pregnancy Index = (No. females pregnant / No. females with evidence of mating) x 100

Post-implantation Loss = ((No. implantations - No. viable foetuses) / No. implantations) x 100

Pre-implantation Loss = ((No. corpora lutea - No. implantations) / No. corpora lutea) x 100

Historical control data:

No. of studies: 2
No. females: 135
No. pregnant: 124
No. not pregnant: 1
No. died pregnant: 0
No. females with all resorptions: 0
No. pregnant by stain: 0
No. females with viable foetuses: 124
No. of litters evaluated: 124
No. external foetuses evaluated: 1 588
No. visceral foetuses evaluated: 794
No. skeletal foetuses evaluated: 794

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No adverse effect of the test material at 0.75 and 3.0 mg/kg/day was observed from the detailed clinical examinations. Red material around the nose was observed at least once in 3/25 and 5/25 animals in the 0.75 and 3.0 mg/kg/day dose groups, respectively and was also observed in 6/25 animals in the 6.0 mg/kg/day group. A similar finding was not observed among the control animals. Due to its sporadic occurrence among the treated animals generally observed only on 1 or 2 days during the study and in many instances only during the first week, its presence while likely test material related, was not considered adverse. Other clinical findings observed in the 0.75 and 3.0 mg/kg/day animals occurred at low incidence or with similar frequency to controls and were considered unrelated to the test material. In the 6.0 mg/kg/day group, clinical findings observed with increased frequency and considered test material related and adverse included low body carriage, red material around the nose, thin appearance, loss of skin elasticity, and pale body colour.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

All animals in the control, 0.75, and 3.0 mg/kg/day groups survived to scheduled termination on GD 20. In the 6.0 mg/kg/day group, animals 4512 and 4525 were euthanised in extremis on GD 12 and GD 9, respectively. Both animals had clinical signs of toxicity that included decreased activity, low body carriage, red material around the nose/mouth, hunched posture, pale body color, and thin unkempt appearance. Likewise both animals lost weight from the start of treatment and had low food consumption. The moribundity observed in these animals was considered test material-related. All other animals in the 6.0 mg/kg/day group survived to scheduled termination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No effect of the test material at dose levels of 0.75 and 3.0 mg/kg/day was observed on gestation body weights and body weight change. At 6.0 mg/kg/day, mean body weights were statistically lower than mean control values on GD 18 (-8 %) and 20 (-9 %). Mean body weight change in this group was lower than mean control values over much of gestation and statistically lower over GD 0 to 3 (-24 %), GD 6 to 9 (-34 %) and over the entire GD 0 to 20 treatment period (-19 %). Considerable variability in gestation body weight change was observed in the 6.0 mg/kg/day dose group. This was attributed to low weight gain and/or weight loss in several animals (4510, 4511, 4516, and 4524) that failed to retain pregnancies with foetuses and at GD 20 had uterine implantations comprised entirely of resorbing foetuses (100 % post-implantation loss). These effects on gestation body weights and body weight change at 6.0 mg/kg/day were considered test material related correlating with adverse pregnancy outcomes in several animals.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

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):

Mean gravid uterine weights, adjusted GD 20 body weights and adjusted weight change GD 0 to 20 in the test material-treated groups were comparable to mean control values.

