bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-05 - 2018-01-05 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-05 - 2018-01-05 (experimental phase)

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:

OECD 422 guideline for testing of chemicals adopted 29 July 2016: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2 to 8°C) and protected from the light.

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

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 Crl:CD(SD) 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.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Males 69 to 76 days old
Females 83 to 90 days old
- Weight at study initiation:
Males 338 to 408 g
Females 239 to 298 g
- Fasting period before study: no
- Housing: Number of animals per cage:
Pre-pairing: up to five animals of one sex
Pairing: one male and one female
Males after mating: up to five animals
Gestation: one female
Lactation: one female + litter
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Environmental Enrichment:
A soft white untreated aspen wood block; provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced when necessary.
Plastic shelter was provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced at the same time as the cages.
- 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 prior to the commencement of treatment.
Females: 20 days prior to the commencement of treatment

DETAILS OF FOOD AND WATER QUALITY: The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

ENVIRONMENTAL CONDITIONS
Rodent Facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
- Temperature (°C): / Humidity (%): Temperature and relative humidity were monitored and maintained within the range of 20-24ºC and 40-70%. There were no deviations from these ranges.
- 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: 16 August 2017 (males), 2 August 2017 (females)
To: 26 September 2017 (males), 11 to 15 October 2017 (females) (F0 necropsy)

Route of administration:

oral: gavage

Details on route of administration:

The oral gavage route of administration was chosen as the preferred route for human risk assessment.

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2-8°C).

- VEHICLE : Corn oil
- Concentration in vehicle: 0, 20, 66, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis
Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. These investigations demonstrated that formulations in the 10 to 200 mg/mL concentration range are homogenous and stable following ambient storage (15-25°C) for one day and following refrigerated storage (2-8°C) for 15 days.
Achieved concentration: Samples of each formulation prepared for administration in Week1 of treatment and on Day 10-12 of lactation were analyzed for achieved concentration of the test item.

Homogeneity and Stability in Corn Oil Formulations
The homogeneity and stability of the test item in corn oil formulations were assessed at nominal concentrations of 10 mg/mL and 200 mg/mL, during ambient and refrigerated storage. Freshly prepared specimen formulations (400 mL) were equally sub divided into 4 amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Homogeneity and Stability of Dose Formulations
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 days and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 5% of the initial time zero value and the coefficient of variation was less than 5%. Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method.

Concentration of Dose Formulations
The mean concentrations of the test item in test formulations analyzed during the study. The original Week 1 samples were extracted but due to repeated system issues, no results can be reported. The contingency samples were analyzed in replacement of the original samples. These results are all within 4% of nominal concentration, confirming accurate formulation. The coefficient of variation values are within 2%, confirming precise analysis. In Day 10-12 of lactation (females), the Group 2 and 4 results were within 5% of nominal concentration, and the coefficient of variation values were within 3%, confirming precise analysis. The Group 3 samples were -36.4% of nominal concentration with a coefficient of variation of 58.11%. Excluding the low ‘bottom’ result as an outlier due to the very low result, the Group 3 samples were -15.8% of nominal concentration with a difference from mean of ±12.56%. The Group 3 contingency samples were analyzed outside of the confirmed stability period, and therefore can only be reported for information purposes only. These contingency results were -8.5% from nominal concentration with a coefficient of variation of 5.59%.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.
The homogeneity and stability was confirmed for The test item in corn oil formulations at nominal concentrations of 10 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days. The mean concentrations of The test item in test formulations analyzed for the study were within the applied limits of +10/-15% of nominal concentrations, confirming accurate formulation, with the exception of Day 10-12 of lactation Group 3 samples which were 15.8% of nominal concentration.

Duration of treatment / exposure:

Three groups of ten male and ten female rats received the test item at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration at a volume dose of 5 mL/kg/day. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 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, corn oil, at the same volume dose as treated groups.

Frequency of treatment:

Once daily, at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

330 mg/kg bw/day (nominal)

Dose / conc.:

1 000 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:
The dose levels selected for investigation in this OECD 422 combined repeated dose toxicity study and reproductive/developmental toxicity screening study (0, 100, 330 and 1000 mg/kg/day) were chosen in conjunction with the Sponsor and were based on the results of a 14-day preliminary study conducted at these laboratories.
In the preliminary study dose levels of 250, 500 and 1000 mg/kg/day were investigated. There were no test item-related premature deaths, no signs observed in relation to dose administration, no test item-related changes in clinical condition, body weight, water consumption or spleen weights. Group mean food intake was slightly reduced during the treatment period when compared with pre-treatment values for all dose groups. Mean absolute and body weight relative liver weights were slightly high for females receiving 1000 mg/kg/day and for males receiving 500 or 1000 mg/kg/day but one male at 250 mg/kg/day had a small liver. Kidney weights also exhibited a modest dose dependent reduction for body weight relative data in males. Macroscopic findings were limited to adhesions involving multiple organs of the thoracic cavity for two animals receiving 250 mg/kg/day which were considered to be related to mis-dosing. In addition, one animal receiving 1000 mg/kg/day was found to have cysts on the kidneys.
Therefore, a high dose level of 1000 mg/kg/day was selected for this study as the limit dose for the OECD 422 guideline. The intermediate and low dose levels of 330 and 100 mg/kg/day were chosen to achieve a dose response and/or aid in the determination of a No-Observed-Adverse-Effect-Level (NOAEL).
- Rationale for animal assignment (if not random):
Allocation: On arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced to ensure variations in body weight of animals did not exceed ± 20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation:
Irregular estrous cycle: Six females
Unexpected death: One female

Positive control:

not required

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality: A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
Clinical and Behavioral Observations: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. 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.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
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 onwards - once each week
F0 females: Week 1 – daily; Week 2 – once, 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; As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see above

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

FOOD CONSUMPTION
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows for F0 animals: Weekly before pairing, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Days 15 to 22), but recommenced for males in Week 4.
For females after mating food consumption was recorded as follows: Days 0-6, 7-13 and 14-19 after mating; Days 1-3, 4-6 and 7-12 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: not stated

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at termination.
- Anaesthetic used for blood collection: Yes, Animals were held under light general anesthesia induced by isoflurane.
- Animals fasted: No
- How many animals: The five lowest numbered surviving males and the first five surviving lactating females with a litter per group
- Parameters examined: Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)
Platelet count (Plt)
* Derived value calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at termination.
- Animals fasted: No
- How many animals: The five lowest numbered surviving males and the first five surviving lactating females with a litter per group
- Parameters examined: Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)
Globulin (Glob)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: see below
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.
Sensory Reactivity and Grip Strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7 to 9 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were moved into individual cages prior to transport to the testing room. The cage labels on these individual cages showed only the study and animal number. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.
The following measurements, reflexes and responses were recorded:
Approach Response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna Reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

Auditory Startle Reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail Pinch Response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip Strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.
Motor Activity
During Week 5 of treatment for males and on Days 7 to 9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER: See related entry of the present OECD 422 in section “Toxicity to Reproduction”

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals after week five
- Maternal animals: All surviving animals on day 14 post partum
In Detail:
F0 males: After final investigations completed (after at least four weeks of treatment).
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females whose litter died before Day 13: On or after day the last offspring died.
F0 females: Day 14 of lactation (following terminal blood sampling).
F1 offspring: Selected offspring for thyroid hormone analysis - Day 4 of age; Scheduled kill - Day 13 of age.

