Registration Dossier

Administrative data

Description of key information

In the systemic toxicity element of an OECD 422 study in rats, the repeated dose oral NOAEL for Oxooil LS9 is 100 mg/kg bw/day.
In a 90-day repeated dose toxicity study it can be concluded that the high dose of 200 mg/kg/day may be considered as the NOAEL.

The substance is considered to be a low toxic risk by both the dermal and inhalation routes of exposure.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose:
reference to other study
Qualifier:
according to
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
Species:
rat
Strain:
other: Crl: CD(SD)
Sex:
male/female
Details on test animals 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)
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.
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
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
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.


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.
Critical effects observed:
not specified
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.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08.09.2017 - 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietroal Natisone (UD), Italy
- Age at study initiation: 27-29 days
- Weight at study initiation: 75-99 g
- Housing: The animals were housed up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages. Nesting material was provided inside suitable bedding bags and changed at least twice a week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:12 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±2°C
- Humidity (%): 55%±15%
- Air changes (per hr):15 to 20
- Photoperiod (hrs dark / hrs light): The rooms were lit by artificial light for 12 hours each day.

IN-LIFE DATES: From: 12 September 2017 To:15 January 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
All animals were dosed once a day, 7 days a week, for a minimum of 13 consecutive weeks,
followed by a recovery period of 4 weeks for 5 males and 5 females from Groups 1 and 4.
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Each main group comprised 10 male and 10 female rats. Satellite groups from control and high dose groups included 5 animals per sex.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose selection is based on a sub-acute 28-day study according to OECD 422.
- Rationale for animal assignment (if not random): random
- Satellite group doses: 0, 200 mg/kg
- Rationale for selecting satellite groups: Investigation of the recovery from any treatment-related effects
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): random
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All clinical signs were recorded for individual animals. Once before commencement of treatment (data not tabulated) and at least once daily during the study, each animal was observed and any clinical signs recorded.
Observations were performed at the same time interval each day.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once per week during the study from the start of treatment, each animal was given a detailed clinical examination.
Each animal was observed in an open arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment group, on the day that
treatment commenced, weekly thereafter and just prior to necropsy.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
Both eyes of all animals were examined prior to the commencement of treatment by means of an ophthalmoscope, and by a slit-lamp microscope, after the instillation of 0.5% Tropicamide
(Visumidriatic®, Visufarma, Rome, Italy). Where possible, animals with non-resolving lesions were replaced with spare animals showing no ocular abnormality, from the batch initially
ordered for the study. The eyes of all animals from high-dose and control groups were re-examined duringWeek 13 of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During week 13
Further blood samples were withdrawn from all surviving animals under conditions of food
deprivation at the end ofWeek 4 of the recovery period in order to re-evaluate the clinical
chemistry parameters,
- Anaesthetic used for blood collection: Yes under isoflurane anaesthesia
- Animals fasted: Yes
- How many animals:all male and female animals from each main phase group

- Parameters checked were examined:
– Haematocrit
– Haemoglobin
– Red blood cell count
– Reticulocyte count
– Mean red blood cell volume
– Mean corpuscular haemoglobin
– Mean corpuscular haemoglobin concentration
– White blood cell count
– Differential leucocyte count
· Neutrophils
· Lymphocytes
· Eosinophils
· Basophils
· Monocytes
· Large unstained cells
– Platelets
– Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Animals fasted: Yes
- How many animals: see haematology

- Parameters checked were examined:
– Alkaline phosphatase
– Alanine aminotransferase
– Aspartate aminotransferase
– Gamma-glutamyltransferase
– Urea
– Creatinine
– Glucose
– Triglycerides
– Inorganic phosphorus
– Total bilirubin
– Total cholesterol
– Total protein
– Albumin
– Globulin
– A/G Ratio
– Sodium
– Potassium
– Calcium
– Chloride