Lower thymus weights, absolute and relative to the adjusted GD 20 body weight, were observed in the test material-treated groups. Mean absolute thymus weights were 19 %, 34 %, and 44 % lower than the mean control value in the 0.75, 3.0, and 6.0 mg/kg/day dose groups, respectively and relative to the adjusted GD 20 body weights, were 20 %, 35 %, and 37 % lower, respectively. These differences in thymus weights, absolute and relative to adjusted GD 20 body weight in the test material-treated groups relative to controls were statistically significant and considered test material related. In the context of this study, the toxicological significance of this decrease in thymus weight in the absence of histopathological examination is unclear.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No effect of the test material at 0.75 and 3.0 mg/kg/day was observed from the maternal macroscopic examinations. The few findings observed among these animals occurred at low incidence or with similar frequency as controls and were considered unrelated to the test material. At 6.0 mg/kg/day, small thymus was observed with increased frequency. This was observed in the two animals euthanised in extremis in this group and 4/23 animals that survived to scheduled termination. Other maternal macroscopic findings observed in the 6.0 mg/kg/day group occurred at low incidence or similar to controls and were considered unrelated to the test article.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- Food consumption: No effect of the test material at dose levels of 0.75 and 3.0 mg/kg/day was observed on gestation food consumption. At 6.0 mg/kg/day, mean food consumption was statistically lower than mean control values over GD 6 to 9 (-14 %), GD 9 to 12 (-13 %), and GD 15 to 18 (-17 %). The considerable variability in food consumption observed in this group was largely attributable to low food consumption in those females with failed pregnancies as discussed previously. Mean food consumption in the 6.0 mg/kg/day group over the entire treatment period (GD 0 to 20) was 15.47g/animal/day and 9 % lower (not statistically significant) than the 17.09 g/animal/day observed in controls.

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

No effect of the test material at 0.75 and 3.0 mg/kg/day was observed on GD 20 uterine implantation parameters.
Statistically significant decreases in mean post-implantation loss in the 0.75 mg/kg/day dose group relative to controls were considered incidental, not adverse, and unrelated to the test material.
In the 6.0 mg/kg/day group, no statistically significant changes in corpora lutea count or uterine implantation data were observed relative to controls.
In the 6.0 mg/kg/day group, notable increases in mean post-implantation loss (25.70 %) were observed. Changes in this parameter in the 6.0 mg/kg/day dose group were also outside the range of recent historical control data for the laboratory maximum study values were 5.35 % for post-implantation loss. Changes were largely attributable to the four females in the group with all resorption sites in utero (100 % post-implantation loss).

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

Four females in the 6.0 mg/kg/day group (4510, 4511, 4516, and 4524) had uterine implantations comprised entirely of resorbing foetuses (100 % post-implantation loss).
The increased incidence of females with all resorption sites in utero was considered test material related and adverse.
In the 6.0 mg/kg/day group, notable increases in mean number of resorption sites (total and early)/dam (2.7) relative to controls (5.40 % and 0.7, respectively) were observed. Changes in these parameters in the 6.0 mg/kg/day dose group were also outside the range of recent historical control data for the laboratory maximum study values were 0.7 resorption sites (total and early)/dam. Changes were largely attributable to the four females in the group with all resorption sites in utero (100 % post-implantation loss).

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increases in mean number of resorption sites (total and early) in the 0.75 mg/kg/day dose group relative to controls were considered incidental, not adverse, and unrelated to the test material.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increases in mean number of viable foetuses and litter size in the 0.75 mg/kg/day dose group relative to controls were considered incidental, not adverse, and unrelated to the test material.
In the 6.0 mg/kg/day group a decrease in number of viable foetuses/dam (9.7 vs. 12.5 in controls) were observed. Changes in these parameters in the 6.0 mg/kg/day dose group were also outside the range of recent historical control data for the laboratory maximum study values were 13.5 viable foetuses/dam). Changes were largely attributable to the four females in the group with all resorption sites in utero (100 % post-implantation loss).

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

Pregnancy index in the control, 0.75, 3.0, and 6.0 mg/kg/day groups was 96 %, 96 %, 92 %, and 88 %, respectively. There were one, one, two, and three nonpregnant females in the control, 0.75, 3.0, and 6.0 mg/kg/day groups, respectively. Two of the nonpregnant females in the 6.0 mg/kg/day group were euthanised in extremis.
Overall, there were 24, 24, 23, and 18 litters with GD 20 foetuses for evaluation in the control, 0.75, 3.0, and 6.0 mg/kg/day dose groups, respectively.

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

mortality

pre and post implantation loss

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No effect of the test material was observed on foetal body weights. Mean foetal body weights distinguished by sex and for the combined sexes, in the test material-treated groups were comparable to mean control values.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No effect of the test material was observed on foetal sex ratios (percent male foetuses/animal). Mean sex ratios ranged from 45.2 % to 48.8 % in the treated groups and were comparable to the mean of 47.1 % in controls.