GROSS NECROPSY
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. Examination included a detailed assessment and documentation of any color changes in the internal organs, adipose tissue or skin. If color changes were observed, representative photographs were taken before retained tissues were placed in fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the table in the free text field were prepared for microscopic examination and weighed, respectively.

Other examinations:

See "Any other information on materials and methods incl. tables.

Statistics:

Due to limitations of this free-text field, see "Any other information on materials and methods incl. tables.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Administration of the test item at doses of 100, 330 and 1000 mg/kg/day during the 5 week treatment period for males, and during the 2-week pre-pairing period, gestation and lactation periods for females was well tolerated.
Group 4 female (No. 97) receiving 1000 mg/kg/day was killed for welfare reasons after dosing on Day 3 of treatment: the female was clinically normal before dosing but showed signs of decreased activity, was cold to touch, had thin build, piloerection, partially closed eyelids and whole body pallor after dosing. Macroscopic examination did not reveal any cause for the poor condition of this animal and there was no sign of mis-dosing. With the exclusion of the mortality, there were no test item-related signs observed following dose administration or signs at routine clinical examination that were considered to be associated with treatment.
On Day 1 of lactation Group 3 female (No. 67) receiving 330 mg/kg/day was terminated early due to total litter loss. Macropathology findings included abnormal pale coloration of the mammary gland and it was noted the gland was inactive.

Mortality:

mortality observed, treatment-related

Description (incidence):

Group 4 female (No. 97) receiving 1000 mg/kg/day was killed for welfare reasons after dosing on Day 3 of treatment: the female was clinically normal before dosing but showed signs of decreased activity, was cold to touch, had thin build, piloerection, partially closed eyelids and whole body pallor after dosing. Macroscopic examination did not reveal any cause for the poor condition of this animal and there was no sign of mis-dosing.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight performance of animals receiving the test item at doses up to and including 1000 mg/kg/day was considered to be unaffected by treatment throughout the 5 week dosing period for males and the 2-week pre-pairing period, gestation and lactation periods for females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption for males receiving the test item at 330 or 1000 mg/kg/day was marginally higher than Controls during the treatment period with males receiving 330 mg/kg/day affected predominantly in the latter part of the treatment period, only. The mean food intake of females at 1000 mg/kg/day was slightly higher during the second week of the pre-pairing treatment period, throughout gestation and lactation when compared to Controls.
Mean food consumption for males receiving 100 mg/kg/day and for females receiving 100 or 330 mg/kg/day were generally similar to that of the Controls and was therefore unaffected by the test item administration.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The haematological examinations at scheduled termination revealed, when compared with Controls, a dose-dependent reduction in activated partial thromboplastin time in females given 100, 330 or 1000 mg/kg/day, which attained statistical significance at all dose levels. Activated partial thromboplastin time for males receiving the test item, however, were generally similar to Control values.
All groups of treated males showed slightly lower than Control reticulocyte concentration, however there was no dose relationship apparent. Among females given 1000 mg/kg/day haemoglobin concentration was slightly lower than Control, with statistical significance attained; this difference was attributable to a single female (No. 83) with an atypical low haemoglobin concentration and no effect of treatment was inferred. In addition, mean cell haemoglobin concentration was slightly low for females at all dose levels with statistical significance being attained for those females receiving 330 or 1000 mg/kg/day, however there was no dose relationship apparent.
All other haematological differences from control observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The haematological examinations at scheduled termination revealed, when compared with Controls, a dose-dependent reduction in activated partial thromboplastin time in females given 100, 330 or 1000 mg/kg/day, which attained statistical significance at all dose levels. Activated partial thromboplastin time for males receiving the test item, however, were generally similar to Control values.
All groups of treated males showed slightly lower than Control reticulocyte concentration, however there was no dose relationship apparent. Among females given 1000 mg/kg/day haemoglobin concentration was slightly lower than Control, with statistical significance attained; this difference was attributable to a single female (No. 83) with an atypical low haemoglobin concentration and no effect of treatment was inferred. In addition, mean cell haemoglobin concentration was slightly low for females at all dose levels with statistical significance being attained for those females receiving 330 or 1000 mg/kg/day, however there was no dose relationship apparent.
All other haematological differences from control observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The sensory reactivity observations conducted during Week 5 of treatment for males and Days 7 to 9 of lactation for females revealed no findings which were considered treatment related.

Motor activity assessment of males during Week 5 of treatment revealed a statistically significant higher incidence of overall high beam scores for animals receiving 1000 mg/kg/day when compared to Controls. Males receiving 100 or 330 mg/kg/day had also shown a slightly higher incidence of overall high beam scores when compared to Controls however, did not attain statistical significance. In addition, males receiving 100, 330 or 1000 mg/kg/day when compared to Controls, have shown a higher overall incidence of low beam scores. Overall low beam scores were statistically significantly higher for males receiving 100 or 1000 mg/kg/day. The increase in motor activity observed in males receiving the test item was considered unrelated to treatment as both overall high and low beam scores lacked a dose-dependent trend and Control performance is lower than in the Historical Control data.
Motor activity assessment of females at Days 7 to 9 of lactation has revealed a slight dose dependent increase in the overall scores for both high and low beam break activity but differences did not attain statistical significance. The higher incidence of high beam scores attained statistical significance for females receiving 1000 mg/kg/day at the 36 minute interval whereby the incidence of low beam scores attained statistical significance at both the 30 and 36 minute intervals. The increase in motor activity observed in the females was not associated with any clinical signs or signs recorded during the arena observation and therefore these differences were considered fortuitous in nature and were unrelated to treatment with the test item.

