Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
OS3600 undergoes rapid hydrolysis in aqueous to MPKO and the corresponding silanol. Silanols undergo continuous condensation reactions to produce higher molecular weight siloxanes which are considered biologically unavailable. Therefore, the observed toxicity is likely due to MPKO and their values are comparable. The study has been conducted according to OECD guidance under GLPs.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 71 days
- Weight at study initiation: 337 g to 394 g (males), 220 g to 271 g (females)
- Fasting period before study: none
- Housing: Animals were housed inside a barriered rodent facility. The gridded cages used during pairing were suspended over trays covered with absorbent paper which was changed daily. For cages with solid floors, wood based material was used as bedding and was sterilised by autoclaving and changed at least twice each week. Cages, cage-trays, food hoppers and water bottles were changed at appropriate intervals. The cages were distributed on the racking to equalise, as far as possible, environmental influences amongst the groups.
- Diet (e.g. ad libitum): ad libitum (SDS VRF1 Certified Diet) except overnight before routine blood sampling. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): ad libitum from the public supply via polycarbonate or polypropylene bottles fitted with sipper tubes.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40 to 70
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: To: 21 March 2012 to 23 May 2012
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance, MPKO, was prepared for administration as a series of graded concentrations in the vehicle, by dilution of individual weightings of the test substance. Small amounts of vehicle were added to the test substance and mixed until a solution was formed. This was made up to the required volume with vehicle and then magnetically stirred until homogenous. The test substance was used as supplied. All formulations were prepared weekly and stored refrigerated before use.


VEHICLE
- Concentration in vehicle: 7.5, 25, 75 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to two weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually in solid-bottom polycarbonate cages with bedding.
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the liquid matrix. Specimen formulations (typically 400 mL) were prepared at concentrations of 1 mg/mL and 100 mg/mL and equally split between four amber screw-capped bottles. Prior to initial sampling on each day, the formulation was mixed by 20-fold inversion. A control vehicle sample was stored with each batch of stability samples. The stability was confirmed for at least 24 hours at ambient temperature and for up to 15 days when refrigerated (2-8¿C).
Samples of each formulation prepared for administration on the first and last occasion of treatment were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups. Two of the samples from each group were analysed. The remainder were retained as contingency for analysis if any result required confirmation.
Duration of treatment / exposure:
The test substance, MPKO, was administered for two weeks before pairing up to necropsy (at least five weeks) for males and two weeks before pairing then throughout pairing and gestation until Day 6 of lactation for females. Recovery animals were treated for approximately six weeks and completed a further 14 days without treatment.

Animals of the F1 generation were not dosed.
Frequency of treatment:
All animals were dosed once each day at approximately the same time each day, seven days per week.
Remarks:
Doses / Concentrations:
15, 50, 150 mg/kg bw/day
Basis:
other: actual administered
No. of animals per sex per dose:
10 males per group (Groups 1 to 4)
15 females (Groups 1 and 4), of these 5 females per group were not mated and were used for the recovery group
10 females (Groups 2 and 3)
Control animals:
yes, concurrent vehicle
Details on study design:
Ten males and 10 females per group were treated for two weeks at dose levels of 15, 50 or 150 mg/kg/day before pairing. Treatment continued to a total of at least 5 weeks. A control group of 10 male and 10 female rats received the vehicle, corn oil, at the same volume-dose throughout the same period. Males were killed after at least 5 weeks of treatment and females were killed on Day 7 of lactation.
Recovery, over 14 days without treatment, was assessed in five of the control and five of the high dose males and in an extra five unmated females in the same groups which were treated for 6 weeks before start of recovery.
The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk.
During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, gestation length and parturition observations, haematology, blood chemistry, organ weight, macropathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily during the first week of treatment and weekly thereafter for all animals and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation for mated females only

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery periods for each adult and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation for Main study females only.