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No effect of the test material was observed from the foetal external examinations. No malformations were observed among foetuses in the 0.75 and 6.0 mg/kg/day groups. In the 3.0 mg/kg/day group, the only external malformation observed was misshapen tongue. The litter incidence for this finding was 17.4 % (4/23 litters affected) and comparable to the 12.5 % (3/24 litters affected) in controls. Filamentous tail and absence of an anus were observed in one control foetus. No external developmental variations were observed among the control and test material-treated foetuses.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No effect of the test material was observed from the foetal skeletal examinations. No skeletal malformations were observed among foetuses from the control and the test material-treated groups. Litter incidences for ossification variations observed in the test material-treated groups were comparable to control and no effect of treatment was observed from these data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No effect of the test material was observed from the foetal visceral examinations. No malformations were observed among foetuses in the control, 0.75, and 6.0 mg/kg/day dose groups. In the 3.0 mg/kg/day group the only visceral malformation observed was folded retina. This was observed in a single foetus and in the absence of a similar malformation among foetuses in the 6.0 mg/kg/day group, its occurrence on study was considered spontaneous and unrelated to the test material. The litter incidence of an irregular palatal rugae pattern, a visceral variation, in the control, 0.75, and 3.0 mg/kg/day treated groups was 4.2 %, 12.5 %, and 26.1 %, respectively. The higher incidence of this variation in these treated groups did not differ statistically from controls and in the absence of a similar finding among foetuses in the 6.0 mg/kg/day group, was not considered test material related. The occurrence of a smaller than normal thyroid, a visceral variation in one foetus at 6.0 mg/kg/day was considered incidental and unrelated to test article.

Key result

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

6 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

Relevant for humans:

not specified

Summary of Maternal and Developmental Observations at Uterine Examination

Endpoint	0 mg/kg/day	0.75 mg/kg/day	3.0 mg/kg/day	6.0 mg/kg/day
No. females on study	25	25	25	25
No. not pregnant	1	1	2	3
No. pregnant	24	24	23	22
Pregnancy index percent	96.0	96.0	92.0	88.0
No. females with resorptions	0	0	0	4
No. females with viable foetuses GD 20	24	24	23	18

Summary of Maternal and Developmental Observations at Uterine Examination

Endpoint		0 mg/kg/day	0.75 mg/kg/day	3.0 mg/kg/day	6.0 mg/kg/day
Corpora lutea No. per animal	Mean	15.4	16.1	16.0	15.4
	SD	2.30	2.02	3.01	2.43
	N	24	24	23	18
Implantation sites No. per animal	Mean	13.2	14.3	14.2	12.5
	SD	1.89	2.35	1.67	2.42
	N	24	24	23	22
Pre-implantation loss % per animal	Mean	12.91	11.03	9.80	14.66
	SD	14.050	9.759	10.112	15.271
	N	24	24	23	18
Viable foetuses	Mean	12.5	14.1*	13.5	9.7
	SD	1.96	2.36	1.78	5.42
	N	24	24	23	22
Foetal sex ration % males per animal	Mean	47.1	45.2	46.6	48.8
	SD	16.45	15.21	11.34	11.35
	N	24	24	23	18
Post-implantation loss % per anima	Mean	5.40	1.46*	4.89	25.70
	SD	5.626	2.952	5.687	39.370
	N	24	24	23	22
Nonviable Foetuses No. per animal	Mean	0	0	0	0
	SD	0	0	0	0
	N	24	24	23	22
Litter size No. per animal	Mean	12.5	14.1*	13.5	9.7
	SD	1.96	2.36	1.78	5.42
	N	24	24	23	22
Resorptions: early + late No. per animal	Mean	0.7	0.2*	0.7	2.7
	SD	0.75	041	0.82	4.26
	N	24	24	23	22
Resorptions: early No. per animal	Mean	0.7	0.2*	0.7	2.7
	SD	0.75	0.41	0.83	4.22
	N	24	24	23	22
Resorptions: late No. per animal	Mean	0	0	0	0
	SD	0	0	0.21	0.21
	N	24	24	23	22

* Significantly different from control (p<0.05).