Immunological findings:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis: There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age. The slightly lower mean T4 concentration in adult males in the 1000 mg/kg/day group is within the historical control data range so no effect of treatment is inferred.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Following five weeks of treatment, males receiving 1000 mg/kg/day showed a body weight adjusted slight increase in mean liver weights when compared to Control (increased by 1.11X Control).
In addition, body weight adjusted and absolute thymus weights were decreased for all groups of males receiving the test item although this difference was considered to be attributable to two high individual weights in the Control group and therefore no effect of treatment was inferred.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals Killed After 5 Weeks of Treatment
The macroscopic examination performed after 5 weeks of treatment revealed the following changes in the mesenteric lymph nodes.
Mesenteric Lymph Nodes
Dark mesenteric lymph nodes were observed in one male given 100 mg/kg/day, one male given 330 mg/kg/day and in four males given 1000 mg/kg/day.
Summary of findings in the mesenteric lymph nodes for animals killed after 5 weeks of treatment
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 330 1000 0 100 330 1000
Abnormal color, Dark 0 1 1 4 0 0 0 0
Number of tissues examined 10 10 10 10 10 10 10 9
The incidence and distribution of all other findings were considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology
Decedents
Female No. 97 (given 1000 mg/kg/day) was killed for welfare reasons on Day 3 of treatment due to poor clinical condition. At macroscopic examination, the adrenals were dark and the duodenum, jejunum and stomach were distended with green and greasy fluid. Dark colour of the adrenals correlated microscopically with sinusoidal dilatation/congestion. There was also slight hypertrophy of the zona fasciculata of the adrenal cortex. The stomach showed diffuse inflammation of the submucosa/mucosa and erosion of the mucosa of the glandular and non-glandular regions. Apoptosis was present in the cortex of the thymus and mesenteric lymph node.

Microscopic Pathology
Animals Killed After 5 Weeks of Treatment
Treatment Related Findings
Changes related to treatment with the test item were seen in the mesenteric lymph nodes and liver.
Mesenteric Lymph Nodes
Erythryocytosis/erythrophagocytosis (minimal or slight severity) was present in the sinuses of the mesenteric lymph nodes of several males given 330 or 1000 mg/kg/day, one female given 1000 mg/kg/day and one control male. Increased cellularity of the follicles (minimal severity) was present in the mesenteric lymph nodes of all males given 1000 mg/kg/day, two males given 100 mg/kg/day and one control male.

Summary of treatment related findings in the mesenteric lymph nodes for animals killed after 5 weeks of treatment
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 330 1000 0 100 330 1000
Erythrocytosis/Erythrophagocytosis
Minimal 1 0 3 0 0 0 0 1
Slight 0 0 0 4 0 0 0 0
Total 1 0 3 4 0 0 0 1
Cellularity Increased, Follicles
Minimal 1 2 0 5 0 0 0 0
Total 1 2 0 5 0 0 0 0
Number of tissues examined 5 5 5 5 5 5 5 5

Liver
Centrilobular hypertrophy was present as a diffuse change in the liver of most males given 100 mg/kg/day and all males given 330 or 1000 mg/kg/day.
Summary of treatment related findings in the liver for animals killed after 5 weeks of treatment
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 330 1000 0 100 330 1000
Hypertrophy, Centrilobular
Minimal 0 3 5 1 0 0 0 0
Slight 0 0 0 4 0 0 0 0
Total 0 3 5 5 0 0 0 0
Number of tissues examined 5 5 5 5 5 5 5 5

Incidental Findings
Mineralisation and tubular degeneration were present in the tubular epithelium of predominantly proximal convoluted tubules in several females given 1000 mg/kg/day and in controls. The distribution of mineralisation in control and treated females indicates alteration in calcium/phosphorus homeostasis. However, the incidence and severity were similar in control and treated females so the findings are considered incidental and unrelated to treatment.
All other findings were considered incidental and not related to administration of the test item.

Histopathological findings: neoplastic:

not examined

Details on results:

Formulation Analysis
The homogeneity and stability of the test item in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 10 mg/mL and 200 mg/mL.
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 days and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 5% of the initial time zero value and the coefficient of variation was less than 5%.
Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method.
The mean concentrations of the test item in test formulations from the original Week 1 samples were extracted but due to repeated system issues, no results can be reported. The contingency samples were analyzed in replacement of the original samples. These results are all within 4% of nominal concentration, confirming accurate formulation. The coefficient of variation values are within 2%, confirming precise analysis.
In Day 10-12 of lactation (females), the Group 2 and 4 results were within 5% of nominal concentration, and the coefficient of variation values were within 3%, confirming precise analysis. The Group 3 samples were -36.4% of nominal concentration with a coefficient of variation of 58.11%. Excluding the low ‘bottom’ result as an outlier due to the very low result, the Group 3 samples were -15.8% of nominal concentration with a difference from mean of ±12.56%. The Group 3 contingency samples were analyzed outside of the confirmed stability period, and therefore can only be reported for information purposes only. These contingency results were -8.5% from nominal concentration with a coefficient of variation of 5.59%.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

immunology

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

other: reproductive performance

Remarks on result:

other: no adverse effects at the highest dose tested

Key result

Critical effects observed:

no

Conclusions:

The study was performed under GLP according to OECD TG 422 without deviations. Hence, the results can be considered sufficiently reliable to assess the potential repeated dose toxicity (and reproductive toxicity) of the test item, especially taking into account the prolonged exposure duration (minimum 5 weeks) compared to a regular subacute 28-day study and the fact that pregnant animals are in general more susceptible for toxic effects.
Oral administration of the test item to parental Sprague Dawley (Crl:CD(SD) rats at dose levels of 100, 330 or 1000 mg/kg/day for two weeks prior to pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 13 of lactation was well tolerated.
There were two premature deaths, one female receiving 1000 mg/kg/day was killed for welfare reasons on Day 3 of treatment after dose administration and one female given 330 mg/kg/day was terminated early as result of a total litter loss. Microscopic changes observed for the female killed for welfare reasons included distension of the gastrointestinal tract and inflammation/erosion in the glandular and non-glandular regions of the stomach. The cause of these lesions could not be determined from pathological examination, however there was no clear evidence in those animals surviving to terminal kill of local gastrointestinal toxicity and therefore it was considered that both deaths were unrelated to treatment.
Test item related histopathological changes were observed in the liver which consisted of diffuse centrilobular hypertrophy in most males given 100 mg/kg/day and all males given 330 or 1000 mg/kg/day. This correlated with a statistically significant increase in body weight adjusted group mean liver weights in males given 1000 mg/kg/day animals compared to controls. Centrilobular hypertrophy commonly reflects increased endoplasmic reticulum as a consequence of the induction of P450-related enzymes, but the precise mechanism of hypertrophy cannot be determined by histopathological examination alone (Sahota et al, 2013). Biochemical examination of blood plasma after 5 weeks of treatment also identified changes in the liver enzymes for males receiving 1000 mg/kg/day. The histopathological changes identified in the liver were of minimal to slight severity and were not accompanied by other significant test item-related changes in the liver and therefore are likely to be adaptive and non-adverse.
Test item-related microscopic changes were observed in the mesenteric lymph nodes of males given 1000 mg/kg/day, comprised of erythrocytosis/erythrophagocytosis and an increase in the cellularity of the follicles. Erythrocytosis/erythrophagocytosis was present in most males given 1000 mg/kg/day and correlated macroscopically with dark colouration of the lymph nodes. This finding can be observed in lymph nodes draining a region of haemorrhage (Elmore, S.A., 2006). In this study, haemorrhage was not observed in the gastrointestinal tract of animals exhibiting this change in the mesenteric lymph nodes. It is considered to be test item-related in males, however the cause of the lesions is uncertain. An increase in cellularity of the follicles of the mesenteric lymph nodes was observed in all males given 1000 mg/kg/day. It was also observed in two males given 100 mg/kg/day and in one control male, but not in males given 330 mg/kg/day, and was considered to be test item-related at 1000 mg/kg/day, only. An increase in cellularity of the follicles is typically observed as a response to immune stimulation. In a study such as this where the test item is delivered by oral gavage, immune stimulation can be observed in the mesenteric lymph nodes, which are the local lymph nodes draining the gastrointestinal tract and therefore these findings are considered non-adverse.
The cause for the dose-dependent reduction in activated partial thromboplastin time in plasma of females given 100, 330 or 1000 mg/kg/day was not established but this was considered a non-adverse finding due to the degree/severity of the change and the fact that clinical condition and survival were unaffected by treatment.
Mean food consumption recordings for males receiving 330 or 1000 mg/kg/day were marginally higher during the treatment period when compared to Control. Similarly, the mean food intake of females receiving 1000 mg/kg/day was slightly higher during the second week of the pre-pairing treatment period, throughout gestation and lactation when compared to Controls. Instances of increased food consumption values were in the absence of a dose dependent response and any test item-related effect upon body weight values.
The assessment of endocrine disruptor relevant endpoints comprised i.a the measurement of circulating thyroxine levels (T4) in adult males. As there were no findings in the endocrine disruptor endpoints and there were no changes observed in the male reproductive system, the test item is considered not to be an EDC.
Mineralisation and tubular degeneration were present in the tubular epithelium of predominantly proximal convoluted tubules in the kidneys of several females given 1000 mg/kg/day and in Controls. However, the incidence and severity were similar in Control and treated females, and there were considered to be no test item related differences in plasma urea and creatinine concentrations and no test item related microscopic changes were detected in the kidneys of males or females.
In conclusion, within the context of this combined repeated dose toxicity and reproductive/ developmental toxicity screening study it was concluded that a dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity and reproductive/developmental toxicity in the CD rat. The test item showed no evidence of being an endocrine disruptor. Further, no evidence for classification as STOT RE was given.

