Octene, hydroformylation products, low-boiling

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 December 2009 (animal arrival) -24 March 2010 (pathology completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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:

The protocol states that animals will be bled for haematology and blood chemistry following overnight deprivation of food. Due to the excessive toxicity observed for males receiving 1000 mg/kg/day, all males receiving 1000 mg/kg/day were killed during Week 4 of treatment (Amendment 4). The haematology and blood chemistry samples were taken without the animals having overnight deprivation of food. As haematology and blood chemistry data were obtained for males receiving 1000 mg/kg/day, this deviation from protocol is not considered to affect the integrity of the study.

Deviations:

yes

Remarks:

The toxicity subgroup for this study comprised 5 male and 5 female rats per treatment group and the reproductive subgroup comprise 5 male and 10 female rats per treatment group. This deviation was undertaken to enhance the robustness of the study.

Principles of method if other than guideline:

The toxicity subgroup for this study comprised 5 male and 5 female rats per treatment group and the reproductive subgroup comprise 5 male and 10 female rats per treatment group. The five male rats in each treatment group of the toxicity subgroup were used for mating with the female reproductive subgroup animals of the corresponding treatment group. The ten female rats in each treatment group of the reproductive subgroup were used exclusively for the reproductive/developmental toxicity phase of the study. This deviation was undertaken to enhance the robustness of the study.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octene, hydroformylation products, low-boiling

EC Number:

273-110-1

EC Name:

Octene, hydroformylation products, low-boiling

Cas Number:

68938-03-4

IUPAC Name:

Octene, hydroformylation products, low-boiling

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Oxooil LS9
- Substance type: Industrial
- Physical state: clear liquid
- Analytical purity: 95.31 %
- Impurities (identity and concentrations): 4.69% higher boiling components (including ethers, alcohols and aldehydes)
- Composition of test material, percentage of components: C-8 hydrocarbons (saturated and unsaturated)
- Isomers composition: no data
- Purity test date: June 2009
- Lot/batch No.: 0649/82531
- Expiration date of the lot/batch: 8 June 2010
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

other: Crl: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 350-397 g males and 204-251 g females
- Fasting period before study: none
- Housing: 5 animals/P2000 polycarbonate cage: males were housed singly with one female in RB3 modified polypropylene cages during mating.
- Diet (e.g. ad libitum): ad libitum (SDS VRF 1 certified diet)
- Water (e.g. ad libitum): ad libitum (Potable water from the public supply)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: 9 December 2009 (animal arrival) To: 1 February 2010 (necropsy completion)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was used as supplied. All formulations were prepared freshly weekly and were stored refrigerated (2-8°C) in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 20, 60 or 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): no data
- Purity: no data

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. The stability was assessed following storage at ambient temperature (nominally 21°C) for 0 hours, 4 hours (continual stirring) and 2 days, and refrigeration (nominally 2-8°C) for 2, 8 and 15 days. Prior to initial sampling on each day, the formulation was mixed by 20-fold inversion and magnetic stirring for a minimum of 5 minutes. At each time point, single samples (nominally 1 mL) were taken for assay from the top, middle and bottom of the magnetically stirred formulation. Stability was determined from the mean concentration of the analyte in the vehicle at each sampling point. Specimen formulations (typically 400 mL) were prepared at concentrations of 2 and 200 mg/mL and equally split between four amber glass screw-capped bottles and were confirmed for 15 days when refrigerated and for 48 hours at room temperature.
Samples of each formulation prepared for administration in the first week of the dosing procedure were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed); 2 assays from each group and 1 assay. The remainder was frozen (nominally -20°C) and retained as contingency for analysis if any result required confirmation.

The GC analytical procedure was validated with respect to linearity of detector response, precision of injection, specificity of chromatographic analysis, limit of detection, accuracy and precision.

The homogeneity and stability was confirmed for Oxooil LS9 in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage for 2 days and refrigerated storage for up to 15 days. The storage times represented the maximum time from preparation to completion of administration.

The mean concentrations of Oxooil LS9 in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation

Duration of treatment / exposure:

5 weeks

Frequency of treatment:

All animals were dosed once each day, at approximately the same time each day, seven days per week for five weeks.