URINALYSIS: Yes
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified

- Parameters checked were examined:
– Appearance
– Volume
– Specific gravity
– pH
– Protein
– Glucose
– Ketones
– Bilirubin
– Urobilinogen
– Blood
– Epithelial cells
– Leucocytes
– Erythrocytes
– Crystals
– Spermatozoa and precursors
– Other abnormal components

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before commencement of treatment and once per week during the study from the
start of treatment, each animal was given a detailed clinical examination.
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No



Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables
the significance of the differences amongst groups was assessed by analysis of variance.
Differences between each treated group and the control group were assessed by Dunnett’s
test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s
test before Dunnett’s test. If the data were found to be inhomogeneous, a Modified t test
(Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual
values in the computer without rounding off. Statistical analysis of histopathological finding
was carried out by means of a nonparametric Kolmogorov-Smirnov test.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs related to treatment were recorded at the daily observations performed
throughout the study. One female animal in Group 4 showed a palpable mass and was
isolated in cage.
No toxicologically relevant changes were observed at the weekly detailed clinical signs. Slight
decreases or increases in the number of rearing, statistically significant, were occasionally
observed in treated males and females, when compared to controls. Since these decreases
were minimal and only occasional, they were not considered to be toxicologically significant.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in body weight during the treatment and
recovery periods.
The slight but statistically significant decrease in body weight (up to 7% starting fromWeek
9) observed in the high dose males (200mg/kg/day), when compared to controls, was considered
incidental.
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant increase of white blood cells recorded in females dosed at 100mg/kg/day
was not dose-related, therefore it was considered incidental.
A statistically significant decrease of prothrombin time was recorded in males receiving
50mg/kg/day. Due to the absence of dose-relation, this change was considered unrelated to
treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant fluctuations of some biochemical parameters were recorded throughout
the study. The severity of the findings observed was not considered to be suggestive of tissue/organ injury
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Slight statistically significant decreases were observed at functional tests: landing foot splay
in mid- and high dose males and grip strength in high dose males, at the end of treatment;
landing foot splay and motor activity in high dose males after 4 weeks of recovery.
All these effects on functional tests and motor activity were considered incidental and
of no toxicological relevance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At completion of the dosing, there was a minimal decrease in the mean terminal body weight
of males treated at the high dose, when compared to the control group. A statistically significant
mild increase in liver mean weight was recorded in males treated at 200 (absolute and relative weights) and 100mg/kg/day (relative weight only), compared to controls.
This increase was dose-related (9% and 15% in mid- and high dose groups for absolute weights;
12% and 24% in mid- and high dose groups for relative weights) and accompanied by corroborative
histopathological changes such as centrilobular hypertrophy in the high dose males
only, which most likely represents an effect of treatment, possibly due to hepatic microsomal
enzyme induction. A statistically significant increase (+10%) in relative kidneys mean weight
was seen in males treated at 200mg/kg/day, compared to controls. This alteration correlated
microscopically with up to moderate hyaline droplets accumulation in renal proximal tubules
and was interpreted to most likely represent an effect due to enzyme induction, as well. No
other treatment-related organ weight changes were reported. Some other minor variations
in mean absolute or relative organ weights were noted in the males treated at 200mg/kg/day,
some of them being statistically significant (decrease in absolute brain mean weight and
increase in relative spleen mean weight). These variations were not considered to be a direct
effect of the test item, but to be incidental or due to the lower terminal body weights recorded
in the high dose male group.
Terminal body and organ weights of females were in the range of controls and showed no
deviations related to treatment.