Executive summary:

The purpose of this OECD 422 study under GLP was to assess the general systemic toxic potential in Crl:CD(SD) rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item (a vulcanisation agent for industrial use) by oral gavage administration for at least five weeks.

Three groups of ten male and ten female rats received the test item at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration at a volume dose of 5 mL/kg/day. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 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, corn oil, at the same volume dose as treated groups.

During the study, for adult animals assessments of 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 and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Blood samples were collected from all adult animals at termination and from selected offspring on Day 4 and Day 13 of age for thyroid hormone analysis.

 

Results

Parental (F0) responses

Oral administration of the test item to parental Sprague Dawley (Crl:CD(SD)) rats at dose levels of 100, 330 or 1000 mg/kg/day for two weeks prior to pairing, during pairing and then up to termination of the males after 5 weeks of treatment and females on Day 14 of lactation was well tolerated. 

There were two premature deaths, one female receiving 1000 mg/kg/day was killed for welfare reasons after dosing on Day 3 of treatment and one female receiving 330 mg/kg/day was terminated early due to total litter loss on Day 1 of lactation. Macropathology findings included abnormal pale coloration of the mammary gland and it was noted the gland was inactive. Both premature deaths were considered unrelated to treatment.

With the exclusion of the mortality, there were no test item-related signs observed during the detailed physical examination and arena observations, no post-dosing observations, no effects on sensory reactivity, grip strength and no effects on body weight performance. There was considered to be no effect on motor activity.

Mean food consumption for males receiving the test item at 330 or 1000 mg/kg/day was marginally higher than Controls during the treatment period. The mean food intake of females at 1000 mg/kg/day was slightly higher during the second week of the pre-pairing treatment period, throughout gestation and lactation when compared to Controls.

Estrous cyclicity, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment with The test item. One female receiving 1000 mg/kg/day was found to be acyclic during treatment and one female receiving 330 mg/kg/day (3F No. 62) was found to be not pregnant.

The haematological examinations at scheduled termination revealed, when compared with Controls, a dose-dependent reduction in activated partial thromboplastin time in females at all dose levels. Activated partial thromboplastin time for males receiving the test item, however, were generally similar to Control values.

Blood chemistry investigations after five weeks of treatment revealed statistically significant changes in the liver enzymes for males receiving 1000 mg/kg/day where alkaline phosphatase was lower compared to Controls and both alanine aminotransferase and aspartate aminotransferase concentration were higher compared to Controls. A dose dependent response was observed for alanine aminotransferase and aspartate aminotransferase only. Liver enzyme changes were observed in females with slightly higher alanine aminotransferase (330 or 1000 mg/kg/day only) and aspartate aminotransferase concentrations compared to Controls. Unlike the males however, these values were in the absence of both a dose dependent response and statistical significance.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring.

Changes in organ weights consisted of slightly high body weight adjusted mean liver weights for males at 1000 mg/kg/day when compared to Control.  

Macroscopic examination of the adult males revealed abnormal dark colour of the mesenteric lymph node in four males that received 1000 mg/kg/day and observed in one male at dose levels of 100 and 330 mg/kg/day. 

Histopathological evaluation of retained tissues revealed erythryocytosis/erythrophagocytosis in the sinuses of the mesenteric lymph nodes of several males given 330 or 1000 mg/kg/day, one female given 1000 mg/kg/day and one control male. Increased cellularity of the follicles (minimal severity) was present in the mesenteric lymph nodes of all males given 1000 mg/kg/day, two males given 100 mg/kg/day and one control male. Centrilobular hypertrophy was present in the liver of most males given 100 mg/kg/day and all males given 330 or 1000 mg/kg/day.

 

F1 litter responses

The clinical condition, litter size, sex ratio, body weight and survival of the F1 offspring was unaffected by parental treatment and at scheduled termination, there were no findings associated with treatment. One female given 330 mg/kg/day had a total litter loss. Mean and adjusted mean values for ano-genital distance were marginally higher for males given 330 or 1000 mg/kg/day compared to the Controls. The mean ano-genital distance of female offspring was essentially similar to Controls in all treated groups.

 

Conclusion

In conclusion, within the context of this combined repeated dose toxicity and reproductive/ developmental toxicity screening study it was concluded that a dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity and reproductive/developmental toxicity in the CD rat. The test item showed no evidence of being an endocrine disruptor.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD 422 guideline for testing of chemicals adopted 29 July 2016: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide

EC Number:

605-708-9

Cas Number:

174125-93-0

Molecular formula:

n.a.