Summary of Gestation Body Weight Values (g)

Study Interval (Day)	0 mg/kg/day			0.75 mg/kg/day			3.0 mg/kg/day			6.0 mg/kg/day
Study Interval (Day)	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
0	187.7	13.87	24	188.1	14.5	24	187.3	14.93	23	186.3	13.16	22
3	205.0	15.09	24	205.2	13.75	24	206.1	16.25	23	199.5	15.07	22
6	215.4	14.72	24	216.3	14.23	24	218.3	17.18	23	210.0	16.60	22
9	232.4	15.6	24	232.5	14.84	24	234.7	18.18	23	221.1	22.80	22
12	248.2	16.57	24	247.2	17.2	24	250.4	20.35	23	233.6	29.55	22
15	265.5	16.66	24	265.7	17.17	24	270.0	21.28	23	248.5	38.22	22
18	303.9	17.65	24	310.0	20.51	24	311.5	26.81	23	280.3*	51.34	22
20	334.4	19.82	24	344.6	24.61	24	344.9	30.34	23	305.4*	62.95	22

* Significantly different from control (p<0.05).

Summary of Gestation Body Weight Change Values (g)

Study Interval (Day)	0 mg/kg/day			0.75 mg/kg/day			3.0 mg/kg/day			6.0 mg/kg/day
Study Interval (Day)	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
0 - 3	17.3	4.80	24	17.1	4.10	24	18.8	3.55	23	13.2*	5.95	22
3 – 6	10.5	4.57	24	11.1	3.60	24	12.2	2.96	23	10.5	4.68	22
6 – 9	17.0	3.85	24	16.3	3.12	24	16.4	4.18	23	11.2*	8.96	22
9 – 12	15.8	3.20	24	14.7	4.19	24	15.7	3.70	23	12.5	8.40	22
12 – 15	17.4	5.43	24	18.5	4.65	24	19.5	6.71	23	15.0	11.03	22
15 – 18	38.4	6.25	24	44.4	6.66	24	41.6	7.71	23	31.7	15.13	22
18 – 20	30.5	4.75	24	34.5	6.61	24	33.3	4.71	23	25.1	13.74	22
0 - 20	146.7	11.73	24	156.5	17.38	24	157.5	21.08	23	119.1†	56.38	22

*Significantly different from control (p<0.05).

† Significantly different from control (p<0.01).

Summary of Gestation Food Consumption Values (g/animal/day)

Study Interval (Day)	0 mg/kg/day			0.75 mg/kg/day			3.0 mg/kg/day			6.0 mg/kg/day
Study Interval (Day)	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
0 - 3	12.28	2.037	24	12.89	1.562	24	13.16	1.875	23	12.21	4.377	22
3 – 6	14.97	2.567	24	15.22	1.817	24	16.54	2.886	23	15.20	3.306	22
6 – 9	16.74	2.852	24	16.81	1.702	24	16.19	1.729	23	14.44*	4.538	22
9 – 12	16.93	1.624	24	16.69	1.322	24	17.22	1.719	23	14.77*	5.286	22
12 – 15	17.61	1.531	24	17.53	1.985	24	18.38	1.876	23	15.89	6.273	22
15 – 18	22.25	2.497	24	21.97	2.113	24	22.14	3.270	23	18.55†	7.008	22
18 – 20	19.69	2.453	24	20.83	2.268	24	21.15	2.822	23	18.11	7.265	22
0 - 20	17.09	1.530	24	17.25	1.182	24	17.66	1.798	23	15.47	4.692	22

* Significantly different from control (p<0.05).

† Significantly different from control (p<0.01).

Summary of GD 20 Maternal Thymus Weights

Group (mg/kg/day)	Thymus Weight (g)	Thymus/ Adjusted GD20 Body Weight (%)
0	0.239	0.0891
0.75	0.193*	0.0716*
3.0	0.158*	0.0581*
6.0	0.134*	0.0558*

* Significantly different from control (p < 0.01).