BODY WEIGHT: Yes
- Time schedule for examinations: On the day that treatment commenced (Week 0), weekly thereafter for the treatment and recovery periods and before necropsy. The weight of each Main study female was also recorded on Days 0, 6, 13 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded on a weekly basis. The males had no food consumption performed in Week 3 due to pairing. The Main study females were recorded on a weekly basis until they were paired for mating. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.
For each Main study female, the weight of food supplied, that remaining and an estimate of any spilled was also recorded for the periods Days 0-5, 6-12 and 13-19 after mating and Days 1-3 and 4-6 of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 2 and Recovery Week 2
- Anaesthetic used for blood collection: Yes, isofluorane
- Animals fasted: Yes
- How many animals: 5/sex
- Parameters checked included those listed in the OECD guidance.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 2 and Recovery Week 2
- Animals fasted: Yes
- How many animals: 5/sex
- Parameters checked included those listed in the OECD guidance.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 5 (males), Days 4-6 of lactation (females)
- Dose groups that were examined: Groups 1, 2, 3, and 4
- Battery of functions tested: sensory activity / grip strength / motor activity
Estrous cyclicity (parental animals):
Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa and the stage of the oestrous cycle.
Sperm parameters (parental animals):
Parameters examined in P0 male parental generations: testis weight, epididymis weight, a detailed qualitative histologi al examination taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: 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

GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities (and internal abnormalities for those pups with an external abnormality); possible cause of death was/was not determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were killed after completion of Week 5 investigations
- Maternal animals: All surviving animals were killed on Day 7 of lactation, with the exception of the two females which were not observed to produce a live litter and were killed on Day 25 after mating.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the OECD422 guidance were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on Day 7.
- These animals were subjected to postmortem examinations (macroscopic) as follows:

GROSS NECROPSY
- For offspring surviving to scheduled termination, a careful external examination was performed for gross abnormalities and externally normal offspring were discarded without internal examination. Externally abnormal offspring were examined internally and any abnormal tissues were retained in an appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed.
Statistics:
All statistical analyses were carried out separately for males and females. For litter/fetal findings the litter was taken as the treated unit and the basis
for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
Reproductive indices:
Percentage mating, conception rate (%), fertility index (%), gestation index (%)
Offspring viability indices:
Post-implantation survival index (%), live birth index (%), viability index(%), lactation index
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased spleen weights in males and females at >= 50 mg/kg bw/day and increased heart weight (slight) in females at 150 mg/kg bw/day. After the 14day recovery period, spleen weights were still slightly higer in the 150 mg/kg bw/day males and females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlarged (>=50 mg/kg bw/day) and dark (>=15 mg/kg bw/day) colored spleens in males and females. Dark colored kidneys in females at >=50 mg/kg bw/day. The dark colored spleens were also observed at 150 mg/kg bw/day in recovery animals.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Spleen: Both sexes - hemosiderosis >=15 mg/kg bw/day, congestion >=50 mg/kg bw/day, extramedullary hematopoiesis 150 mg/kg bw/day. Liver: Both sexes - extramedullary hematopoiesis at 150 mg/kg bw/day. Full recovery of extramedullary hematopoiesis.
Reproductive function: estrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths and no post dosing signs.

BODY WEIGHT AND WEIGHT GAIN
There were no adverse bodyweight effects in males or females before or after mating, during lactation and during recovery.

FOOD CONSUMPTION
Food consumption was not affected by treatment with MPKO.

HAEMATOLOGY
Treatment with MPKO at 50 and 150 mg/kg/day was associated with major adverse effects upon the red blood cells and the following changes were attributed to treatment. Males and females receiving 50 and 150 mg/kg/day showed low haematocrit and haemoglobin levels, low red blood cell counts, high reticulocytes and low mean cell haemoglobin concentrations, and for males only at those dose levels high mean cell volumes. A dose relationship was also apparent. The high mean cell volume in females was restricted to those receiving 150 mg/kg/day. High mean cell haemoglobin levels and high platelet levels in males and females receiving 150 mg/kg/day were also evident.

After the 14 day off dose period many of the parameters noted to be different to control during treatment had shown complete recovery. Those changes which had not completely resolved included high mean cell haemoglobin level, low mean cell haemoglobin concentration and high mean cell volumes for males previously receiving 150 mg/kg/day, low red blood cell counts, high mean cell haemoglobin level and high mean cell volume for females previously receiving the same dose.

CLINICAL CHEMISTRY
High bilirubin concentrations were recorded for males and females at 50 and 150 mg/kg/day during the week 2 of the treatment period. There was a suggestion of an increase in potassium and phosphorus in males receiving 150 mg/kg/day. Total protein was low for males attaining statistical significance for those receiving 50 and 150 mg/kg/day and albumin for males at 150 mg/kg/day was also low. Females receiving 150 mg/kg/day had a high Albumin/Globulin ratio.