IUPAC Name:

bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide

Test material form:

liquid

Remarks:

orange

Specific details on test material used for the study:

URCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Refrigerated (2 to 8°C) and protected from the light.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

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 Crl:CD(SD) 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.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Males 69 to 76 days old
Females 83 to 90 days old
- Weight at study initiation:
Males 338 to 408 g
Females 239 to 298 g
- Fasting period before study:
no
- Housing:
Number of animals per cage:
Pre-pairing: up to five animals of one sex
Pairing: one male and one female
Males after mating: up to five animals
Gestation: one female
Lactation: one female + litter
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Environmental Enrichment:
A soft white untreated aspen wood block; provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced when necessary.
Plastic shelter was provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced at the same time as the cages.
- 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 prior to the commencement of treatment.
Females: 20 days prior to the commencement of treatment

DETAILS OF FOOD AND WATER QUALITY:
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

ENVIRONMENTAL CONDITIONS
Rodent Facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
- Temperature (°C): / Humidity (%):
Temperature and relative humidity were monitored and maintained within the range of 20-24ºC and 40-70%. There were no deviations from these ranges.
- 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: 16 August 2017 (males), 2 August 2017 (females)
To: 26 September 2017 (males), 11 to 15 October 2017 (females) (F0 necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2-8°C).

- VEHICLE: Corn oil
- Concentration in vehicle: 0, 20, 66, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Details on mating procedure:

Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis
Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. These investigations demonstrated that formulations in the 10 to 200 mg/mL concentration range are homogenous and stable following ambient storage (15-25°C) for one day and following refrigerated storage (2-8°C) for 15 days.
Achieved concentration: Samples of each formulation prepared for administration in Week1 of treatment and on Day 10-12 of lactation were analyzed for achieved concentration of the test item.

Homogeneity and Stability in Corn Oil Formulations
The homogeneity and stability of the test item in corn oil formulations were assessed at nominal concentrations of 10 mg/mL and 200 mg/mL, during ambient and refrigerated storage. Freshly prepared specimen formulations (400 mL) were equally sub divided into 4 amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Homogeneity and Stability of Dose Formulations
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 days and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 5% of the initial time zero value and the coefficient of variation was less than 5%. Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method.

Concentration of Dose Formulations
The mean concentrations of the test item in test formulations analyzed during the study. The original Week 1 samples were extracted but due to repeated system issues, no results can be reported. The contingency samples were analyzed in replacement of the original samples. These results are all within 4% of nominal concentration, confirming accurate formulation. The coefficient of variation values are within 2%, confirming precise analysis. In Day 10-12 of lactation (females), the Group 2 and 4 results were within 5% of nominal concentration, and the coefficient of variation values were within 3%, confirming precise analysis. The Group 3 samples were -36.4% of nominal concentration with a coefficient of variation of 58.11%. Excluding the low ‘bottom’ result as an outlier due to the very low result, the Group 3 samples were -15.8% of nominal concentration with a difference from mean of ±12.56%. The Group 3 contingency samples were analyzed outside of the confirmed stability period, and therefore can only be reported for information purposes only. These contingency results were -8.5% from nominal concentration with a coefficient of variation of 5.59%.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.
The homogeneity and stability was confirmed for The test item in corn oil formulations at nominal concentrations of 10 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days. The mean concentrations of The test item in test formulations analyzed for the study were within the applied limits of +10/-15% of nominal concentrations, confirming accurate formulation, with the exception of Day 10-12 of lactation Group 3 samples which were 15.8% of nominal concentration.

Duration of treatment / exposure:

Three groups of ten male and ten female rats received the test item at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration at a volume dose of 5 mL/kg/day. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 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, corn oil, at the same volume dose as treated groups.

Frequency of treatment:

Once daily, at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

330 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 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:
The dose levels selected for investigation in this OECD 422 combined repeated dose toxicity study and reproductive/developmental toxicity screening study (0, 100, 330 and 1000 mg/kg/day) were chosen in conjunction with the Sponsor and were based on the results of a 14-day preliminary study conducted at these laboratories.
In the preliminary study dose levels of 250, 500 and 1000 mg/kg/day were investigated. There were no test item-related premature deaths, no signs observed in relation to dose administration, no test item-related changes in clinical condition, body weight, water consumption or spleen weights. Group mean food intake was slightly reduced during the treatment period when compared with pre-treatment values for all dose groups. Mean absolute and body weight relative liver weights were slightly high for females receiving 1000 mg/kg/day and for males receiving 500 or 1000 mg/kg/day but one male at 250 mg/kg/day had a small liver. Kidney weights also exhibited a modest dose dependent reduction for body weight relative data in males. Macroscopic findings were limited to adhesions involving multiple organs of the thoracic cavity for two animals receiving 250 mg/kg/day which were considered to be related to mis-dosing. In addition, one animal receiving 1000 mg/kg/day was found to have cysts on the kidneys.
Therefore, a high dose level of 1000 mg/kg/day was selected for this study as the limit dose for the OECD 422 guideline. The intermediate and low dose levels of 330 and 100 mg/kg/day were chosen to achieve a dose response and/or aid in the determination of a No-Observed-Adverse-Effect-Level (NOAEL).
- Rationale for animal assignment (if not random):
Allocation: On arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced to ensure variations in body weight of animals did not exceed ± 20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation:
Irregular estrous cycle: Six females
Unexpected death: One female

Positive control:

not required

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality: A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
Clinical and Behavioral Observations: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. 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.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
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 onwards - once each week
F0 females: Week 1 – daily; Week 2 – once, 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; As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see above

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

FOOD CONSUMPTION
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows for F0 animals: Weekly before pairing, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Days 15 to 22), but recommenced for males in Week 4.
For females after mating food consumption was recorded as follows: Days 0-6, 7-13 and 14-19 after mating; Days 1-3, 4-6 and 7-12 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: not stated

OTHER: See related entry of the present OECD 422 in section “Repeated Dose Toxicity”

Oestrous cyclicity (parental animals):

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 four days before scheduled termination (nominally Days 11 to 14 of lactation).

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight
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: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

Parturition Observations and Gestation Length
Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Records Made During Littering Phase
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 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Thyroid Hormone Analysis: Blood samples were collected as follows:
Day 4 of age: Offspring: two females selected for sampling per litter (if possible):
If four female offspring available - one female was selected for T4 (serum)#
If five or more female offspring available - one female was selected for T4 (serum) and one female for TSH (plasma)
No females were selected for sampling if:
The resultant live litter size would fall below ten pups.
The resultant number of live females would fall to less than three (for subsequent allocation to the Day 13 procedures) e.g. three female offspring in the litter - no offspring selected
Day 13 of age: Offspring: two males and two females per litter (where possible):
two for T4 (serum); where possible one male and one female (Priority was given to serum sample)
two for TSH (plasma); where possible one male and one female

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after week five
- Maternal animals: All surviving animals on day 14 post partum
In Detail:
F0 males: After final investigations completed (after at least four weeks of treatment).
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females whose litter died before Day 13: On or after day the last offspring died.
F0 females: Day 14 of lactation (following terminal blood sampling).
F1 offspring: Selected offspring for thyroid hormone analysis - Day 4 of age; Scheduled kill - Day 13 of age.