Historical Control Data

Parameter	Mean	Minimum	Maximum
Pregnancy Index (%)	99.50	99.0	100.0
Corpora Lutea (Mean No. per Animal)	15.35	15.1	15.6
Implantation Sites (Mean No. per Animal)	13.7	13.3	14.1
Preimplantation Loss (Mean % per Animal)	9.950	8.82	11.08
Viable Foetuses (Mean No. per Animal)	13.05	12.6	13.5
Foetal Sex Ratio (Mean % Males per Animal)	49.20	47.4	51.0
Post-implantation Loss (Mean % per Animal)	4.755	4.16	5.35
Nonviable Foetuses (Mean No. per Animal)	0	0	0
Litter Size (Mean No. per Animal)	13.05	12.6	13.5
Resorptions: Early and Late (Mean No. per Animal)	0.65	0.6	0.7
Resorptions: Early (Mean No. per Animal)	0.65	0.6	0.7
Resorptions: Late (Mean No. per Animal)	0	0	0
Mean Foetal Body Weight Male (g)	4.05	4.02	4.08
Mean Foetal Body Weight Female (g)	3.850	3.83	3.87
Mean Foetal Body Weight Males and Females (g)	3.950	3.93	3.97

Historical Control Data - Litter Evaluations

Parameter	Total No.		Max % / Study
Parameter	Foetuses	Litters	Foetuses	Litters
External malformation
Body, umbilicus, ophalocele	2	2	0.2	2.0
Skeletal malformation
Cervical vertebra€, neural arch(es), misshapen	1	1	0.6	4.0
Visceral malformations
Head, retina, folded retina	1	1	0.2	1.0
Skeletal variation
Rib(s), rudimentary	158	65	29.8	68.0
Rib(s), unilateral full rib	5	4	1.8	8.0
Skull, hyoid, not ossified	1	1	0.2	1.0
Skull, palatine, irregular ossification	62	20	11.3	24.0
Sternum, sternebra(e), misaligned	10	10	1.6	10.1
Sternum, sternebra(e), not ossified	11	11	1.8	11.1

Analytical Calibration Results

Analytical Run	Standard Identification	Nominal Concentration (μg/mL)	Calculated Concentration (μg/mL)	% Recovery	Coefficient of Determination (R^2)
1	Standard 1	2.003113	2.053228	102.5	0.9984
	Standard 2	3.004669	2.925588	94.7
	Standard 3	6.00939	6.206982	103.3
	Standard 4	10.015564	9.868528	98.5
	Standard 5	12.519455	12.220998	97.6
	Standard 6	15.023346	15.300160	101.8
3	Standard 1	2000934	2.039972	102.0	0.9992
	Standard 2	3.001401	3.036667	101.2
	Standard 3	6.002802	6.047250	100.7
	Standard 4	10.004669	9.7400897	97.9
	Standard 5	12.505837	12.400897	99.2
	Standard 6	15.007004	15.206865	101.3

Analysis of the Test Material in Dosing Formulations – Week 1

Dose Level (mg/kg/day)	Sample Identification	Nominal Concentration (mg/mL)	Calculated Concentration (mg/mL)	Average Calculated Concentration (mg/mL)	% Recovery	Average % Recovery	% Relative Standard Deviation
0	Replicate 1	0.00	BLQ	BLQ	NA	NA	NA
0	Replicate 2	0.00	BLQ	BLQ	NA	NA	NA
0.75	Top replicate 1	0.15	0.1501	0.1416	100.1	94.4	5.976
	Top replicate 2		0.1514		101.0
	Middle replicate 1		0.1296		86.4
	Middle replicate 2		0.1386		92.4
	Bottom replicate 1		0.1360		90.7
	Bottom replicate 2		0.1437		95.8
3.0	Replicate 1	0.60	0.6070	0.5916	101.2	98.6	3.691
3.0	Replicate 2	0.60	0.5761	0.5916	96.0	98.6	3.691
6.0	Top replicate 1	1.20	1.1520	1.1083	96.0	92.4	3.485
	Top replicate 2		1.1598		96.6
	Middle replicate 1		1.0905		90.9
	Middle replicate 2		1.0709		89.2
	Bottom replicate 1		1.1020		91.8
	Bottom replicate 2		1.0745		89.5

BLQ: Below the Limit of Quantitation (< 20.0000 μg/mL).