The affected parameters were similar to control following two weeks of recovery.

NEUROBEHAVIOUR
Sensory reactivity findings and grip strength values for males and females were unaffected by treatment with MPKO.
There was no effect of MPKO on motor activity scores.

PRE-COITAL INTERVAL
All animals mated within the first four days following pairing

MATING PERFORMANCE AND FERTILITY
Percentage mating, conception rate and fertility index scores for all groups were 100%.

GESTATION LENGTH, PARTURITION, AND GESTATION INDEX
Gestation length was within the expected range for this strain for of rat at these laboratories. Two females, one each receiving 50 and 150 mg/kg/day, found to have implantation scars at necropsy on Day 25 of gestation but were not observed to give birth either due to total litter resorption or cannibalising pups born overnight prior to the first check of the day: a relationship to treatment is not inferred.

ORGAN WEIGHTS
Adjusted mean spleen weight was higher than Control in males and females receiving 50 or 150 mg/kg/day (X1.6 or X3.2 of Control for males and X1.3 and X2.5 of Control for females) with a dose response evident. In females receiving 150 mg/kg/day there was a slight increase in adjusted mean heart weight (X1.1 of Control).

After 14 days recovery, the spleen weights were still slightly higher than Control values for males and females which had received 150 mg/kg/day (X1.5 for males and X1.2 for females of Control); although no statistical significance was attained for females, and the values were lower than the main study animals. The heart weights of females receiving 150 mg/kg/day were similar to control values. Kidney weights were slightly higher than Control after the recovery period in males that had received 150 mg/kg/day (X1.1 of Control) this was not seen in the main phase animals.

GROSS PATHOLOGY
Enlarged spleen was seen in all males and females treated with 150 mg/kg/day and in three out of 10 males treated with 50 mg/kg/day. Dark colouration of the spleen was also seen in all males and females treated with 150 mg/kg/day, in nine out of 10 males and females treated with 50 mg/kg/day and in one out of 10 males treated with 15 mg/kg/day.

Dark colouration of the kidneys (left and right) was seen in nine females treated with 150 mg/kg/day and in three females treated with 50 mg/kg/day.

After the recovery period, dark colouration of the spleen was seen in three males and two females treated with 150 mg/kg/day

HISTOPATHOLOGY: NON-NEOPLASTIC
Spleen: Haemosiderosis was observed in males and females treated at 15, 50 and 150 mg/kg/day. Congestion was also seen in males and females treated at 50 and 150 mg/kg/day. An increase in the incidence of extramedullary haemopoiesis was also observed in males and females treated at 150 mg/kg/day. These changes revealed dose-relationship.
Liver: Extramedullary haemopoiesis was observed in males and females treated at 50 and 150 mg/kg/day.

After Recovery Period:
Spleen: An increase in the severity of haemosiderosis was observed in males and females previously treated at 150 mg/kg/day. A decrease in severity of congestion was also observed in males previously treated at 150 mg/kg/day



Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
16 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Read-across from an analogue for which NOAEL = 15 mg/kg bw/day. Basis for effect: red blood cell destruction with associated findings in the spleen, liver or kidneys at an estimated dose levels of 53 mg/kg bw/day and above.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING): The post implantation survival was slightly low in females receiving 50 or 150 mg/kg/day this is attributed to the single female which failed to litter at each dose level. Offspring survival up to Day 7 of age was unaffected by parental treatment.

BODY WEIGHT (OFFSPRING): Offspring bodyweight on Day 1 of age and subsequent bodyweight gain up to Day 7 of age was unaffected by parental treatment with MPKO at levels up to 150 mg/kg/day.

GROSS PATHOLOGY (OFFSPRING): Macroscopic examination of offspring dying before scheduled termination or killed at scheduled termination on Day 7 of age did not reveal any findings that were attributed to parental treatment.
Dose descriptor:
NOEL
Remarks:
(toxicity to reproduction)
Generation:
F1
Effect level:
159 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Read-across from an analogue for which NOEL = 150 mg/kg bw/day. There were no effects on any of the reproductive parameters or developmental endpoints evaluated.
Reproductive effects observed:
not specified

The data matrix is included in the reporting format attached.