GROSS NECROPSY
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. Examination included a detailed assessment and documentation of any color changes in the internal organs, adipose tissue or skin. If color changes were observed, representative photographs were taken before retained tissues were placed in fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the table in the free text field were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
Selected offspring for thyroid hormone analysis - Day 4 of age. / Scheduled kill - Day 13 of age.
Method of kill:
Offspring - selected for thyroid hormone sampling on Day 4 and 13 of age: Decapitation.
Offspring - all other: Intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content and particular attention paid to external genitalia was performed. Abnormal tissues were retained.
F1 offspring on Day 4 of age: Blood sampling required.
Externally normal offspring discarded without examination.
Externally abnormal offspring examined, and retained pending possible future examination.
F1 offspring on Day 13 of age: Blood sampling required.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination.
Thyroid glands were preserved from two offspring per litter, one male and one female in each litter, where possible.

Statistics:

Due to limitations of this free-text field, see "Any other information on materials and methods incl. tables.

Reproductive indices:

Estrous Cycle
The percentage females showing the following classifications of estrous cycles before treatment commenced and during treatment are presented:
Regular: All observed cycles of 4 or 5 days (divided into cycles of 4, 4 and 5 and 5 days)
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus

Vaginal smearing prior to termination is presented in terms of numbers of females that showed estrus during this period and the cycle stage at termination

Pre-Coital Interval
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.

Mating Performance and Fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = (Number of animals mating / Animals paired) * 100
Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) * 100
Fertility index (%) = (Number of animals achieving pregnancy / Animals paired) * 100

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = (Number of live litters born / Number pregnant) * 100

Offspring viability indices:

Litter Size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 (before and after blood sampling) and 13 of age. Group mean litter size and SD were calculated from the individual litter values.

Survival Indices
The following were calculated for each litter:
Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) * 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) * 100
Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) * 100
Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) * 100
Group mean values were calculated from individual litter values.

Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after blood sampling) and 13 of age.
Percentage males = (Number of males in litter / Total number of offspring in litter) * 100
Group mean values were calculated from individual litter values.

Offspring Examinations
Ano-genital distance were presented both as absolute/unadjusted and adjusted for body weight, using the weight recorded on Day 1 of age.
A check was performed to assess for the presence or absence of nipple/areolae for the male offspring.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Administration of the test item at doses of 100, 330 and 1000 mg/kg/day during the 5 week treatment period for males, and during the 2-week pre-pairing period, gestation and lactation periods for females was well tolerated.
Group 4 female (No. 97) receiving 1000 mg/kg/day was killed for welfare reasons after dosing on Day 3 of treatment: the female was clinically normal before dosing but showed signs of decreased activity, was cold to touch, had thin build, piloerection, partially closed eyelids and whole body pallor after dosing. Macroscopic examination did not reveal any cause for the poor condition of this animal and there was no sign of mis-dosing. With the exclusion of the mortality, there were no test item-related signs observed following dose administration or signs at routine clinical examination that were considered to be associated with treatment.
On Day 1 of lactation Group 3 female (No. 67) receiving 330 mg/kg/day was terminated early due to total litter loss. Macropathology findings included abnormal pale coloration of the mammary gland and it was noted the gland was inactive.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Group 4 female (No. 97) receiving 1000 mg/kg/day was killed for welfare reasons after dosing on Day 3 of treatment: the female was clinically normal before dosing but showed signs of decreased activity, was cold to touch, had thin build, piloerection, partially closed eyelids and whole body pallor after dosing. Macroscopic examination did not reveal any cause for the poor condition of this animal and there was no sign of mis-dosing

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight performance of animals receiving the test item at doses up to and including 1000 mg/kg/day was considered to be unaffected by treatment throughout the 5 week dosing period for males and the 2-week pre-pairing period, gestation and lactation periods for females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption for males receiving the test item at 330 or 1000 mg/kg/day was marginally higher than Controls during the treatment period with males receiving 330 mg/kg/day affected predominantly in the latter part of the treatment period, only. The mean food intake of females at 1000 mg/kg/day was slightly higher during the second week of the pre-pairing treatment period, throughout gestation and lactation when compared to Controls.
Mean food consumption for males receiving 100 mg/kg/day and for females receiving 100 or 330 mg/kg/day were generally similar to that of the Controls and was therefore unaffected by the test item administration.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The haematological examinations at scheduled termination revealed, when compared with Controls, a dose-dependent reduction in activated partial thromboplastin time in females given 100, 330 or 1000 mg/kg/day, which attained statistical significance at all dose levels. Activated partial thromboplastin time for males receiving the test item, however, were generally similar to Control values.
All groups of treated males showed slightly lower than Control reticulocyte concentration, however there was no dose relationship apparent. Among females given 1000 mg/kg/day haemoglobin concentration was slightly lower than Control, with statistical significance attained; this difference was attributable to a single female (No. 83) with an atypical low haemoglobin concentration and no effect of treatment was inferred. In addition, mean cell haemoglobin concentration was slightly low for females at all dose levels with statistical significance being attained for those females receiving 330 or 1000 mg/kg/day, however there was no dose relationship apparent.
All other haematological differences from control observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The haematological examinations at scheduled termination revealed, when compared with Controls, a dose-dependent reduction in activated partial thromboplastin time in females given 100, 330 or 1000 mg/kg/day, which attained statistical significance at all dose levels. Activated partial thromboplastin time for males receiving the test item, however, were generally similar to Control values.
All groups of treated males showed slightly lower than Control reticulocyte concentration, however there was no dose relationship apparent. Among females given 1000 mg/kg/day haemoglobin concentration was slightly lower than Control, with statistical significance attained; this difference was attributable to a single female (No. 83) with an atypical low haemoglobin concentration and no effect of treatment was inferred. In addition, mean cell haemoglobin concentration was slightly low for females at all dose levels with statistical significance being attained for those females receiving 330 or 1000 mg/kg/day, however there was no dose relationship apparent.
All other haematological differences from control observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The sensory reactivity observations conducted during Week 5 of treatment for males and Days 7 to 9 of lactation for females revealed no findings which were considered treatment related.