Recovery sacrifice
Organ weight deviations related to treatment were not seen. The treatment-related organ
weight changes, previoulsy observed in the liver and kidneys, were completely reversible.
All organ weight variations between control and treated animals were considered to be within
the physiological range of Sprague Dawley SD rats of this age.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were noted, following gross pathology examination at final
and recovery sacrifices. All observed changes were considered spontaneous and incidental,
having a comparable incidence in control and treated groups and/or are characteristically
seen in untreated Sprague Dawley SD rats of the same age.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes considered to be associated with the oral administration of Oxooil LS9 were present in the liver of males dosed with 200 mg/kg/day (high dose) and kidneys of males treated at all dosages (50, 100 and 200 mg/kg/day).
In the liver, minimal or mild centrilobular hepatocellular hypertrophy was seen in all males (10/10) treated at the high dose. The hepatocytes were minimally enlarged with intense eosinophilic cytoplasm, compared to controls and were distributed mainly in centrilobular areas. This change correlated with an increased liver mean weight observed in males and was most likely considered related to microsomal enzyme induction. As no hepatocellular degenerative findings were present, the centrilobular hypertrophy was considered an adaptive change and not adverse. The further evaluation of the intermediate and low dose treated animals revealed no treatment-related pathomorphological changes.
In the kidneys, there was an increased severity of hyaline droplets accumulation in the proximal tubular epithelial cell in males from all treated groups in comparison to controls. The severity of this change ranged from mild to marked and showed a dose-response relationship.
Hyaline droplets in the cortical tubular cytoplasm of the kidneys resulted from a concentration of short chain hydrocarbon molecules bound to alpha 2µ-globulin. This globulin is androgen regulated, and synthesized in copious amounts in the liver of male rats only.
Hyaline droplets generally represent low molecular weight protein accumulation within lysosomes, due to disturbance of the normal balance of tubular reabsorption and hydrolysis as a result of either increased filtered protein loads or decreased catabolism. Reversible binding of chemicals or their metabolites to this abundant male-rat-specific protein is causally related to the induction of nephropathy. In fact, if the tubular cells become overloaded with the accumulation of alpha 2µ-globulin, loss of cytoplasmic integrity may occur leading to cell death, regeneration and repair. In this study, hyaline droplets accumulation was accompanied by moderate exacerbation of spontaneous nephropathy characterized by multifocal tubular basophilia, intratubular hyaline or granular casts, and/or tubular dilatation, in the high and mid-dose males, exhibiting a dose response-relationship. On the other hand, the incidence and the severity of the degenerative changes in the renal tubules of the low dose males were comparable to the concurrent controls.
All other reported findings were considered spontaneous and incidental, including the basal cell tumor originating from the dorsal region of the skin of a single high dose treated female (no. A2990085), having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

Recovery sacrifice
After 4 weeks of non-dosing, the treatment-related changes in the liver were completely reversible.
In the kidneys, the amount of hyaline droplets in proximal tubular epithelial cells was comparable between controls and high dose treated males. However, when compared to controls, there was still an increase for incidence and severity of nephropathy, characterized by multifocal cortical tubules basophilia and casts. In the treated animals, yellow-brown, intracytoplasmic granular pigment was detected in the degenerated tubular cells which may represent most likely lipofuscin deposition.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
Further details of results in tabular form are attached under "Attached background material".
Key result
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no toxicologically relevant effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
On the basis of the above mentioned results, it can be concluded that the high dose of 200 mg/kg/day may be considered as the No Observed Adverse Effect Level (NOAEL) for females and the low dose of 50 mg/kg/day the NOAEL for males.
However, since alpha 2u-globulin response, following chemical administration, appears to be unique to the male rat and this observation is not relevant for humans, the high dose of 200 mg/kg bw/d is considered the overall NOAEL and will be used for chemical safety assessment.
Executive summary:

The oral toxicity of Oxooil LS9 in Sprague Dawley rats, following daily oral administration at dose levels of 50, 100 and 200 mg/kg/day for 13 consecutive weeks, and recovery from any treatment-related effect during a period of 4 weeks, were investigated in this study.

No clinical signs related to treatment were recorded at the daily and weekly observations performed throughout the study.

Body weight and food consumption were not affected by treatment.

Statistically significant decreases were observed at functional tests performed at the end of treatment: landing foot splay in mid- and high dose males and grip strength in high dose males. Landing foot splay was still statistically significantly decreased at the end of the recovery period, as well as a significant decrease was noted in motor activity measurements after 4 weeks of recovery.

No lesions were recorded at ophthalmological examination.

No changes of toxicological relevance were observed in haematology, clinical chemistry and urinalysis.