NA: Not Applicable/Not Available

Analysis of the Test Material in Dosing Formulations – Week 3

Dose Level (mg/kg/day)	Sample Identification	Nominal Concentration (mg/mL)	Calculated Concentration (mg/mL)	Average Calculated Concentration (mg/mL)	% Recovery	Average % Recovery	% Relative Standard Deviation
0	Backup replicate 1*	0.00	BLQ	BLQ	NA	NA	NA
	Backup replicate 2*		BLQ		NA
0.75	Backup replicate 1*	0.15	0.1362	0.1445	90.8	96.4	9.135
	Backup replicate 2*		0.1529		101.9
3.00	Backup replicate 1*	0.60	0.5932	0.6007	98.9	100.1	1.764
	Backup replicate 2*		0.6082		101.4
6.00	Backup replicate 1*	1.20	1.2518	1.2083	104.3	100.7	5.088
	Backup replicate 2*		1.1649		97.1

BLQ: Below the Limit of Quantitation (< 20.0000 μg/mL).

NA: Not Applicable/Not Available

* Backup samples were analysed due to the initial run failing to meet acceptance criteria for performance check 2.

Analysis of Dosing Formulations

- Homogeneity: Analyses of the low- and high-dose formulations (0.15 and 1.20 mg/mL) used for dosing the first week of study confirmed they were homogeneous as prepared meeting the laboratory’s acceptance criteria of 100 ± 15 % of nominal for the average percent recovery and percent Relative Standard Deviation (RSD) ≤ 10.

Dose Level

(mg/kg/day)

Nominal Concentration

(mg/mL)

Average Calculated Concentration

(mg/mL)

Average % Recovery*

% Relative Standard Deviation

0.75

0.15

0.1416

94.4

5.976

6.0

1.20

1.1083

92.4

3.485

* Average % recovery was calculated from the nominal concentration.

- Concentration: Mean concentrations of formulations used for dosing in Weeks 1 and 3 ranged between 92.4 % and 100.7 % of nominal with percent RSDs ranging between 1.764 and 8.135, confirming that animals were receiving the appropriate dose levels when the formulations were administered at 5 mL/kg. No test material was detected in the control samples.

Dose Level (mg/kg/day)	Nominal Concentration (mg/mL)	Average Calculated Concentration* (mg/mL)	*Average % Recovery†**	% Relative Standard Deviation*
0	0.00	BLQ	NA	NA
0.75	0.15	0.1416 – 0.1445	94.4 – 96.4	5.976 – 8.135
3.0	0.60	0.5916 – 0.6007	98.6 – 100.1	1.764 – 3.691
6.0	1.20	1.1083 – 1.2083	92.4 – 100.7	3.485 – 5.088

* Results are the range of values determined during Weeks 1 and 3.

† Average % recovery was calculated from the nominal concentration.

BLQ: Below the Limit of Quantitation (< 20.0000 μg/mL).

NA: Not applicable.

Conclusions:

Under the conditions of the study the no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity with the test material was 3.0 mg/kg/day. At dose levels of 0.75, 3.0, and 6.0 mg/kg/day, the test material was not teratogenic in the rat.

Executive summary:

This study was conducted for to evaluate the possible adverse effects of the test article following repeated oral exposure to pregnant animals according to OECD Test Guideline 414 and in compliance with GLP.

This evaluation included systemic toxicity, female reproductive performance, and evaluation of the fetuses. The vehicle, peanut oil, or test material was administered to time-mated female Hsd:Sprague Dawley® SD® rats once daily via oral gavage from Gestation Day (GD) 0 through 19.

Observations of the animals included clinical signs, body weights, food consumption, and anatomical pathology including a uterine examination. All foetuses were given an external and visceral or skeletal examination.