Motor activity assessment of males during Week 5 of treatment revealed a statistically significant higher incidence of overall high beam scores for animals receiving 1000 mg/kg/day when compared to Controls. Males receiving 100 or 330 mg/kg/day had also shown a slightly higher incidence of overall high beam scores when compared to Controls however, did not attain statistical significance. In addition, males receiving 100, 330 or 1000 mg/kg/day when compared to Controls, have shown a higher overall incidence of low beam scores. Overall low beam scores were statistically significantly higher for males receiving 100 or 1000 mg/kg/day. The increase in motor activity observed in males receiving the test item was considered unrelated to treatment as both overall high and low beam scores lacked a dose-dependent trend and Control performance is lower than in the Historical Control data.
Motor activity assessment of females at Days 7 to 9 of lactation has revealed a slight dose dependent increase in the overall scores for both high and low beam break activity but differences did not attain statistical significance. The higher incidence of high beam scores attained statistical significance for females receiving 1000 mg/kg/day at the 36 minute interval whereby the incidence of low beam scores attained statistical significance at both the 30 and 36 minute intervals. The increase in motor activity observed in the females was not associated with any clinical signs or signs recorded during the arena observation and therefore these differences were considered fortuitous in nature and were unrelated to treatment with the test item.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology
Decedents
Female No. 97 (given 1000 mg/kg/day) was killed for welfare reasons on Day 3 of treatment due to poor clinical condition. At macroscopic examination, the adrenals were dark and the duodenum, jejunum and stomach were distended with green and greasy fluid. Dark colour of the adrenals correlated microscopically with sinusoidal dilatation/congestion. There was also slight hypertrophy of the zona fasciculata of the adrenal cortex. The stomach showed diffuse inflammation of the submucosa/mucosa and erosion of the mucosa of the glandular and non-glandular regions. Apoptosis was present in the cortex of the thymus and mesenteric lymph node.

Microscopic Pathology
Animals Killed After 5 Weeks of Treatment
Treatment Related Findings
Changes related to treatment with the test item were seen in the mesenteric lymph nodes and liver.
Mesenteric Lymph Nodes
Erythryocytosis/erythrophagocytosis (minimal or slight severity) was present in the sinuses of the mesenteric lymph nodes of several males given 330 or 1000 mg/kg/day, one female given 1000 mg/kg/day and one control male. Increased cellularity of the follicles (minimal severity) was present in the mesenteric lymph nodes of all males given 1000 mg/kg/day, two males given 100 mg/kg/day and one control male.

Summary of treatment related findings in the mesenteric lymph nodes for animals killed after 5 weeks of treatment
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 330 1000 0 100 330 1000
Erythrocytosis/Erythrophagocytosis
Minimal 1 0 3 0 0 0 0 1
Slight 0 0 0 4 0 0 0 0
Total 1 0 3 4 0 0 0 1
Cellularity Increased, Follicles
Minimal 1 2 0 5 0 0 0 0
Total 1 2 0 5 0 0 0 0
Number of tissues examined 5 5 5 5 5 5 5 5

Liver
Centrilobular hypertrophy was present as a diffuse change in the liver of most males given 100 mg/kg/day and all males given 330 or 1000 mg/kg/day.
Summary of treatment related findings in the liver for animals killed after 5 weeks of treatment
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 330 1000 0 100 330 1000
Hypertrophy, Centrilobular
Minimal 0 3 5 1 0 0 0 0
Slight 0 0 0 4 0 0 0 0
Total 0 3 5 5 0 0 0 0
Number of tissues examined 5 5 5 5 5 5 5 5

Incidental Findings
Mineralisation and tubular degeneration were present in the tubular epithelium of predominantly proximal convoluted tubules in several females given 1000 mg/kg/day and in controls. The distribution of mineralisation in control and treated females indicates alteration in calcium/phosphorus homeostasis. However, the incidence and severity were similar in control and treated females so the findings are considered incidental and unrelated to treatment.
All other findings were considered incidental and not related to administration of the test item.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to the study showed normal 4/5 day estrous cycles during the acclimatization period.
Estrous cyclicity, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment with the test item. One female receiving 1000 mg/kg/day was found to be acyclic during treatment.
All females were not cycling before termination (Days 11 to 14 of lactation) and were in diestrous at termination.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Estrous cyclicity, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment with the test item. One female receiving 1000 mg/kg/day was found to be acyclic during treatment and one female receiving 330 mg/kg/day (3F No. 62) was found to be not pregnant.

Details on results (P0)

Formulation Analysis
The homogeneity and stability of the test item in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 10 mg/mL and 200 mg/mL.
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 days and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 5% of the initial time zero value and the coefficient of variation was less than 5%.
Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method.
The mean concentrations of the test item in test formulations from the original Week 1 samples were extracted but due to repeated system issues, no results can be reported. The contingency samples were analyzed in replacement of the original samples. These results are all within 4% of nominal concentration, confirming accurate formulation. The coefficient of variation values are within 2%, confirming precise analysis.
In Day 10-12 of lactation (females), the Group 2 and 4 results were within 5% of nominal concentration, and the coefficient of variation values were within 3%, confirming precise analysis. The Group 3 samples were -36.4% of nominal concentration with a coefficient of variation of 58.11%. Excluding the low ‘bottom’ result as an outlier due to the very low result, the Group 3 samples were -15.8% of nominal concentration with a difference from mean of ±12.56%. The Group 3 contingency samples were analyzed outside of the confirmed stability period, and therefore can only be reported for information purposes only. These contingency results were -8.5% from nominal concentration with a coefficient of variation of 5.59%.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

haematology

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: no adverse effects at the highest dose tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed among the offspring which were considered to be related to parental treatment with the test item.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

One Group 3 female (No. 67) receiving 330 mg/kg/day was terminated early on Day 1 of lactation due to a total litter loss.
The mean number of implantation sites and litter size were unaffected by parental treatment at all dose levels, with all litter size values within range of the Historical Control Data. The mean number of implantations for Controls is at the higher range of the Historical Control Data.
The post implantation survival index was unaffected by parental treatment with the test item.
Offspring survival to Day 13 of age and sex ratio appear unaffected by parental treatment up to and including 1000 mg/kg/day.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 13 of age the mean body weights of male and female offspring receiving 1000 mg/kg/day were slightly lower than that of the Control. As this difference was slight, did not attain statistical significance and there was no apparent dose response, it was considered that there was no effect on mean offspring body weights of parental treatment with the test item.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

Offspring Ano-genital Distance: For males receiving 330 or 1000 mg/kg/day, the mean and adjusted mean values for ano genital distance were slightly higher than that of Controls. This slight difference was minimal and as there were no effects observed on the male reproductive system, it is considered this finding was not adverse. The mean ano-genital distance of female offspring was essentially similar to Controls in all treated groups.
Offspring Nipple Count: A check was performed to assess for the presence of nipple/areolae for the male offspring.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Offspring Macropathology: There were no findings in the decedent offspring, or offspring at termination on Day 13 of age that were considered to be related to parental treatment.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

See above
Offspring Ano-genital Distance: For males receiving 330 or 1000 mg/kg/day, the mean and adjusted mean values for ano genital distance were slightly higher than that of Controls. This slight difference was minimal and as there were no effects observed on the male reproductive system, it is considered this finding was not adverse. The mean ano-genital distance of female offspring was essentially similar to Controls in all treated groups.
Offspring Nipple Count: A check was performed to assess for the presence of nipple/areolae for the male offspring.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: no adverse effects at the highest dose tested