At the end of treatment period, liver weight was statistically significantly increased in males treated at 100 and 200 mg/kg/day and kidney weight was statistically significantly increased in males treated at 200 mg/kg/day.

No treatment-related findings were reported at post mortem macroscopic observations performed at final and recovery sacrifices.

At histopathology, changes considered treatment-related were present in the liver of males dosed with 200 mg/kg/day and kidneys of males treated at all dosages (50, 100 and 200

mg/kg/day).

In the liver, hepatocellular hypertrophywas seen in all high dose males. This change correlated with an increased liver mean weight observed in males and was considered an adaptive change and not adverse. In addition, the further evaluation of the intermediate and low dose treated animals revealed no treatment-related pathomorphological changes. After 4 weeks of recovery, the treatment-related changes in the liver were completely reversible.

In the kidneys, there was an increased severity, dose-related, of hyaline droplets accumulation in the proximal tubular epithelial cell in males from all treated groups in comparison to controls. This hyaline droplets accumulation was accompanied by moderate exacerbation of spontaneous nephropathy in the high and mid-dose animals with a dose response relationship. On the other hand, the incidence and the severity of this change in the low dose males was comparable to the controls. After a 4-week recovery period, although the hyaline droplets were not observed, there was still an increase for incidence and severity of nephropathy.

On the basis of the above mentioned results, it can be concluded that the high dose of 200 mg/kg/day may be considered as the No Observed Adverse Effect Level (NOAEL) for females and the low dose of 50 mg/kg/day the NOAEL for male.

In conclusion, Oxooil LS9 induced lesions observed in the proximal tubules of the male rat kidney were considered most likely associated with lysozymal accumulation of alpha 2u-globulin and constitute a distinctive pathological entity. In fact, alpha 2u-globulin response, following chemical administration, appears to be unique to the male rat. This observation is not relevant for humans.

Thus, the high dose of 200 mg/kg bw/d is considered the overall NOAEL and will be used for chemical safety assessment.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and target substance have similar toxicological properties because they
- have similar chemical structures with common functional groups: the target and source substances are mainly branched C8-hydrocarbons.
- have similar physico-chemical properties,
- have the same mode of action, which is well-established for low molecular weight hydrocarbons

This read-across hypothesis corresponds to scenario 2 - different compounds have qualitatively and quantitatively similar properties - of the read-across assessment framework i.e. properties of the target substance are predicted to be similar to those of the source substance.

Based on the available experimental data, the read-across strategy is supported by close structural analogy as well as similar toxicological data independent from the route of exposure.

Therefore, read-across from the existing subchronic toxicity studies via inhalation conducted with the source substances isooctene and light alkylate naphtha distillate is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached information

3. ANALOGUE APPROACH JUSTIFICATION
see attached information

4. DATA MATRIX
see attached information
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: air
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 hours per day / 5 days per week
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:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
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:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no mortality. Clinically, slight ataxia (from day 9 onward) and salivation (from day 8 onward) were observed at 15000 mg/m3 indicating marginal irritation and systemic toxicity of Isooctene.

BODY WEIGHT AND WEIGHT GAIN
In male animals the mean body weight and body weight change were found to be significantly decreased when compared with the controls.

FOOD CONSUMPTION
A transient decrease of food consumption was observed in male and female animals on study day 7, which did not occur again after the animals adjusted to the test substance and the exposure procedure.

FOOD EFFICIENCY
In male animals, food efficiency was found to be significantly decreased when compared with the controls.

HAEMATOLOGY
After inhalation of the substance for about 2 months there was noticed a slight increase of the median of the platelet counts in the parent males exposed to 15000 mg/m3.
The median of the prothrombin time was decreased in the male parents beginning in group 2 (5000 mg/m3), and in the female parents in the group exposed to 15000 mg/m3, only.