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 or 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females per dosage group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels of 100, 300 and 1000 mg/kg/day were selected in conjunction with the Sponsor with reference to previous work with this compound performed in these laboratories (Huntingdon Life Sciences Report Number: BBB0026). In that study, the CD rats received Oxooil LS9 at doses of 100, 300 or 1000 mg/kg/day for seven days, there were no findings which precluded the use of these dose levels on the subsequent 4 week general toxicity and reproductive developmental toxicity screening study.

- Rationale for animal assignment (if not random):
On arrival, the animals were removed from the transit boxes and allocated to study cages. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately. Before the start of treatment mean bodyweights were reviewed. Control males had a low mean bodyweight in comparison to treated male groups. To average the group means to similar values, two males from the Control group were changed with two males from the middle treatment (Group 3).

- Rationale for selecting satellite groups: no satellite groups used
- Post-exposure recovery period in satellite groups: no post-exposure recovery period

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

- Time schedule:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupants. 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.
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Immediately before dosing
Immediately after dosing on return of the animal to its cage
On completion of dosing of each group
Between one and two hours after completion of dosing of all groups
As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Before treatment commenced and during each week of treatment, 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 (as far as practically possible) of the experimental group to which the animal belonged.
After removal from the home cage, animals were assessed for physical condition and behaviour 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 behaviour.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.


BODY WEIGHT: Yes

- Time schedule for examinations:
Toxicity subgroup males and females and reproductive subgroup males were weighed weekly throughout the study. Reproductive subgroup females were weighed weekly until mating was detected, on Days 0, 7, 14 and 20 after mating and Days 1 and 4 of lactation.

FOOD CONSUMPTION: Yes

The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded during weeks 1 and 2 for all male (two cages of 5 animals per treatment group) and all female (three cages of 5 animals per treatment group) toxicity and reproductive subgroups. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION : Yes

Fluid intake was assessed by daily visual observation. During Week 4 of treatment, water consumption was recorded by weight (over a 3 day period on each occasion) for 5 males and 5 females per treatment group.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 5 of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 males and 5 females per treatment group
- Parameters examined.
Blood samples (nominally 0.5 mL) were collected into tubes containing EDTA as anticoagulant and examined for the following characteristics:

The following were measured using a Bayer Advia 120 haematology analyser
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Total leucocyte count (WBC)
Differential leucocyte count
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

Morphology flags were generated by the Advia 120 analyser. The most common morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were recorded as follows:
- = no abnormalities detected
+ = slight
++ = moderate
+++ = marked

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyser. Confirmation or a written description from the blood film was made where appropriate.

Additional blood samples were taken into tubes containing citrate anticoagulant and examined in respect of:
Prothrombin time (PT) - using an ACL 3000 Plus analyser and IL PT-Fibrinogen reagent
Activated partial thromboplastin time (APTT) - using an ACL 3000 Plus Analyser and IL APTT reagent




CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 5 of treatment (at the same time and using the same animals as for haematology)
- Animals fasted: Yes
- How many animals: 5 males and 5 females per treatment group
- Parameters examined.
Using lithium heparin as anticoagulant blood samples were mechanically agitated for at least two minutes and subsequently centrifuged at 2000 g for 10 minutes in order to separate the plasma. After separation, the plasma was examined using a Roche P Modular Analyser: in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Bile acids (BIAC)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb) - by chemical assay

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Sensory reactivity, grip strength and motor activity assessments were performed (before dosing) on five males and five females in each group during Week 5 of treatment. For sensory reactivity and grip strength, animals were tested by an observer who was unaware of the treatment group to which each animal belonged.