The results from the read-across approach are expressed on the basis of the amount of MPKO generated from the hydrolysis reaction of OS3600 and their molecular weights. The amount of MPKO was estimated using the assumption that 1 mole of the target substance OS3600 produces 4 moles of MPKO. No other adaptions are necessary.

Conclusions:
Based on the read-across approach from the analogue substance MPKO, the NOEL for OS3600 was considered to be 159 mg/kg bw/day since no effects were observed on reproductive screening parameters up to the highest dose tested.
Executive summary:

The systemic toxic potential and effects on reproduction were assessed in rats following oral administration of the analogue substance MPKO for at least five weeks in accordance with OECD Guideline 422 (GLP study). 10 animals per sex and per group were treated for two weeks at dose levels of 15, 50 or 150 mg/kg bw/day before pairing. Treatment continued to a total of at least 5 weeks. A control group of 10 animals per sex received the vehicle, corn oil, at the same volume-dose throughout the same period. Males were killed after at least 5 weeks of treatment and females were killed on Day 7 of lactation. Recovery, over 14 days without treatment, was assessed in 5 of the control and 5 of the high dose males and in an extra 5 unmated females in the same groups which were treated for 6 weeks before start of recovery. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, gestation length and parturition observations, haematology, blood chemistry, organ weight, macropathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed. The results observed in this study showed effects of MPKO assessed by haematology, organ weights and macroscopic appearance at dose levels of 50 mg/kg bw/day and above and microscopic tissue appearance were observed at dose levels of 15 mg/kg bw/day and above. After two weeks off dose complete recovery was seen in many clinical pathology parameters and recovery was in progress but not complete in males for high mean cell haemoglobin level, low mean cell haemoglobin concentration and high mean cell volume and in females low red blood cell counts, high mean cell haemoglobin level and high mean cell volume, organ weights, macroscopic and microscopic appearance. There were no adverse effects of treatment on the reproductive/developmental screening parameters assessed at least 150 mg/kg/day. The NOAEL for MPKO was considered to be 15 mg/kg bw/day for general systemic toxicity. The NOEL for reproductive and developmental screening parameters was considered to be 150 mg/kg bw/day. Based on these results, the read-across approach was applied, and the NOAEL for OS3600 was determined to be 16 mg/kg bw/day for systemic toxicity and 159 mg/kg bw/day for toxicity to reproduction.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
159 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is a GLP compliant and has a Klimisch score 2 (read-across from an endpoint with Klimisch score 1).
Additional information

Key study: Read-across approach from experimental data on the analogue substance MPKO:

The systemic toxic potential and effects on reproduction of the analogue MPKO were assessed in rats following oral administration for at least five weeks in accordance with OECD Guideline 422 (GLP study). Based on the read-across approach from the analogue substance MPKO, the hydrolysis product of OS9600 (NOEL = 150 mg/kg bw/day since no adverse effects observed on the reproductive screening parameters at the highest dose level), the NOEL for OS3600 was determined to be 159 mg/kg bw/day for reproductive toxicity in rats.


Short description of key information:
Based on the read-across approach from the analogue substance MPKO, The NOEL (P, fertility or reproductive toxicity) for OS3600 was determined to be 159 mg/kg bw/day since no effects were observed on reprotox parameters at the highest dose tested.

Justification for selection of Effect on fertility via oral route:
Only one study available