CLINICAL CHEMISTRY
In the female parents of the 15000 mg/m3 group the median of the aspartate aminotransferase activity was decreased after 2 months of substance inhalation, in the male parents of the same dose group there was assessed an increased median of the alanine aminotransferase activity. The median of the γ-glutamyltransferase activity was slightly increased in the 15000 mg/m3 group of the rats of both sexes, but this was only statistically significant in the males.
In the male rats the median of the total protein levels was increased beginning in the 5000 mg/m3 dose group. Regarding the medians of the protein fractions albumin and globulin the significant increase was only assessed in the 15000 mg/m3 dose group. The median of the globulin fraction was increased in the female rats of the 15000 mg/m3 dose group, too. The male parents showed increased medians of the bilirubin concentration in the 15000 mg/m3 dose group, and decreased medians of the glucose levels starting in the 5000 mg/m3 dose group. The medians of the cholesterol values were increased in the rats of both sexes beginning in the 5000 mg/m3 dose groups. The median of the triglyceride concentration was increased in the 15000 mg/m3 dose group of the females, only. In this group, there was found a decrease of the urea and the creatinine concentration, too.
The medians of the potassium and calcium serum levels were increased in the rats of both sexes (potassium: males beginning in the 5000 mg/m3, and in the females in the 15000 mg/m3 dose group; calcium: in the 15000 mg/m3 dose group of both sexes). The magnesium levels were increased only in the male parents beginning in the 1000 mg/m3 dose group. The increase in the low-concentration group was still within the range of biological variations. Therefore, this is not regarded as a toxicologically relevant effect.

ORGAN WEIGHTS
Absolute- and relative liver and kidney weights of males were significantly increased in animals exposed to 5000 and 15000 mg Isooctene/m3

HISTOPATHOLOGY: NON-NEOPLASTIC
The treatment-related microscopic changes observed were increased hyaline cast(s) and basophilic cortical tubules in the kidneys of male animals from group 2 (5000 mg/m3) onward. In addition, in males there was suspected greater propensity for the development of large hyaline droplets related to the proximal tubules compared to controls. Mallory-Heidenhain stain of all male kidneys and immunhistochemistry of selected male kidneys with an alpha 2u globulin antibody revealed a concentration-dependent accumulation of alpha 2u globulin in cortical tubular cells in all treated groups. However, in the absence of any treatment-related morphological signs of cytotoxicity, the minimal to slight accumulation of alpha 2u globulin in the group 1000 mg/m3 males is regarded to be adaptive and non-adverse in character.
In the respiratory tract no signs of toxicity were observed.

Dose descriptor:
NOAEC
Effect level:
1 000 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
no
Conclusions:
The NOAEC in this study was 1000 mg/m³ for male and female rats. The male kidney was identified as the target organ as increased hyaline casts and basophilic cortical tubules were observed in the kidneys of male animals exposed to 5000 mg/m³ onwards. These changes were associated with accumulation of alpha 2u globulin in cortical tubular cells. The presence of increased hyaline casts and basophilic cortical tubules are an indication of proximal tubular cell damage and a classical effect of α2µ-nephropathy which is male rat-specific effect not considered relevant human hazard assessment.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1 000 mg/m³
Study duration:
subchronic
Experimental exposure time per week (hours/week):
30
Species:
rat
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
15 000 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral expoure

The repeated dose oral toxicity of Oxooil LS9 was investigated in a GLP subacute study in rats (OECD 422). Three groups, each comprising five male and five female CD rats, received Oxooil LS9 by gavage 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 treatment group were used to investigate the reproductive toxicity potential of Oxooil LS9. Pertinent observations and findings in these latter animals are included in the assessment of the general systemic toxicity of Oxooil LS9.

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 group, treatment of all males receiving 1000 mg/kg/day was terminated in week 4 and all surviving males were subsequently killed. These males were observed to be of thin build and had brown staining on their body surface; these signs were indicative of their poor condition.  

Overall mean bodyweight gain was reduced 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. These effects are considered to represent a non-specific toxic response to treatment with Oxooil LS9. 