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The study was performed under GLP according to OECD TG 422 without deviations. Hence, the results can be considered sufficiently reliable to assess the potential reproductive toxicity (and repeated dose toxicity) of the test item.
Oral administration of the test item to parental Sprague Dawley (Crl:CD(SD) rats at dose levels of 100, 330 or 1000 mg/kg/day for two weeks prior to pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 13 of lactation was well tolerated.
There were two premature deaths, one female receiving 1000 mg/kg/day was killed for welfare reasons on Day 3 of treatment after dose administration and one female given 330 mg/kg/day was terminated early as result of a total litter loss. Microscopic changes observed for the female killed for welfare reasons included distension of the gastrointestinal tract and inflammation/erosion in the glandular and non-glandular regions of the stomach. The cause of these lesions could not be determined from pathological examination, however there was no clear evidence in those animals surviving to terminal kill of local gastrointestinal toxicity and therefore it was considered that both deaths were unrelated to treatment.
Test item related histopathological changes are likely to be adaptive and non-adverse.
Test item-related microscopic changes were observed in the mesenteric lymph nodes of males given 1000 mg/kg/day,and are considered non-adverse.
Estrous cycle length, mating performance, fertility, reproductive capacity and gestation length were unaffected by treatment with The test item at all dose levels investigated, only one female receiving 330 mg/kg/day was found not to be pregnant.
The assessment of endocrine disruptor relevant endpoints comprised the measurement of circulating thyroxine levels (T4) in adult males and in Day 13 offspring, measurement of the ano-genital distance of offspring on Day 1 of age and nipple counts for male offspring on Day 13 of age. Body weight adjusted mean values for ano-genital distance were slightly higher for males given 330 or 1000 mg/kg/day than that of Controls. As there were no findings in the other endocrine disruptor endpoints and there were no changes observed in the male reproductive system it is considered this change is not treatment-related.
There was no effect of parental treatment on the mean number of uterine implantations, litter size, sex ratio, offspring birth weights and subsequent body weight gain or on offspring development up to Day 13 of age.
All Control females on study showed regular oestrus cycles during treatment, mated and became pregnant with a live litter. The litter size and offspring survival indices in the Control group were not adversely affected by the mineralisation with mean values within or marginally above those in the Historical Control Data range. All of these observations support the power of the study to detect test item related effects.
In conclusion, within the context of this combined repeated dose toxicity and reproductive/ developmental toxicity screening study it was concluded that a dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity and reproductive/developmental toxicity in the CD rat. The test item showed no evidence of being an endocrine disruptor. No necessity for classification as reproductive toxicant is given.

Executive summary:

The purpose of this OECD 422 study under GLP was to assess the general systemic toxic potential in Crl:CD(SD) rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item (a vulcanisation agent for industrial use) by oral gavage administration for at least five weeks.

Three groups of ten male and ten female rats received the test item at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration at a volume dose of 5 mL/kg/day. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 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, corn oil, at the same volume dose as treated groups.

During the study, for adult animals assessments of 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 and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Blood samples were collected from all adult animals at termination and from selected offspring on Day 4 and Day 13 of age for thyroid hormone analysis.

 

Results

Parental (F0) responses

Oral administration of the test item to parental Sprague Dawley (Crl:CD(SD)) rats at dose levels of 100, 330 or 1000 mg/kg/day for two weeks prior to pairing, during pairing and then up to termination of the males after 5 weeks of treatment and females on Day 14 of lactation was well tolerated. 

There were two premature deaths, one female receiving 1000 mg/kg/day was killed for welfare reasons after dosing on Day 3 of treatment and one female receiving 330 mg/kg/day was terminated early due to total litter loss on Day 1 of lactation. Macropathology findings included abnormal pale coloration of the mammary gland and it was noted the gland was inactive. Both premature deaths were considered unrelated to treatment.

With the exclusion of the mortality, there were no test item-related signs observed during the detailed physical examination and arena observations, no post-dosing observations, no effects on sensory reactivity, grip strength and no effects on body weight performance. There was considered to be no effect on motor activity.

Mean food consumption for males receiving the test item at 330 or 1000 mg/kg/day was marginally higher than Controls during the treatment period. The mean food intake of females at 1000 mg/kg/day was slightly higher during the second week of the pre-pairing treatment period, throughout gestation and lactation when compared to Controls.

Estrous cyclicity, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment with The test item. One female receiving 1000 mg/kg/day was found to be acyclic during treatment and one female receiving 330 mg/kg/day (3F No. 62) was found to be not pregnant.

The haematological examinations at scheduled termination revealed, when compared with Controls, a dose-dependent reduction in activated partial thromboplastin time in females at all dose levels. Activated partial thromboplastin time for males receiving the test item, however, were generally similar to Control values.

Blood chemistry investigations after five weeks of treatment revealed statistically significant changes in the liver enzymes for males receiving 1000 mg/kg/day where alkaline phosphatase was lower compared to Controls and both alanine aminotransferase and aspartate aminotransferase concentration were higher compared to Controls. A dose dependent response was observed for alanine aminotransferase and aspartate aminotransferase only. Liver enzyme changes were observed in females with slightly higher alanine aminotransferase (330 or 1000 mg/kg/day only) and aspartate aminotransferase concentrations compared to Controls. Unlike the males however, these values were in the absence of both a dose dependent response and statistical significance.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring.

Changes in organ weights consisted of slightly high body weight adjusted mean liver weights for males at 1000 mg/kg/day when compared to Control.  

Macroscopic examination of the adult males revealed abnormal dark colour of the mesenteric lymph node in four males that received 1000 mg/kg/day and observed in one male at dose levels of 100 and 330 mg/kg/day. 

Histopathological evaluation of retained tissues revealed erythryocytosis/erythrophagocytosis in the sinuses of the mesenteric lymph nodes of several males given 330 or 1000 mg/kg/day, one female given 1000 mg/kg/day and one control male. Increased cellularity of the follicles (minimal severity) was present in the mesenteric lymph nodes of all males given 1000 mg/kg/day, two males given 100 mg/kg/day and one control male. Centrilobular hypertrophy was present in the liver of most males given 100 mg/kg/day and all males given 330 or 1000 mg/kg/day.

 

F1 litter responses

The clinical condition, litter size, sex ratio, body weight and survival of the F1 offspring was unaffected by parental treatment and at scheduled termination, there were no findings associated with treatment. One female given 330 mg/kg/day had a total litter loss. Mean and adjusted mean values for ano-genital distance were marginally higher for males given 330 or 1000 mg/kg/day compared to the Controls. The mean ano-genital distance of female offspring was essentially similar to Controls in all treated groups.

 

Conclusion

In conclusion, within the context of this combined repeated dose toxicity and reproductive/ developmental toxicity screening study it was concluded that a dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity and reproductive/developmental toxicity in the CD rat. The test item showed no evidence of being an endocrine disruptor.