Effects on developmental toxicity

Description of key information
Based on the read-across approach from the analogue substance MPKO, the NOEL (F1, developmental toxicity) for OS3600 was determined to be 159 mg/kg bw/day since no effects were observed on developmental parameters at the highest dose tested.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
OS3600 undergoes rapid hydrolysis in aqueous to MPKO and the corresponding silanol. Silanols undergo continuous condensation reactions to produce higher molecular weight siloxanes which are considered biologically unavailable. Therefore, the observed toxicity is likely due to MPKO and their values are comparable. The study has been conducted according to OECD guidance under GLPs.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 71 days
- Weight at study initiation: 337 g to 394 g (males), 220 g to 271 g (females)
- Fasting period before study: none
- Housing: Animals were housed inside a barriered rodent facility. The gridded cages used during pairing were suspended over trays covered with absorbent paper which was changed daily. For cages with solid floors, wood based material was used as bedding and was sterilised by autoclaving and changed at least twice each week. Cages, cage-trays, food hoppers and water bottles were changed at appropriate intervals. The cages were distributed on the racking to equalise, as far as possible, environmental influences amongst the groups.
- Diet (e.g. ad libitum): ad libitum (SDS VRF1 Certified Diet) except overnight before routine blood sampling. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): ad libitum from the public supply via polycarbonate or polypropylene bottles fitted with sipper tubes.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40 to 70
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: To: 21 March 2012 to 23 May 2012
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance, MPKO, was prepared for administration as a series of graded concentrations in the vehicle, by dilution of individual weightings of the test substance. Small amounts of vehicle were added to the test substance and mixed until a solution was formed. This was made up to the required volume with vehicle and then magnetically stirred until homogenous. The test substance was used as supplied. All formulations were prepared weekly and stored refrigerated before use.

VEHICLE
- Concentration in vehicle: 7.5, 25, 75 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the liquid matrix. Specimen formulations (typically 400 mL) were prepared at concentrations of 1 mg/mL and 100 mg/mL and equally split between four amber screw-capped bottles. Prior to initial sampling on each day, the formulation was mixed by 20-fold inversion. A control vehicle sample was stored with each batch of stability samples. The stability was confirmed for at least 24 hours at ambient temperature and for up to 15 days when refrigerated (2-8¿C).
Samples of each formulation prepared for administration on the first and last occasion of treatment were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups. Two of the samples from each group were analysed. The remainder were retained as contingency for analysis if any result required confirmation.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 2 weeks
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
The test substance, MPKO, was administered for two weeks before pairing up to necropsy (at least five weeks) for males and two weeks before pairing then throughout pairing and gestation until Day 6 of lactation for females. Recovery animals were treated for approximately six weeks and completed a further 14 days without treatment.

Animals of the F1 generation were not dosed.
Frequency of treatment:
All animals were dosed once each day at approximately the same time each day, seven days per week.
Remarks:
Doses / Concentrations:
15, 50, 150 mg/kg bw/day
Basis:
other: actual administered
No. of animals per sex per dose:
10 males per group (Groups 1 to 4)
15 females (Groups 1 and 4), of these 5 females per group were not mated and were used for the recovery group
10 females (Groups 2 and 3)
Control animals:
yes, concurrent vehicle
Details on study design:
Ten males and 10 females per group were treated for two weeks at dose levels of 15, 50 or 150 mg/kg/day before pairing. Treatment continued to a total of at least 5 weeks. A control group of 10 male and 10 female rats received the vehicle, corn oil, at the same volume-dose throughout the same period. Males were killed after at least 5 weeks of treatment and females were killed on Day 7 of lactation.
Recovery, over 14 days without treatment, was assessed in five of the control and five of the high dose males and in an extra five unmated females in the same groups which were treated for 6 weeks before start of recovery.
The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk.
During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, gestation length and parturition observations, haematology, blood chemistry, organ weight, macropathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily during the first week of treatment and weekly thereafter for all animals and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation for mated females only

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery periods for each adult and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation for Main study females only.

BODY WEIGHT: Yes
- Time schedule for examinations: On the day that treatment commenced (Week 0), weekly thereafter for the treatment and recovery periods and before necropsy. The weight of each Main study female was also recorded on Days 0, 6, 13 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded on a weekly basis. The males had no food consumption performed in Week 3 due to pairing. The Main study females were recorded on a weekly basis until they were paired for mating. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.
For each Main study female, the weight of food supplied, that remaining and an estimate of any spilled was also recorded for the periods Days 0-5, 6-12 and 13-19 after mating and Days 1-3 and 4-6 of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 2 and Recovery Week 2
- Anaesthetic used for blood collection: Yes, isofluorane
- Animals fasted: Yes
- How many animals: 5/sex
- Parameters checked included those listed in the OECD guidance.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 2 and Recovery Week 2
- Animals fasted: Yes
- How many animals: 5/sex
- Parameters checked included those listed in the OECD guidance.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 5 (males), Days 4-6 of lactation (females)
- Dose groups that were examined: Groups 1, 2, 3, and 4
- Battery of functions tested: sensory activity / grip strength / motor activity
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: No
- Other:
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
All statistical analyses were carried out separately for males and females. For litter/fetal findings the litter was taken as the treated unit and the basis
for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
Indices:
Post-implantation survival index (%), live birth index (%), viability index(%)
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths and no post dosing signs.