There was a dose-related increase in the incidence of minimal hepatocellular centrilobular hypertrophy in the livers of males that received 100, 300 or 1000 mg/kg/day and in females that received 1000 mg/kg/day, which correlated with significant increase of the mean absolute organ weight. Hypertrophy is generally indicative of metabolic adaptation and is commonly encountered in rats following exposure to high levels of a xenobiotic and, as such, is not of direct toxicological importance. The lower levels of alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase activities and higher cholesterol level amongst animals receiving 300 or 1000 mg/kg/day are considered to reflect the minimal cellular changes of the liver, but are not indicative of liver damage.

Microscopic examination of the kidneys revealed a dose-related increase in the incidence of hyaline droplets, cortical tubular basophilia and dilatation in malesgiven 100, 300 or 1000 mg/kg/day. Hyaline droplets in the cortical tubular cytoplasm of the kidneysresult from a concentration of short chain hydrocarbon molecules bound to α2µ globulin. This globulin is specific to male rats and is produced by the liver. It is not produced in female rats and there is no similar risk for human exposure.  

The abnormal pink 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.

There were no effects of treatment on the sensory reactivity, grip strength or motor activity.  

Based on the results of this study, it was concluded that for male and female CD rats treated with Oxooil LS 9 for up to 5 weeks, the No-Observed-Adverse-effect-level (NOAEL) for systemic toxicity is 100 mg/kg/day. 

 

 

The oral toxicity of Oxooil LS9 in Sprague Dawley rats, following daily oral administration at dose levels of 50, 100 and 200 mg/kg/day for 13 consecutive weeks, and recovery from any treatment-related effect during a period of 4 weeks, were investigated in this study.

No clinical signs related to treatment were recorded at the daily and weekly observations performed throughout the study.

Body weight and food consumption were not affected by treatment.

Statistically significant decreases were observed at functional tests performed at the end of treatment: landing foot splay in mid- and high dose males and grip strength in high dose males. Landing foot splay was still statistically significantly decreased at the end of the recovery period, as well as a significant decrease was noted in motor activity measurements after 4 weeks of recovery.

No lesions were recorded at ophthalmological examination.

No changes of toxicological relevance were observed in haematology, clinical chemistry and urinalysis.

At the end of treatment period, liver weight was statistically significantly increased in males treated at 100 and 200mg/kg/day and kidney weight was statistically significantly increased in males treated at 200 mg/kg/day.

No treatment-related findings were reported at post mortem macroscopic observations performed at final and recovery sacrifices.

At histopathology, changes considered treatment-related were present in the liver of males dosed with 200 mg/kg/day and kidneys of males treated at all dosages (50, 100 and 200

mg/kg/day).

In the liver,hepatocellular hypertrophywas seen in all high dose males. This change correlated with an increased liver mean weight observed in males and was considered an adaptive change and not adverse. In addition, the further evaluation of the intermediate and low dose treated animals revealed no treatment-related pathomorphological changes. After 4 weeks of recovery, the treatment-related changes in the liver were completely reversible.

In the kidneys, there was an increased severity, dose-related, of hyaline droplets accumulation in the proximal tubular epithelial cell in males from all treated groups in comparison to controls. This hyaline droplets accumulation was accompanied by moderate exacerbation of spontaneous nephropathy in the high and mid-dose animals with a dose response relationship. On the other hand, the incidence and the severity of this change in the low dose males was comparable to the controls. After a 4-week recovery period, although the hyaline droplets were not observed, there was still an increase for incidence and severity of nephropathy.

On the basis of the above mentioned results, it can be concluded that the high dose of 200 mg/kg/day may be considered as the No Observed Adverse Effect Level (NOAEL) for females and the low dose of 50 mg/kg/day the NOAEL for male.

In conclusion, Oxooil LS9 induced lesions observed in the proximal tubules of the male rat kidney were considered most likely associated with lysozymal accumulation of alpha 2u-globulin and constitute a distinctive pathological entity. In fact, alpha 2u-globulin response, following chemical administration, appears to be unique to the male rat. This observation is not relevant for humans.

Thus, the high dose of 200 mg/kg bw/d is considered the overall NOAEL and will be used for chemical safety assessment.