- Dose groups that were examined: all dose groups examined

- Battery of functions tested: sensory activity / grip strength / motor activity :

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 vibrissae). The animal’s reaction was recorded as:
1 No reaction or ignores probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Abnormally fearful or aggressive reaction

Touch response

The animal was stroked gently on the nape of the neck with a blunt probe and the reaction recorded as:
1 No reaction or ignores probe
2 Normal awareness and reaction e.g. turns towards or moves away
3 Abnormally fearful or aggressive reaction

Auditory startle reflex

The animal’s response to a sudden loud 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 vocalisation without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalisation
4 Exaggerated response e.g. excessive vocalisation, body movement or aggression

Grip strength

Forelimb and hindlimb grip strength was measured using Mecmesin Force Indicators. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity

Motor activity was measured using a Rodent Activity Monitoring System, with hardware supplied by Pearson Technical Services and software developed and maintained by Huntingdon Life Sciences. 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. All animals were not necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Males receiving 1000 mg/kg/day showed excessive toxicity, resulting in the premature sacrifice of three animals. Following the deterioration of this sex at this dose level, the remiaming males in this tretament group were killed during Week 4. Five females that received 1000 mg/kg/day and five males and five females that received 0 (control), 100 or 300 mg/kg/day were killed after 5 weeks of treatment. All animals were killed by carbon dioxide asphyxiation. The sequence in which the animals were killed after completion of the study was selected to allow satisfactory inter-group comparison
.
All animals were subject to a detailed necropsy, which involved the following:

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. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

The following organs, taken from five males and five females per treatment group were dissected free of adjacent fat and other contiguous tissue and the weights recorded:

Adrenal glands Spleen
Brain Thymus
Heart Thyroid with parathyroids*
Kidneys Uterus with cervix
Liver
Ovaries with oviducts (L&R)
Pituitary


* Weighed after partial fixation

Organ weights were also adjusted for terminal bodyweight using the weight recorded before necropsy.


HISTOPATHOLOGY: Yes

Tissues were examined for 5 males and 5 females of the Control and 1000 mg/kg/day treatment groups, sacrificed on completion of the scheduled treatment period and all animals killed during the study.

Adrenals Pituitary
Brain Peyer’s patches
Caecum Rectum
Colon Sciatic nerves+
Duodenum Skin
Heart Spinal cord
Ileum Spleen
Jejunum Sternum (with marrow bone)
Kidneys Stomach
Liver Thymus
Lungs Thyroid with parathyroids
Lymph nodes - mandibular Trachea
- mesenteric Urinary bladder
Mammary area - caudal Uterus with cervix
Marrow smear Vagina
Oesophagus
Ovaries with oviducts (L&R)

The kidneys and liver were considered to exhibit reaction to treatment at the high dose and were examined for the animals that received 100 or 300 mg/kg/day.

Statistics:

See free text field below

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

One male receiving 1000 mg/kg/day was killed for welfare reasons on Day 16 of treatment (Week 3) due to general poor condition and substantial weight loss. The ante-mortem clinical signs consisted of hunched posture, thin build conformation, reduced body tone and temperature, abnormal gait and irregular breathing. Macroscopic examination revealed: thin build, pale areas on the liver, abnormal contents (pale) of and pale jejunum, small spleen and abnormal pink colouration of the skin and sciatic nerve and seminal vesicles. A second male receiving 1000 mg/kg/day was killed for welfare reasons on Day 24 of treatment (Week 4) due to general poor condition, substantial weight loss and limited use of hind limbs. The ante-mortem clinical signs consisted of underactive behaviour, hunched posture; thin build conformation, piloerection and limited use of hind limbs. Macroscopic examination revealed: thin build, small thymus and pale jejunum and abnormal pink colouration of the skin, prostate, urinary bladder, sciatic nerve, adipose tissue and seminal vesicles. A third male receiving 1000 mg/kg/day was killed for welfare reasons on Day 25 of treatment (Week 4) due to general poor condition, persistent weight loss and limited use of hind limbs. The ante-mortem clinical signs consisted of thin build conformation, reduced body tone and temperature, limited use of limbs and underactive behaviour. Macroscopic examination revealed: thin build, thickened and pale jejunum and duodenum, pale mesenteric lymph nodes and adrenals, enlarged mandibular lymph node, distended urinary bladder, small thymus and abnormal pink colouration of the skin, seminal vesicles, adipose tissue and sciatic and peripheral (trigeminal) nerve(s). Due to the deterioration of males receiving 1000 mg/kg/day and the excessive toxicity observed for this group including the sacrifice of 3/10 males, all surviving males receiving 1000 mg/kg/day were killed earlier than scheduled; in Week 4 of treatment.