BODY WEIGHT AND WEIGHT GAIN
There were no adverse bodyweight effects in males or females before or after mating, during lactation and during recovery.

FOOD CONSUMPTION
Food consumption was not affected by treatment with MPKO.

HAEMATOLOGY
Treatment with MPKO at 50 and 150 mg/kg/day was associated with major adverse effects upon the red blood cells and the following changes were attributed to treatment. Males and females receiving 50 and 150 mg/kg/day showed low haematocrit and haemoglobin levels, low red blood cell counts, high reticulocytes and low mean cell haemoglobin concentrations, and for males only at those dose levels high mean cell volumes. A dose relationship was also apparent. The high mean cell volume in females was restricted to those receiving 150 mg/kg/day. High mean cell haemoglobin levels and high platelet levels in males and females receiving 150 mg/kg/day were also evident.

After the 14 day off dose period many of the parameters noted to be different to control during treatment had shown complete recovery. Those changes which had not completely resolved included high mean cell haemoglobin level, low mean cell haemoglobin concentration and high mean cell volumes for males previously receiving 150 mg/kg/day, low red blood cell counts, high mean cell haemoglobin level and high mean cell volume for females previously receiving the same dose.

CLINICAL CHEMISTRY
High bilirubin concentrations were recorded for males and females at 50 and 150 mg/kg/day during the week 2 of the treatment period. There was a suggestion of an increase in potassium and phosphorus in males receiving 150 mg/kg/day. Total protein was low for males attaining statistical significance for those receiving 50 and 150 mg/kg/day and albumin for males at 150 mg/kg/day was also low. Females receiving 150 mg/kg/day had a high Albumin/Globulin ratio.

The affected parameters were similar to control following two weeks of recovery.

NEUROBEHAVIOUR
Sensory reactivity findings and grip strength values for males and females were unaffected by treatment with MPKO.
There was no effect of MPKO on motor activity scores.

PRE-COITAL INTERVAL
All animals mated within the first four days following pairing

MATING PERFORMANCE AND FERTILITY
Percentage mating, conception rate and fertility index scores for all groups were 100%.

GESTATION LENGTH, PARTURITION, AND GESTATION INDEX
Gestation length was within the expected range for this strain for of rat at these laboratories. Two females, one each receiving 50 and 150 mg/kg/day, found to have implantation scars at necropsy on Day 25 of gestation but were not observed to give birth either due to total litter resorption or cannibalising pups born overnight prior to the first check of the day: a relationship to treatment is not inferred.

ORGAN WEIGHTS
Adjusted mean spleen weight was higher than Control in males and females receiving 50 or 150 mg/kg/day (X1.6 or X3.2 of Control for males and X1.3 and X2.5 of Control for females) with a dose response evident. In females receiving 150 mg/kg/day there was a slight increase in adjusted mean heart weight (X1.1 of Control).

After 14 days recovery, the spleen weights were still slightly higher than Control values for males and females which had received 150 mg/kg/day (X1.5 for males and X1.2 for females of Control); although no statistical significance was attained for females, and the values were lower than the main study animals. The heart weights of females receiving 150 mg/kg/day were similar to control values. Kidney weights were slightly higher than Control after the recovery period in males that had received 150 mg/kg/day (X1.1 of Control) this was not seen in the main phase animals.

GROSS PATHOLOGY
Enlarged spleen was seen in all males and females treated with 150 mg/kg/day and in three out of 10 males treated with 50 mg/kg/day. Dark colouration of the spleen was also seen in all males and females treated with 150 mg/kg/day, in nine out of 10 males and females treated with 50 mg/kg/day and in one out of 10 males treated with 15 mg/kg/day.

Dark colouration of the kidneys (left and right) was seen in nine females treated with 150 mg/kg/day and in three females treated with 50 mg/kg/day.