 

Dermal exposure

A repeated dose dermal toxicity study has not been conducted on Oxooil LS9. The delayed contact hypersensitivity observed in the murine local lymph node assay (Section 7.4.1) shows that absorption of Oxooil LS9 occurs through the skin, although the weakness of the response (EC3, 37%) and the absence of systemic effects following acute dermal exposure (Section 7.2.2) indicate that the degree of absorption would not be expected to be any greater than by the oral route. Since relevant specific target organ toxicity was not observed by the oral route at 300 mg/kg/day in an OECD 422 screening study (Section 7.5.1), it is considered unlikely that Oxooil LS9 will represent a long-term systemic toxic risk via the dermal route.

 

Inhalation exposure

No inhalation toxicity studies were conducted with the target substance Oxooil LS9. However, relevant and reliable data are available for structurally related substances. A justification for read-across is attached to the respective target record.

 

Isooctene (CAS 11071-47-9) has been tested in a 90-day inhalation study in rats (combined OECD 422 and 413) at air concentrations of 1000, 5000 and 15,000 mg/m3. Absolute and relative liver and kidney weights were significantly increased in the mid- and high-exposure groups. The only histopathological effects were increased hyaline casts and basophilic cortical tubules, along with accumulation ofalpha-2u-globulinin the cortical tubular cells of male rats at 5000 and 15,000 mg/m3. These male rat-specific kidney effects are indicative of alpha-2u-globulin nephropathy and are not considered relevant to humans. The NOAEL for systemic toxicity is 1000 mg/m3.

 

A 13-week inhalation toxicity study was conducted using whollyvaporized light alkylate naphtha distillate(LAND-2) generated in nitrogen (Schreiner et al., 1998). Male and female rats were exposed by inhalation in whole-body exposure cages 6 hours/day, 5 days/week for 13 weeks at analytical concentrations of 0, 668, 2220, and 6646 ppm. All animals survived the treatment period and were sacrificed according to study design at the end of week 13 or 18 (recovery group). No test-related observations were noted in the exposure chambers during any exposure period for any treatment groups or during non-exposure periods. From weekly clinical observations, the only apparent treatment-related finding was an increased incidence of red facial staining in both male and female rats in the high dose group. At week 13, there were statistically significant dose-related increases in absolute and relative kidney weights in males of all 3 treatment groups. The kidney weights of high-dose males remained elevated after the recovery period. These increases correlated with microscopic observations of hyaline droplet formation in the proximal convoluted tubules considered to contain an alpha2-microglobulin-hydrocarbon complex as well as an increase in incidence and severity of nephropathy and dilated tubules at the corticomedullary junction. These microscopic finding are characteristic of ‘light hydrocarbon nephropathy” also known as hyaline droplet nephropathy and are male rat specific. Therefore, these effects are not considered to be relevant to humans. Statistically significant increases in absolute and relative liver weights were observed in high-dose male and female rats at week 13 after sacrifice. Differences were not present after the recovery period and had no microscopic correlate.

The no-observed-effects level (NOEL) for the test substance was 2220 ppm (8100 mg/m³)for subchronic toxicity and >/ = 6646 ppm (24300 mg/m³) for neurotoxicity.

 

The NOAEC obtained with Isooctene was lower than the NOAEC obtained with light alkylate naphtha distillate, therefore, the NOAEC of 1000 mg/m³ will be used as key value for inhalation toxicity.

 

Since 1000 mg/m³ is equivalent to a dosage of 205 mg/kg/day (ECHA Technical Guidance, Appendix R.8-2, May 2008), and the subacute oral NOAEL is 100 mg/kg bw/day (Section 7.5.1), based on these data, Oxooil LS9 is not expected to be a long-term toxic risk by the inhalation route.

Justification for classification or non-classification

No classification for repeated exposure, specific target organ toxicity is therefore indicated according to the general classification and labelling requirements for dangerous substances and preparations (Directive 67-548-EEC) or the classification, labelling and packaging (CLP) regulation (EC) No 1272/2008.