Clinical signs for males receiving 1000 mg/kg/day consisted of a high incidence of brown staining on the dorsal body/ whole body surface and thin build. Post dose signs comprised of repetitive movement of the forepaws (paddling) on Days 21 and 24 of treatment, and chin rubbing and salivation was also observed periodically after dosing for males at this dose level.

There were no clinical signs for females receiving 1000 mg/kg/day or animals of either sex receiving 300 or 100 mg/kg/day that were considered to be related to treatment with Oxooil LS9.


BODY WEIGHT AND WEIGHT CHANGE (Table 7 appended)

For males receiving 1000 mg/kg/day, bodyweight gain was statistically significantly low during the first week of treatment, and bodyweight loss was apparent during Weeks 2 and 3 when compared with Controls. Mean bodyweight gain was low for males receiving 100 or 300 mg/kg/day during Weeks 3-4 of treatment when compared with Controls. Overall mean weight gain was slightly lower (84% of Control) for males receiving 300 mg/kg/day.

Mean bodyweight gain was slightly lower for females receiving 100 or 300 mg/kg/day during Weeks 2-3 and 3-4 of treatment when compared with Controls, with mean bodyweight loss during Weeks 4-5 of treatment. Overall mean weight gains were moderately lower (69% of Control) for females receiving 300 mg/kg/day and markedly lower for females receiving 1000 mg/kg/day (56% of Contro

There was no overall effect on bodyweight gain for animals receiving 100 mg/kg/day when compared with Controls.

FOOD CONSUMPTION

The food consumption of males receiving 1000 mg/kg/day was low during weeks 1 and 2, when compared with Controls. . There was no effect on food consumption during this period for females receiving 1000 mg/kg/day or rats receiving 100 or 300 mg/kg/day.

WATER CONSUMPTION

Water intake assessed in Week 4 of treatment was increased for males receiving 100 mg/kg/day or above and females receiving 1000 mg/kg/day when compared with Controls.

HAEMATOLOGY

Prolonged activated partial prothrobin time was recorded during week 5 for females receiving 1000 mg/kg/day, when compared with the female Controls.

All other inter-group differences from Controls were minor or confined to one sex and were therefore considered to be due to normal biological variation.

CLINICAL CHEMISTRY (Table 13 appended)

The biochemical examination of the blood plasma in Week 5 (for all animals except males receiving 1000 mg/kg/day, which were obtained in Week 4 (prior to early termination)) indicated that alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase activities were statistically significantly lower for females receiving 1000 mg/kg/day when compared to those of the Controls. Alanine aminotransferase was also low in females receiving 300 mg/kg/day and aspartate aminotransferase was low in males receiving 300 mg/kg/day.

Total plasma cholesterol levels were higher in males and females receiving 1000 mg/kg/day, when compared with that of the Controls.

In males receiving 300 mg/kg/day and males and females receiving 1000 mg/kg/day, there was an increase of plasma albumin concentration, compared with the Controls, but only in males receiving 1000 mg/kg/day was there an increase of the albumin to globulin ratio.

All other inter-group differences from Controls were minor, lacked dose-relationship, were confined to one sex or were considered not of any toxicological importance.

NEUROBEHAVIOUR

There was no effect of treatment on the sensory reactivity, grip strength or motor activity.

Males receiving 1000 mg/kg/day were killed in Week 4 of treatment, so were not subject to assessment

ORGAN WEIGHTS (Table 20 appended)

The analysis of organ weights after 5 weeks of treatment, indicated that mean absolute and adjusted liver and kidney weights were slightly high among males that received 300 mg/kg/day and females that received 1000 mg/kg/day, with adjusted weights attaining statistical significance when compared to Controls.

Seminal vesicles absolute and adjusted weights were slightly low in males that received 300 mg/kg/day when compared with Controls, with adjusted weights attaining statistical significance


GROSS PATHOLOGY

Macroscopic examination of males that were killed or died early at 1000 mg/kg/day revealed a high number of animals with abnormal pink colouration of the peripheral nerve(s), sciatic nerve, seminal vesicles, pale jejunum and pale and thickened duodenum, they were also of thin build and their fur was stained (7/10, 10/10, 9/10, 8/10, 7 and 4/10 and 7/10, respectively). A few males had abnormal colouration of the skin, mesenteric lymph node, adipose tissue, prostate and urinary bladder which was also distended for two males. Dark and enlarged mandibular lymph nodes, small thymus, non glandular thickened stomach, thickened and abnormal contents of the jejunum was also observed at a lower incidence for males that received 1000 mg/kg/day.