After the recovery period, dark colouration of the spleen was seen in three males and two females treated with 150 mg/kg/day

HISTOPATHOLOGY: NON-NEOPLASTIC
Spleen: Haemosiderosis was observed in males and females treated at 15, 50 and 150 mg/kg/day. Congestion was also seen in males and females treated at 50 and 150 mg/kg/day. An increase in the incidence of extramedullary haemopoiesis was also observed in males and females treated at 150 mg/kg/day. These changes revealed dose-relationship.
Liver: Extramedullary haemopoiesis was observed in males and females treated at 50 and 150 mg/kg/day.

After Recovery Period:
Spleen: An increase in the severity of haemosiderosis was observed in males and females previously treated at 150 mg/kg/day. A decrease in severity of congestion was also observed in males previously treated at 150 mg/kg/day
Dose descriptor:
NOAEL
Remarks:
(parental systemic toxicity)
Effect level:
16 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no effects on any of the developmental endpoints assessed.
Dose descriptor:
NOEL
Effect level:
159 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

The data matrix is included in the reporting format attached.

The results from the read-across approach are expressed on the basis of the amount of MPKO generated from the hydrolysis reaction of OS9600 and their molecular weights. The amount of MPKO was estimated using the assumption that 1 mole of the target substance OS9600 produces 3 moles of MPKO. No other adaptions are necessary.

Conclusions:
Based on the read-across approach from the analogue substance MPKO, the NOEL for OS3600 was considered to be 159 mg/kg bw/day since no effects were observed on the developmental screening parameters up to the highest dose tested.
Executive summary:

The systemic toxic potential and effects on reproduction were assessed in rats following oral administration of the analogue substance MPKO for at least five weeks in accordance with OECD Guideline 422 (GLP study). 10 animals per sex and per group were treated for two weeks at dose levels of 15, 50 or 150 mg/kg bw/day before pairing. Treatment continued to a total of at least 5 weeks. A control group of 10 animals per sex received the vehicle, corn oil, at the same volume-dose throughout the same period. Males were killed after at least 5 weeks of treatment and females were killed on Day 7 of lactation. Recovery, over 14 days without treatment, was assessed in 5 of the control and 5 of the high dose males and in an extra 5 unmated females in the same groups which were treated for 6 weeks before start of recovery. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, gestation length and parturition observations, haematology, blood chemistry, organ weight, macropathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed. The results observed in this study showed effects of MPKO assessed by haematology, organ weights and macroscopic appearance at dose levels of 50 mg/kg bw/day and above and microscopic tissue appearance were observed at dose levels of 15 mg/kg bw/day and above. After two weeks off dose complete recovery was seen in many clinical pathology parameters and recovery was in progress but not complete in males for high mean cell haemoglobin level, low mean cell haemoglobin concentration and high mean cell volume and in females low red blood cell counts, high mean cell haemoglobin level and high mean cell volume, organ weights, macroscopic and microscopic appearance. There were no adverse effects of treatment on the reproductive/developmental screening parameters assessed at least 150 mg/kg/day. The NOAEL for MPKO was considered to be 15 mg/kg bw/day for general systemic toxicity. The NOEL for reproductive and developmental screening parameters was considered to be 150 mg/kg bw/day. Based on these results, the read-across approach was applied, and the NOAEL for OS3600 was determined to be 16 mg/kg bw/day for systemic toxicity and 159 mg/kg bw/day for toxicity to reproduction.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
159 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is a GLP compliant and has a Klimisch score 2 (read-across from an endpoint with Klimisch score 1).
Additional information

Key study: Read-across approach from experimental data on the analogue substance MPKO:

The systemic toxic potential and effects on reproduction of the analogue MPKO were assessed in rats following oral administration for at least five weeks in accordance with OECD Guideline 422 (GLP study). Based on the read-across approach from the analogue substance MPKO, the hydrolysis product of OS2600 (NOEL = 150 mg/kg bw/day since no adverse effects observed on the developmental screening parameters at the highest dose level), the NOEL for OS3600 was determined to be 159 mg/kg bw/day for developmental toxicity in rats


Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available.

Justification for classification or non-classification

Taking into account the results obtained in the combined repeat dose and reproductive/developmental toxicity screening study, the substance OS3600 is not classified in accordance with CLP Regulation (EC) no. 1272/2008 since no adverse effects were observed on reproductive and/or developmental parameters both in P and F1 generations.