N-pentan-2-ylidenehydroxylamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

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:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of the Government of the United Kingdom

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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 Certifie 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 weighings 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.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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:

10 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 thoses 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 int eh 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

Oestrous 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 histological 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

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment with MPKO at 50 and 150 mg/kg/day was associated with major adverse effects upon the red blood cells. Many of the affected paramters showed complete recovery after the 14-day recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Several parameters at 50 and 150 mg/kg/day were affected in males and/or females during Week 2 of the treatment period. The affected parameters were similar to control after the 14-day recovery period.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous 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., aA 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

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

blood

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

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.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no effects on any reproductive parameters assessed.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

There were no adverse effects of treatment on the reproductive screening parameters assessed up to 150 mg/kg bw/day, the highest dose tested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

484-470-6

EC Name:

-

Cas Number:

623-40-5

Molecular formula:

C5H11NO

IUPAC Name:

N-pentan-2-ylidenehydroxylamine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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: ad libitum (SDS VRF1 Certifie Diet) except overnight before routine blood sampling. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: 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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral 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 weighings 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.

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

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.

Examinations

Observations and examinations performed and frequency:

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 thoses 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 int eh 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment with MPKO at 50 and 150 mg/kg bw/day was associated with major adverse effects upon the red blood cells. Many of the affected paramters showed complete recovery after the 14-day recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Several parameters at 50 and 150 mg/kg/day were affected in males and/or females during Week 2 of the treatment period. The affected parameters were similar to control after the 14-day recovery period.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

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 14-day recovery period, splpeen 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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Spleen: Both sexes - haemosiderosis >=15 mg/kg bw/day, congestion >=50 mg/kg bw/day, extramedullary haematopoiesis 150 mg/kg bw/day. Liver: Both sexes - extramedullary haematopoiesis at 150 mg/kg bw/day. Full recovery of extramedullary haematopoiesis.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

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 bw/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 bw/day showed low haematocrit (-12 and -17% (m); -9.6% and -11% (f)) and haemoglobin levels (-15.2% and -22% (m); -12% and -16.7% (f)), low red blood cell counts (-15.5% and -31.5% (m);-12.1% and -29.2% (f)), high reticulocytes (+184% and +383% (m); +159% and +346% (f)) and low mean cell haemoglobin concentrations (-3.6% and -6.4% (m); -2.7% and -6.3% (f)) compared to control, and for males only at those dose levels high mean cell volumes (+4.5% and +21%). A dose relationship was also apparent. The high mean cell volume in females was restricted to those receiving 150 mg/kg/day(+26.5%). High mean cell haemoglobin levels (+13.2% (m); +18.1% (f)) and high platelet levels (+40.8% (m) and +23.3% (f)) in animals receiving 150 mg/kg bw/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 (+11.2%), low mean cell haemoglobin concentration (-2.8%) and high mean cell volumes (+13.5%) for males previously receiving 150 mg/kg bw/day, low red blood cell counts (-7.8%), high mean cell haemoglobin level (+12.2%) and high mean cell volume (+13.4%) for females previously receiving the same dose.

CLINICAL CHEMISTRY
High bilirubin concentrations were recorded for males and females at 50 and 150 mg/kg bw/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 bw/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 bw/day was also low. Females receiving 150 mg/kg bw/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 bw/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 (+59.9% and +218%) and females (+25.4% and +147.2%) receiving 50 or 150 mg/kg bw/day with a dose response evident. In females receiving 150 mg/kg bw/day there was a slight increase in adjusted mean heart weight.

After 14 days recovery, the spleen weights were still slightly higher than Control values for males and females which had received 150 mg/kg bw/day; 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 bw/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 bw/day 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 bw/day and in three out of 10 males treated with 50 mg/kg bw/day. Dark colouration of the spleen was also seen in all males and females treated with 150 mg/kg bw/day, in nine out of 10 males and females treated with 50 mg/kg bw/day and in one out of 10 males treated with 15 mg/kg bw/day.

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

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

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

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

Effect levels

1

Key result

Dose descriptor:

LOAEL

Remarks:

(toxicity to F0 generation)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Remarks:

(developmental/reproductive effects)

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: There were no effects on any of the reproductive parameters or developmental endpoints evaluated.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

blood

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Due to the multiple effects in the blood and spleen related to haemolytic anaemia 2-PO is classified for STOT RE Cat 2.

Executive summary:

The effects observed in this study included changes in haematological parameters and organ weights starting at a dose level of 50 mg/kg bw/day and above and in microscopic tissue appearance (haemosiderosis) at dose levels of 15 mg/kg bw/day and above. After two weeks of 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 for 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 bw/day.

The Low Observed Adverse Effect Level (LOAEL) for 2-PO (MPKO) is considered to be 15 mg/kg bw/day for general systemic toxicity (the lowest dose tested), taken into account that haemosiderosis, which is regarded as an adverse effect related to haemolytic anaemia, was already observed in the lowest dose group of 15 mg/kg bw/day. At the two higher dose groups multiple effects related to haemolytic anaemia developed, showing the dose-dependent progress of effects related to haemolytic anaemia. The NOAEL for reproductive and developmental screening parameters is considered to be 150 mg/kg bw/day.