Females that received 1000 mg/kg/day and males that received 300 mg/kg/day were observed to have abnormal pink colouration of the sciatic nerve(s) and peripheral nerve(s), when compared with Controls. Males that received 300 mg/kg/day were observed to have abnormal pink colouration of the seminal vesicles.


HISTOPATHOLOGY: NON-NEOPLASTIC

Decedents and animals killed during week 4 of treatment

Treatment related findings
In the liver minimal hepatocellular centrilobular hypertrophy was observed in 4/6 males given 1000 mg/kg/day.
In the kidneys cortical tubular basophilia, cortical tubular dilatation and hyaline droplets were respectively observed in 4/6, 3/6 and 6/6 males given 1000 mg/kg/day.

Animals killed after 4 weeks of treatment

Treatment related findings
Changes related to treatment with Oxooil LS 9 were seen in liver and kidneys.

Liver
Microscopic observations revealed the presence of hepatocellular centrilobular hypertrophy at a dose-related incidence in treated males (100 and 300 mg/kg/day) and females (1000 mg/kg/day) which correlated with significant increase of the mean absolute organ weight in males (300 mg/kg/day) and females (1000 mg/kg/day).

Group/sex 1M 2M 3M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 300 0 100 300 1000

Hypertrophy, Centrilobular
Minimal 0 2 5 0 0 0 5
Total 0 2 5 0 0 0 5
Number of tissues examined 5 5 5 5 5 5 5


Kidneys
Renal findings included a dose related increase in the incidence and severity of hyaline droplets in the proximal convoluted tubules in the male groups given 100 or 300 mg/kg/day of Oxooil LS 9.

Cortical tubular basophilia and tubular dilatation were seen in the male groups given 100 or 300 mg/kg/day.

Group/sex 1M 2M 3M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 300 0 100 300 1000
Cortical Tubular Basophilia
Slight 0 1 2 0 0 0 0
Minimal 0 4 3 0 0 0 0
Total 0 5 5 0 0 0 0

Cortical Tubular Dilatation
Slight 0 1 1 0 0 0 0
Minimal 0 3 3 0 1 1 0
Total 0 4 4 0 1 1 0

Cortical Tubules with Hyaline Droplets
Marked 0 0 1 - - - -
Moderate 0 2 2 - - - -
Slight 0 2 2 - - - -
Minimal 1 2 0 - - - -
Total 1 6 5 - - - -

Number of tissues examined 5 6 5 5 7 5 5


Incidental findings

Testes

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

A single case, characterized by unilateral severe atrophy of the seminiferous tubular epithelium and germ cell depletion, and was seen in the testis of one control male. Based on the single occurrence this finding was considered incidental.

The abnormal coloration of the sciatic nerve, adipose tissue, seminal vesicles, prostate and urinary bladder macroscopically observed in the treated animals given 300 or 1000 mg/kg/day did not correlate with any microscopic changes. The toxicological importance of this finding is unknown.

All other microscopic findings were considered to be incidental and not of toxicological importance.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced bodyweight gain at 300 mg/kg/day and frank toxicity at 1000 mg/kg/day.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity in adult male and female rats treated with Oxooil LS9 for up to 5 weeks is 100 mg/kg/day.

Executive summary:

A study was performed to GLP standard at the Laboratories of Huntingdon Life Sciences, Eye, on behalf of Evonik Oxeno GmbH., to investigate the subacute toxicity of the test substance Oxooil LS9 when administered to rats by oral gavage (OECD 422). Three groups, each comprising five male and five female CD rats, received Oxooil LS9 once daily at dosages of 100, 300 or 1000 mg/kg/day for a period of five complete weeks before termination. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose for the same duration. An additional five male and ten female rats per treatement group were used to investigate the reproductive toxicity potential of Oxooil LS9. Pertinent observations and findings in these animals are included in the assessment of the general sytemic toxicity of Oxooil LS9..