Analytical results for formulations used for dosing in Weeks 1 and 3 confirmed homogeneity and concentration levels ranged between 92.4 % and 100.7 % of nominal. No test material was detected in the control samples.

All animals in the control, 0.75, and 3.0 mg/kg/day groups survived to scheduled termination on GD 20. At 6.0 mg/kg/day, two animals were euthanized in extremis with clinical signs of toxicity, low body weights, low body weight change, and low food consumption. A low incidence of red material around the mouth was observed in the test material-treated groups but not considered adverse due to its sporadic occurrence generally early in the study. No other maternal effects at dose levels ≤3.0 mg/kg/day were observed from clinical findings, gestation body weights, body weight change, food consumption, or maternal macroscopic findings. At 6.0 mg/kg/day, maternal effects were apparent from clinical findings (low body carriage, red material around the nose, thin appearance, loss of skin elasticity, and pale body color), lower gestation body weights, lower body weight change, and lower food consumption. No effect at dose levels ≤3.0 mg/kg/day was observed on pregnancy or uterine implantation data. At 6.0 mg/kg/day, 4/23 females at GD 20 had uterine implantation sites comprised entirely of resorbing foetuses (100 % post-implantation loss) and this contributed to decreases in mean number of viable foetuses and increases in mean post-implantation loss and mean number of resorption sites for the group. No effect of the test material was observed on foetal sex ratio, foetal body weight, or foetal external, visceral, or skeletal examinations. Lower maternal thymus weights were observed at all test material-treatment levels and an increased incidence of small thymus observed macroscopically in the 6.0 mg/kg/day animals.

In the context of this study, the toxicological significance of test material-related effects on thymus weights (decrease) in the absence of histopathological examination is unclear.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 3 mg/kg bw/day
Study duration:: subacute
Species:: rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Additional information

There is an oral developmental dose toxicity study available for DBTO:

Schroeder, 2017: This study was conducted for to evaluate the possible adverse effects of the test article following repeated oral exposure to pregnant animals according to OECD Test Guideline 414 and in compliance with GLP.

In the context of this study, the toxicological significance of test material-related effects on thymus weights (decrease) in the absence of histopathological examination is unclear.

Furthermore, the study according to OECD 422 (Morley, 2023) provides information on potential developmental toxicity of DBTO. 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.

In addition, a publicly available study also addressed the potential teratogenicity of dibutyltin compounds including DBTO in the rat (Noda et al., 1993). In this non-guideline study, pregnant rats were exposed to a single dose of the test item on Gestation Day 8 (GD) only. For DBTO, only one dose level was examined, and the observation of fetal malformations is reported in this publication. However, no detailed information is available allowing the assessment of potential maternal toxicity and no information on test item purity is provided, hampering the evaluation of the basis for the diverging results.

In conclusion, the well-documented recent studies conducted according to current OECD Testing Guidelines under GLP conditions are considered more reliable and the results obtained in these studies are given more weight in the overall assessment of the registered substance.

Justification for classification or non-classification

The available study according to OECD 422 clearly shows that the registered substance DBTO does not adversely affect fertility. Both guideline conform studies (OECD 422 and OECD 414) show some indications for potential effects of the registered substance DBTO on fetuses/ofspring at maternally toxic dose levels. In the OECD 422 study, a reduced pup body weight was observed at 5 mg/kg/day, whereas the OECD 414 study found no impact on foetal body weight up to 6 mg/kg/day. Furthermore, an increased number of post-implantation losses was observed at the high dose level (6 mg/kg/day) in the OECD 414 study, while the OECD 422 study found no effect of DBTO on post-implantation losses up to 5 mg/kg/day. Importantly, both studies did not find any indication for the induction of malformations by DBTO, and no external, visceral or skeletal effects were observed in the foetuses.

Taken together, these results do not justify any classification of DBTO for adverse effects on sexual function and fertility according to Regulation (EC) No 1272/2008 (CLP Regulation). Based on the developmental findings observed at maternally toxic dose levels in both studies conducted according to OECD TG and compliant with GLP, the registered substance DBTO is classified as Repr. 2 (H361d) as conservative and precautionary measure.

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