Throughout the study, data was recorded on clinical condition, detailed physical and arena observations and bodyweight. Food consumption was recorded during Weeks 1 and 2 and sensory reactivity, grip strength, motor activity, haematology and blood chemistry during Week 4. Organ weight, macroscopic and microscopic pathology investigations were undertaken at termination. Three of ten males receiving 1000 mg/kg/day were killed for reasons of animal welfare during weeks 3 or 4 of treatment. Due to the sudden deterioration of individuals within this treatment group, treatment of all males receiving 1000 mg/kg/day was terminated in week 4 and all males (including reproductive group animals) were subsequently killed. These males were observed to be of thin build and had brown staining on their body surface. Bodyweight gain and food consumption was low during the first week of treatment, and bodyweight loss was apparent during weeks 2 and 3 when compared with Controls. Macroscopic examination revealed a high number of males with abnormal pink colouration of the peripheral nerve(s), sciatic nerve, seminal vesicles, pale jejunum and pale and thickened duodenum, they were also of thin build and their fur was stained. A few males had abnormal colouration of the skin, mesenteric lymph node, adipose tissue, prostate and urinary bladder which was also distended for two males. Dark and enlarged mandibular lymph nodes, small thymus, non glandular thickened stomach, thickened and abnormal contents of the jejunum was also observed at a lower incidence. Minimal hepatocellular centrilobular hypertrophy was observed in the liver of 4/6 males and cortical tubular basophilia, cortical tubular dilatation and hyaline droplets were respectively observed in 4/6, 3/6 and 6/6 males given 1000 mg/kg/day. There were no clinical signs for females receiving 1000 mg/kg/day or animals receiving 300 or 100 mg/kg/day and sensory reactivity observation, grip strength values and motor activity scores were unaffected by treatment with Oxooil LS9. Overall mean bodyweight gain was slightly low for males (84%) and females (69%) treated at 300 mg/kg/day and females (56%) treated at 1000 mg/kg/day, when compared with Controls. Water intake assessed during Week 4 of treatment was increased for males receiving 100 mg/kg/day or above, and females receiving 1000 mg/kg/day when compared with Controls. Haematological investigations performed during Week 5 of treatment revealed, when compared with Controls, prolonged activated partial prothrombin time (APTT) in females receiving 1000 mg/kg/day. Biochemical examination of the blood plasma indicated when compared with Controls that alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase activities were lower for females receiving 1000 mg/kg/day. Alanine aminotransferase activity was low in females receiving 300 mg/kg/day and aspartate aminotransferase activity was low in males receiving 300 mg/kg/day. Total plasma cholesterol levels were higher in animals receiving 1000 mg/kg/day. In males receiving 300 mg/kg/day and animals receiving 1000 mg/kg/day, there was an increase of plasma albumin concentration and albumin to globulin ratio was increased in males receiving 1000 mg/kg/day. The analysis of organ weights after 5 weeks of treatment indicated, when compared with Controls, high mean absolute and adjusted liver and kidney weights among males that received 300 mg/kg/day and females that received 1000 mg/kg/day. Seminal vesicles absolute and adjusted weights were slightly low in males that received 300 mg/kg/day. Females that received 1000 mg/kg/day and males that received 300 mg/kg/day were observed to have abnormal pink colouration of the sciatic nerve(s) and peripheral nerve(s), when compared with Controls. Males that received 300 mg/kg/day were observed to have abnormal pink colouration of the seminal vesicles. The abnormal colouration of these tissues did not correlate with any microscopic changes, and the toxicological importance of this finding is unknown. Microscopic examination revealed the presence of hepatocellular centrilobular hypertrophy at a dose-related incidence in males that received 100 or 300 mg/kg/day and females that received 1000 mg/kg/day. Renal findings consisted of a dose related increase in the incidence and severity of hyaline droplets in the proximal convoluted tubules in the males given 100 or 300 mg/kg/day and cortical tubular basophilia and tubular dilatation were seen in the males given 100 or 300 mg/kg/day. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted. Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity in adult male and female rats treated with Oxooil LS9 for up to 5 weeks is 100 mg/kg/day.

The study is considered acceptable for classification and satisfies the guideline requirements for a rat repeated dose oral toxicity screening test.